Seawater-Based Biocatalytic Strategy: Stereoselective Reductions of Ketones with Marine Yeasts

Article abstract of DOI:10.1002/cctc.201600947

The large consumption of freshwater in fermentations and bio-transformations is a matter of concern for the sustainability of many bio-processes. The use of seawater to perform bio-processes is a sustainable alternative. In this work, we used marine yeasts from deep-sub-seafloor sediments grown in seawater as bio-catalysts to perform the stereoselective reduction of different ketones, and the bio-transformations were accomplished in seawater as well. Strains of Meyerozyma guilliermondii and Rhodotorula mucilaginosa were able to reduce different aromatic ketones with high molar conversions and moderate-to-high enantioselectivity with no significant differences between bio-catalysis performed in seawater and freshwater. Finally, the selected marine yeasts were used for the reduction of key intermediates in seawater for the synthesis of molecules of pharmaceutical interest (desogestrel, norgestrel, gestodene, pramipexole).

Full text of DOI:10.1002/cctc.201600947

DOI: 10.1002/cctc.201600947
Full Papers
Seawater-Based Biocatalytic Strategy: Stereoselective
Reductions of Ketones with Marine Yeasts
Immacolata Serra,[a] Benedetta Guidi,[b] Gaetan Burgaud,[c] Martina L. Contente,[a]
Patrizia Ferraboschi,[b] Andrea Pinto,*[d] Concetta Compagno,[a] Francesco Molinari,[a] and
Diego Romano*[a]
The large consumption of freshwater in fermentations and bio-
transformations is a matter of concern for the sustainability of
many bio-processes. The use of seawater to perform bio-pro-
cesses is a sustainable alternative. In this work, we used marine
yeasts from deep-sub-seafloor sediments grown in seawater as
bio-catalysts to perform the stereoselective reduction of differ-
ent ketones, and the bio-transformations were accomplished
in seawater as well. Strains of Meyerozyma guilliermondii and
Rhodotorula mucilaginosa were able to reduce different aro-
matic ketones with high molar conversions and moderate-to-
high enantioselectivity with no significant differences between
bio-catalysis performed in seawater and freshwater. Finally, the
selected marine yeasts were used for the reduction of key in-
termediates in seawater for the synthesis of molecules of
pharmaceutical interest (desogestrel, norgestrel, gestodene,
pramipexole).
Introduction
Seawater is a widely available and inexpensive resource, which
is a promising and sustainable alternative to freshwater in
water-saving bio-processes (fermentations and bio-catalytic
processes),[1] especially if we consider that integrated filtration
techniques may help to circumvent possible regulatory con-
straints for food and pharmaceutical applications.[2] Integrated
methods (filtration techniques, nanoparticles) can be applied
to remove by-products from the sea(waste)water bio-process
effluents. Consequently, difficult-to-degrade chemicals are not
sent back to the sea.[1b] Marine microorganisms are a logical
but still largely untapped resource for use as cell factories in
seawater-based bio-processes.[3] Enzymes produced by marine
microorganisms are naturally endowed with an excellent stabil-
ity towards high salinity, as a consequence of different struc-
tural adjustments adopted to cope with high ionic strength
conditions.[3a,4] Interestingly, adaptation to high salinity
corresponds to an increased tolerance towards organic sol-
vents, a requisite for many enzymes to be used as bio-catalysts
in organic chemistry. Marine yeasts have been seldom exploit-
ed so far for bio-technological applications, especially to per-
form bio-processes under medium- or high-salinity conditions,
such as those found in seawater (a typical salt concentration in
seawater is 25–35 practical salinity unit, PSU).[5,6] Culture-based
analysis of fungi isolated from deep-sub-surface sediments
showed that isolates affiliated with Meyerozyma and Rhodotor-
ula spp. were the most common yeasts encountered.[7]
The stereoselective reduction of pro-chiral ketones into
chiral alcohols catalysed by ketoreductases (KREDs) and alcohol
dehydrogenases (ADHs) is an established tool for organic
chemists.[8] KREDs and ADHs with improved selectivity and ro-
bustness are needed to develop bio-processes under different
constraints. In this context, beside the quintessential yeast
Saccharomyces cerevisiae,[9] a number of non-conventional
yeasts have been exploited as sources of new stereoselective
KREDs,[10] whereas only a few studies have considered the use
of marine fungi for the reduction of ketones.[11]
[a] Dr. I. Serra, Dr. M. L. Contente, Prof. C. Compagno, Prof. F. Molinari,
Dr. D. Romano
Department of Food, Environmental and Nutritional Science (DeFENS)
University of Milan
The bio-prospecting of marine yeasts from untapped deep-
sea extreme habitats with special reference to stereoselective
ketone reduction activity is the main aim of this study. Marine
yeasts isolated from deep-sub-surface sediments were grown
and used in seawater for the stereoselective reduction of dif-
ferent pro-chiral ketones.
