Biocatalytic oxidation of 1,4-diols and γ-lactols into γ-lactones: Application to chemoenzymatic synthesis of drospirenone

Article abstract of DOI:10.1007/s00706-012-0921-3

Oxidation of 1-alkyl-1,4-butanediols with Acetobacter aceti MIM 2000/28 gave the corresponding γ-lactones in good yields. The biotransformation occurred with intermediate formation of γ-lactols, which are also substrates for oxidation with Acetobacter aceti MIM 2000/28, as validated by selective biotransformation of 6β,7β;15β,16β-dimethylene-3- oxo-17α-pregn-4-en-21,17-carbolactol to drospirenone.

Full text of DOI:10.1007/s00706-012-0921-3

Monatsh Chem (2013) 144:735–737
DOI 10.1007/s00706-012-0921-3
ORIGINAL PAPER
Biocatalytic oxidation of 1,4-diols and c-lactols into c-lactones:
application to chemoenzymatic synthesis of drospirenone
•
Diego Romano Martina Contente Tiziana Granato
•
•
•
William Remelli Paolo Zambelli Francesco Molinari
•
Received: 27 September 2012 / Accepted: 31 December 2012 / Published online: 1 March 2013
Ó Springer-Verlag Wien 2013
Abstract Oxidation of 1-alkyl-1,4-butanediols with
Acetobacter aceti MIM 2000/28 gave the corresponding
c-lactones in good yields. The biotransformation occurred
with intermediate formation of c-lactols, which are also
substrates for oxidation with Acetobacter aceti MIM
2000/28, as validated by selective biotransformation of
6b,7b;15b,16b-dimethylene-3-oxo-17a-pregn-4-en-21,17-
carbolactol to drospirenone.
achieved by several methods [2–19]; non-hazardous oxi-
dants, i.e. TEMPO (2,2,6,6-tetramethylpiperidin-1-yloxy)
or derivatives thereof, have also been used [20].
Biological oxidation of alcohols is a potential alterna-
tive, because it occurs under mild pH, temperature, and
pressure conditions without the requirement of toxic
reagents, and often with high chemo, regio, and stereose-
lectivity; dehydrogenases and oxidases (both as isolated
enzymes or bound to microbial cells) can sometimes be
used to perform selective oxidation of primary alcohols in
the presence of secondary alcohols, thus enabling green
and efficient lactonization of diols. Different methods have
been reported for enzymatic [21–26] or microbial [26, 27]
oxidation of alcohols and diols; the cofactor-dependency of
oxidative enzymes has long been regarded as a major
obstacle to preparative application of isolated enzymes,
whereas use of aerobic whole cells has the practical
advantage that reduced cofactors are re-oxidized via the
respiratory chain, therefore shifting the equilibrium toward
oxidation. The preparative use of whole microbial cells is
often hampered by further degradation of the desired
molecule. An exception to the degradative aerobic metab-
olism is the one found in acetic acid bacteria [28–34].
Acetic acid bacteria, which contain several membrane-
bound selective dehydrogenases, can be used with advan-
tage as chemo, regio, and enantioselective biocatalysts for
oxidation of primary and secondary alcohols and diols
[29]; they often ensure high yields, because the product of
oxidation is not further degraded. In previous work, it was
observed that biotransformation of racemic 1,4-nonadiol
with different acetic acid bacteria occurred with formation
of c-nonanoic lactone as final product [30]. In this work,
we have investigated the use of Acetobacter aceti MIM
2000/28 [30, 35] for oxidation of different 1,4-butanediols
and c-lactols.
Keywords Enzymatic oxidation Á Acetobacter
c-Lactones Á Drospirenone
Introduction
Oxidative lactonization of diols can be achieved by dif-
ferent chemical approaches. The reaction usually proceeds
via oxidation of diols to the corresponding hydroxy alde-
hydes, which are in equilibrium with the lactols; finally the
lactols are oxidized to lactones. Functionalised lactones are
important intermediates in the synthesis of biologically
active molecules, including antibiotics, antifungal com-
pounds, flavour components, and pheromones. Oxidation
by use of excess silver carbonate is easy to accomplish, but
a large excess of expensive silver salts is required [1].
Metal-catalysed oxidative lactonization of diols can be
D. Romano Á M. Contente Á T. Granato Á W. Remelli Á
P. Zambelli Á F. Molinari (&)
Department of Food, Environmental and Nutritional Sciences
(DeFENS), University of Milano, Via Celoria 2,
20133 Milan, Italy
e-mail: francesco.molinari@unimi.it
123