via Mangiagalli 25, 20133 Milan (Italy)
E-mail: diego.romano@unimi.it
[b] B. Guidi, Prof. P. Ferraboschi
Department of Medical Biotechnology and Translational Medicine
University of Milan
Via Saldini 50, 20133 Milan (Italy)
[c] Dr. G. Burgaud
Laboratoire Universitaire de Biodiversitꢀ et Ecologie Microbienne
Universitꢀ de Brest
29280 Plouzane (France)
Results and Discussion
[d] Dr. A. Pinto
Marine yeasts (8 strains of Meyerozyma guilliermondii and 12
strains of Rhodotorula mucilaginosa) isolated previously from
different deep-sub-seafloor sediment depths at Canterbury
Basin (New Zealand)[7] were grown in a liquid medium (yeast
Department of Pharmaceutical Sciences (DISFARM)
University of Milan
Via Mangiagalli 25, 20133 Milan (Italy)
E-mail: andrea.pinto@unimi.it
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Figure 1. Growth (OD at 600 nm) of marine yeasts after 48 h in YPD medium based on freshwater or seawater; results are an average of five replicates.
extract, peptone, dextrose; YPD) prepared with purified fresh-
water or micro-filtered seawater (Figure 1).
From the point of view of stereoselectivity, all the tested
strains gave ketone reduction that followed the so-called
Prelog rule with the formation of the alcohols with an S config-
uration. Overall, bio-processes performed entirely in seawater
gave good results, although a slightly poorer activity was gen-
erally observed than for that in freshwater.
Three strains appeared to be particularly halo-tolerant and
showed a similar production of biomass in freshwater- and in
seawater-based media, namely: Meyerozyma guilliermondii
UBOCC-A-214008 isolated at 137 metres below the seafloor
(mbsf), Rhodotorula mucilaginosa UBOCC-A-214025 isolated at
4 mbsf and Rhodotorula mucilaginosa UBOCC-A-214036 isolat-
ed at 765 mbsf. Therefore, the ketoreductase activity of these
strains was checked towards structurally different substrates.
The ketoreductase activities of the yeasts grown in freshwa-
ter- and seawater-based media were first compared by study-
ing the reduction of 1-arylethanones (1a–d) (Table 1). 1-Aryle-
thanones are substrates of choice to test ketoreductase activity
and to find new enzymes with potential as stereoselective
bio-catalysts.[12]
Asymmetric b-hydroxy nitriles are valuable chiral intermedi-
ates for the synthesis of a number of biologically active com-
pounds; hence, we evaluated the reduction of 3-oxo-3-arylpro-
panenitriles 1e–g with marine yeasts in seawater. The bio-cata-
lytic reduction of b-ketonitriles has been explored using both
isolated enzymes (which need systems for co-factor regenera-
tion)[13] and whole cells;[14] the latter approach has the advant-
age of that it can supply co-factors and systems naturally for
their regeneration but it is often hampered by the occurrence
of competitive alkylation,[15] which limits the preparative scope
of these bio-transformations. Two distinct behaviours were ob-
served for the yeasts employed: strains of R. mucilaginosa gave
the reduction products 2e–g (Table 2) as the only detectable
compounds obtained by bio-transformation with a high enan-
tioselectivity to afford optically pure (S)-2e–g. The yields and
enantioselectivity obtained using the two strains are remark-
able; the high selectivity (both because of the avoidance of
by-products and enantioselectivity) in the reduction of aromat-
ic b-ketonitriles with whole cells of yeasts was observed previ-
ously mostly with strains that belong to the genus Rhodotoru-
la.[16] However, M. guilliermondii UBOCC-A-214008 gave the
ethylated ketones 3e–g as major products and traces of (S)-3-
hydroxy-3-arylpropanenitriles 2e–g. Ethylated ketones resulted
from non-enzymatic aldol condensation between aromatic
b-ketonitriles and acetaldehyde (formed by the yeasts in the
presence of glucose) followed by the reduction of activated
C=C by enoate reductases (Scheme 1).[16] This different behav-
iour seems to indicate the presence of pyruvate decarboxylase
activity in M. guillermondii, which is lacking in R. mucilaginosa
strains. The bio-transformations with cells grown and used in
freshwater gave similar results (data not shown).
Whole cells obtained after growth in freshwater-based YPD
medium were suspended in a phosphate buffer medium
(pH 7.5, 0.1m), whereas cells obtained after growth in seawa-
ter-based YPD medium were suspended in seawater; 5% glu-
cose and 10 mm substrate were added to the bio-transforma-
tion media. As the pH of the filtered seawater was 7.5, a phos-
phate buffer at the same pH was employed. No activity was
found towards acetophenone 1a with strains of Meyerozyma
guilliermondii (entries 1–2, Table 1), whereas the selected
strains of Rhodotorula mucilaginosa gave (S)-1-phenylethanol
with high yield and enantioselectivity (entries 9–10 and 17–18,
Table 1). The bio-transformation of 1a occurred with no signifi-
cant differences between freshwater or seawater. The two
strains of Rhodotorula mucilaginosa were not active for 1b (en-
tries 11–12 and 19–20, Table 1) but reduced 1c and 1d to a dif-
ferent extent. 1-(Furan-2-yl)ethanone (1d) was reduced enan-
tioselectively with high yields and high rates by the two strains
of R. mucilaginosa both in freshwater and seawater (en-
tries 15–16 and 23–24, Table 1). With 1c, a higher efficiency in
the reduction to enantiopure (S)-1-(thiophen-2-yl)ethanol (2c)
could be observed in freshwater (entries 13 and 21, Table 1).
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Table 1. Reduction of 1-aryl-2-ethanones 1a–d (10 mm) with marine yeasts.[a]
Entry
Strain
Medium
Substrate
Yield [%]
ee [%]
n.d.
n.d.
97
88
n.d.
t [h]
1
2
3
4
5
6
7
8
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
M. guilliermondii UBOCC-A-214008
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
R. mucilaginosa UBOCC-A-214036
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
freshwater
seawater
1a
1a
1b
1b
1c
1c
1d
1d
1a
1a
1b
1b
1c
1c
1d
1d
1a
1a
1b
1b
1c
1c
1d
1d
<5
<5
90
48
48
24
48
48
48
48
48
8
65
<5
<5
<5
<5
> 97
> 97
<5
<5
72
60
94
87
> 97
> 97
<5
<5
72
n.d.
n.d.
n.d.
9
> 98
> 98
n.d.
n.d.
> 98
> 98
> 98
> 98
> 98
> 98
n.d.
n.d.