736
D. Romano et al.
Results and discussion
with no traces of c-butyrolactone or other final and tran-
sient products (73 % unreacted substrate). The preference
for the oxidation of primary alcohols by Acetobacter aceti
MIM 2000/28 was confirmed for oxidation of 1,4-butane-
diol, which was rapidly (2 h) and completely transformed
into succinic acid.
First, experimental conditions (biocatalyst concentration,
temperature, pH, type of buffer) for oxidation of (R,S)-1,4-
nonadiol were optimised, using cells grown as described
elsewhere [35]. The best result (Table 1, entry 5) was
achieved when the biotransformation was performed with
cells centrifuged and suspended (10 g dry cells/dm3) in
phosphate buffer (0.1 M, pH 6.8). It is interesting to
observe that slight differences in yields were observed in
the pH range 5–8, showing the condition had no important
effect on lactone formation. Oxidation of other 1-alkyl-1,4-
butanediols (Scheme 1) was performed under the opti-
mised conditions (Table 1).
c-Nonalactol was also used as substrate, giving c-non-
alactone (2e, 87 % yield after 4 h) [27]. The ability of
Acetobacter aceti MIM 2000/28 to convert c-lactols into c-
lactones was utilised for oxidation of steroid carbolactol 7
(6b,7b;15b,16b-dimethylene-3-oxo-17a-pregn-4-en-21,17-
carbolactol) into 6b,7b;15b,16b-dimethylene-3-oxo-17a-
pregn-4-ene-21,17-carbolactone, commonly known as
drospirenone (8, Scheme 2). Drospirenone is a synthetic
steroid which is used as a hormonal contraceptive [36].
The biotransformation occurred with no by-product
formation and 76 % yield of the recovered product; the
highest yields were obtained when ethanol was used as co-
solvent (to contribute to substrate solubilisation) and as co-
substrate (to activate metabolism by acetic acid bacteria).
In conclusion, report that Acetobacter aceti MIM
2000/28 was a good biocatalyst for preparation of struc-
turally different c-lactones starting form 1,4-diols and c-
lactols, under mild pH and temperature conditions, in
water.
The reaction occurred rapidly, affording c-lactones as
the only detectable product; the amount of biocatalyst used
(10 g dry weight/dm3) was relatively low compared with
biotransformation performed with isolated enzymes [24,
25]. Enantioselectivity was usually low, as expected,
because the reactive site is quite remote from the stereo-
genic centre involved in the resolution.
The corresponding c-lactols were observed as transient
intermediates (by GC analysis); no 4-hydroxy acids were
observed. The possibility the lactones were formed by
enzymatic lactonization of 4-hydroxy acid was ruled out by
the observation that 4-hydroxybutyric was completely
converted into succinic acid ([97 %), and 4-hydroxypen-
tanoic acid was converted by Acetobacter aceti MIM
2000/28 into 4-oxopentanoic acid only (27 % after 24 h),
Experimental
1H NMR and 13C NMR spectra were recorded on a Bruker
AC 200 instrument. Enantiomeric composition was rou-
tinely determined by gas chromatographic analysis using a
chiral capillary column (diameter 0.25 mm, length 25 m,
thickness 0.25 lm, DMePeBeta-CDX-PS086, MEGA,
Legnano, Italia). The absolute configuration of the products
was determined by comparison with the optical rotation of
authentic samples of the optically pure enantiomers.
Table 1 Molar conversion (after 3 h) and E value of the biotrans-
formation of 4-hydroxy alcohols to c-lactones with whole cells (10 g
dry cells/dm3) of Acetobacter aceti MIM 2000/28
Entry
Substratea
R
Yield/%
Product
E valueb
1
2
3
4
5
6
a
1a
1b
1c
1d
1e
1f
Methyl
Ethyl
46
45
53
59
70
74
2a
2b
2c
2d
2e
2f
2.8 S
2.5 S
2.2 S
2.2 S
2.2 S
2.2 S
n-Propyl
n-Butyl
n-Pentyl
n-Hexyl
Biotransformations
Biotransformations of 1a–1f were performed with 24-h
submerged cultures of Acetobacter aceti MIM 2000/28
(Microbiologia Industriale Milano). Submerged cultures
were conducted in a GlyY medium (glycerol 25 g/dm3,
Reactions were performed using 18 mM of the substrates
b
Enantiomeric composition of c-lactones was determined before
50 % conversion
Scheme 1
R
O
O
R
OH
R
O
OH
OH
1a-1f
2a-2f
123

Biocatalytic oxidation of 1,4-diols and c-lactols into c-lactones
737
Scheme 2
OH
O
O
O
O
O
7
8
yeast extract 10 g/dm3, pH 5), in 1-dm3 Erlenmeyer flasks
containing 150 cm3 medium, on a reciprocal shaker
(100 spm). Biotransformations were performed with
growing cells or with cells centrifuged and suspended in
phosphate buffer at different pH. Neat substrates were
directly added to the suspensions and flasks were shaken on
a reciprocal shaker (100 spm). After centrifugation, the
aqueous phase was acidified with 0.1 M HCl and extracted
with EtOAc; the crude products were purified by flash
chromatography. Biotransformation of carbolactol 7 was
performed with 24 h growing cells by adding 300 mg
substrate in 10 cm3 ethanol; after 24 h, the biotransfor-
mation mixture was centrifuged and the aqueous phase was
extracted with dichloromethane. The organic phase was
dried over Na2SO4, filtered, and the solvent was removed
from the filtrate under reduced pressure. The crude product
was purified by crystallization from isopropyl acetate.
Yield 226 mg (76 %); m.p.: 197–199 °C; [a]2D5 = -182°
dm-1 g-1 cm3 (c = 0.5 %, CHCl3); analytical data agree
with those reported in Ref. [36].
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Acknowledgments The substrate 6b,7b;15b,16b-dimethylene-3-
oxo-17a-pregn-4-en-21,17-carbolactol was kindly furnished by In-
dustriale Chimica (Saronno, Italy).
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