> 98
> 98
>98
>98
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8
48
48
24
24
3
3
8
8
48
48
48
48
3
61
80
72
3
[a] Experimental conditions: Cells were grown in freshwater-YPD or in seawater-YPD medium. Bio-transformations were performed for 24 h with freshly
prepared cells (50 mgdry weight mLÀ1). Cells grown in freshwater-YPD medium were suspended in phosphate buffer (pH 7.5, 0.1M), whereas cells grown in sea-
water-YPD medium were suspended in seawater. Bio-transformations were performed in the presence of 5% glucose.
Table 2. Reduction of b-ketonitriles 1e–g (10 mm) with R. mucilaginosa
grown in YPD-seawater medium.[a]
Table 3. Reduction of ethyl secodione 1h (10 mm) with marine yeasts
grown in YPD-seawater medium.[a]
Entry
Strain
Yield [%]
2h/3h
Entry Strain
Substrate Yield [%] ee [%]
1
2
3
M. guilliermondii UBOCC-A-214008
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214036
<5
97
96
n.d.
63/37
92/8
1
2
3
4
5
6
R. mucilaginosa UBOCC-A-214025 1e
93
85
88
92
93
79
>98
>98
>98
>98
>98
>98
R. mucilaginosa UBOCC-A-214025 1 f
R. mucilaginosa UBOCC-A-214025 1g
R. mucilaginosa UBOCC-A-214036 1e
R. mucilaginosa UBOCC-A-214036 1 f
R. mucilaginosa UBOCC-A-214036 1g
[a] Reaction conditions: Bio-transformations were performed for 24 h with
freshly prepared cells (50 mgdry weight mLÀ1) suspended in seawater in the
presence of 3.5% EtOH. Yields and diastereoselectivity after 24 h.
[a] Reaction conditions: Bio-transformations were performed for 24 h with
freshly prepared cells (50 mgdry weight mLÀ1) suspended in seawater in the
presence of 5% glucose. Yields and enantioselectivity after 24 h.
thesis of a number of hormonal contraceptives (i.e., desoges-
trel, norgestrel, gestodene).[18] The bio-transformation of 1h is
complicated by the low solubility of the substrate in water; re-
cently, we reported the use of different bio-catalysts for the
stereoselective mono-reduction of 1h and noticed that the use
of EtOH (both to increase solubility and favour co-factor regen-
To further explore the substrate scope of the selected
marine yeasts, ethyl secodione derivative 1h was tested as
a substrate (Table 3). The stereoselective reduction of the achi-
ral diketone 1h provides the key chiral precursor for the syn-
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Scheme 1. Formation of 2-benzoylbutanenitrile (3e) from 3-oxo-3-phenylpropanenitrile (1e) in the presence of glucose.[14]
eration) was beneficial for bio-transformations with whole mi-
crobial cells.[19] Thus, the reduction of 1h with marine yeasts
was performed in the presence of different amounts of EtOH
(1–5%), and the best results were found with an EtOH concen-
tration of 3.5%.
formed by using the Multisimplexꢁ 2.0 software[20] for the bio-
reduction with R. mucilaginosa UBOCC-A-214025, which took
into account different parameters but without changing the
medium; the conditions of the sequential experimental trials
aimed at the optimisation of the bio-transformation conditions
were selected; the control variables were the bio-catalyst and
substrate concentrations, co-substrate type and concentrations
and temperature. No relevant improvement of the enantiose-
lectivity was observed, and the best results were observed
using 40 mgdry weight mLÀ1 of cells, 25 mm substrate in the pres-
ence of 7% of iPrOH at 278C; under these conditions the total
conversion of the substrate was detected after 5 h with the
formation of (S)-2i in 72% ee. At higher substrate concentra-
tions (150 mm) the same enantioselectivity was observed and
a recovered yield of 73% was obtained, which shows the pos-
sibility to apply these bio-transformations on a preparative
scale. The reduction of 1i was also performed with all the
strains used in this work, but none of them gave a higher
enantioselectivity than that obtained with R. mucilaginosa
UBOCC-A-214025, also if used in freshwater.
The bio-transformation of 1h in seawater occurred only with
R. mucilaginosa; the reduction of the carbonyl occurred with
high enantioselectivity to form a mixture of the stereoisomers
(13S, 17S)-2h and (13R, 17S)-3h with a different diastereoselec-
tivity that depended on the strain employed. Reduction with R.
mucilaginosa UBOCC-A-214036 was highly diastereoselective
and furnished (13S, 17S)-2h as the main product.
Finally, the reduction of the pro-chiral bicyclic ketone 1i was
evaluated (Table 4). Enantiomerically pure 2-acetylamino-6-hy-
droxy-4,5,6,7-tetrahydrobenzothiazole (2i) is a synthon of the
anti-Parkinson (S)-pramipexole.[20] In this case, seawater was
used at pH 5.6 because of the low stability of the substrate at
a higher pH.
Table 4. Reduction of ketone 1i (10 mm) with marine yeasts grown in
YPD-seawater medium.[a]
Conclusions
The stereoselective reduction of structurally different ketones
using halo-tolerant marine yeasts (Meyerozyma guilliermondii
and Rhodotorula mucilaginosa) was studied using cells grown
and bio-converted in seawater. The preparation of valuable
chemicals through water-saving (bio)processes based on the
direct exploitation of seawater is a significant step towards sus-
tainable bio-catalysis.
Entry
Strain
Yield [%]
ee [%]
1
2
3
M. guilliermondii UBOCC-A-214008
R. mucilaginosa UBOCC-A-214025
R. mucilaginosa UBOCC-A-214036
93
94
96
5
64
38
By choosing a suitable strain, high yields and stereoselectiv-
ity could be achieved in most cases. Notably, high chemoselec-
tivity and enantioselectivity were observed using R. mucilagino-
sa in the reduction of aromatic b-ketonitriles, which allowed
the recovery of the optically pure corresponding alcohols; no-
tably, reduction with whole cells of yeasts generally give a mix-
ture of undesired products, as observed with M. guilliermondii.
Although the examples reported here are still limited to the
laboratory scale, these results show that freshwater can be re-
placed by seawater in a whole bio-catalysis process using
marine yeasts. It can be foreseen that halophilic and/or halo-
tolerant marine yeasts (and more generally halophilic and/or
halo-tolerant microorganisms) could be a plentiful source of
enzymes to accomplish bio-catalysis in seawater.
[a] Reaction conditions: Bio-transformations were performed for 24 h with
freshly prepared cells (50 mgdry weight mLÀ1) suspended in seawater (pH 5.6)
in the presence of 5% glucose. Yields and enantioselectivity after 24 h.
All the tested strains gave a high conversion of 1i into
the desired alcohol with the predominant formation of
(S)-2i; R. mucilaginosa UBOCC-A-214025 gave the highest ee
(64%), whereas reduction with M. guilliermondii was not
enantioselective. The relatively low enantioselectivity observed
in the reduction of 1i performed under standard conditions
led us to investigate the effect of bio-transformation condi-
tions to improve enantioselectivity. Optimisation was per-
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trated solution in DMSO (final concentration of co-solvent 1%). The
bio-transformations of ethyl secodione derivative 1h was per-
formed using freshly cells suspended in seawater in the presence
of 3.5% EtOH both to increase the solubility and to favour co-
factor regeneration. The bio-transformations of 1i were performed
using fresh cells suspended in seawater in the presence of different
co-solvents. The bio-transformations were performed on a 25 mL
scale and kept under reciprocal shaking (150 rpm) at the desired
temperature. Once the reaction was over, EtOAc (12 mL) was
added, and the resulting mixture was shaken and centrifuged; the
aqueous phase was extracted twice more with 12 mL of EtOAc.
The organic phases were collected and dried over Na2SO4 and the
solvent was evaporated. The crude residues were purified by flash
chromatography.
Experimental Section
Materials
All reagents and solvents were obtained from Sigma–Aldrich-Fluka
and used without further purification or drying. TLC was performed
with Merck silica gel 60 F254 pre-coated plates. Silica gel column
chromatography was performed on silica gel 60ꢀ (40–63 mm parti-
cle size). Substrate 1h was kindly gifted by Dr. Roberto Lenna (In-
dustriale Chimica, Italy); substrate 1i (2-acetylamino-6-oxo-4,5,6,7-
tetrahydrobenzothiazole) was prepared following a procedure de-
scribed previously.[20] For each compound, the alcohol racemic mix-
ture was obtained by NaBH4 reduction (0.25 mm of substrate). Nat-
ural seawater (pH 7.5) was collected from the Camogli beach
(Genova, Italy) and maintained at 48C after microfiltration; a water
salinity of 35 PSU was reported by ARPA (Agenzia Regionale Pre-
venzione e Ambiente) for this area.
Purification and chemical characterisation
(S)-1-Phenylethanol (2a)
Characterisation
The reaction progress and ee were determined by using HPLC
1H and 13C NMR spectra were recorded by using a Varian Mercury
300 (300 MHz) spectrometer. Chemical shifts (d) are expressed in
ppm, and coupling constants (J) are expressed in Hz. HPLC analy-
ses were performed by using a Jasco Pu-980 equipped with a
UV/Vis detector Jasco UV-975. Chiral HPLC columns used: Chiralcel
OD (4.6ꢂ250 mm, 5 mm Daicel), Chiralcel OJ-H (4.6ꢂ250 mm,
5 mm, Daicel), Chiralcel OD-H (4.6ꢂ250 mm, 5 mm, Daicel), Chiral-
pak IA (4.6ꢂ250 mm, 5 mm, Daicel), Lux cellulose-2 column (4.6ꢂ
250 mm, 5 mm, Phenomenex) and Lux cellulose 3 column (4.6ꢂ
250 mm, 5 mm, Phenomenex). Rotary power determinations were
performed by using a Jasco P-1010 spectropolarimeter coupled
with a Haake N3-B thermostat. Elemental analyses were performed
by using a Carlo Erba Model 1106 Elemental Analyzer for C, H and
N, and the obtained results are within 0.4% of the theoretical
values.
using a Chiralcel OD column (n-hexane/iPrOH 95:5, 0.7 mLminÀ1
,
254 nm): tr (R)-2a 14.2 min, tr (S)-2a 18.0 min.[22] Oil. Rf =0.4
(hexane/EtOAC=8:2); [a]2D5 =À40.0 (c=0.5 chloroform) lit.[23]
[a]2D5 =À39.2 (c=2.54 chloroform); 1H NMR (300 MHz, CDCl3):
d=7.20–7.35 (m, 5H), 4.85 (q, J=6.4 Hz, 1H), 1.46 ppm (d,
J=6.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=145.9, 128.5, 127.5,
125.4, 70.4, 25.2 ppm; elemental analysis calcd (%) for C8H10O
(122.07): C 78.65, H 8.25; found: C 78.38, H 8.01.
(S)-1-Pyridin-3yl-ethanol (2b)
The reaction progress and ee were determined by using HPLC
using a Phenomenex Lux cellulose 2 column (n-hexane/iPrOH
90:10, 0.7 mLminÀ1
, 254 nm): tr (R)-2b 20.46 min, tr (S)-2b
25.30 min.[24] Oil. Rf =0.3 (EtOAc); [a]2D5 =À38.04 (c=0.4 ethanol)
lit.[26] =À39.0 (c=0.93 ethanol); 1H NMR (300 MHz, CDCl3):
d=8.46–8.33 (m, 2H), 7.75–7.70 (m, 1H), 7.24 (dd, J=8.0, 5.0 Hz,
1H), 4.90 (q, J=6.5 Hz, 1H), 4.5 (br s, 1H, OH), 1.49 ppm (d,
J=6.5 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=148.0, 147.0, 141.8,
133.5, 123.5, 67.5 25.1 ppm; elemental analysis calcd (%) for
C7H9ON (123.07): C 68.27, H 7.37, N 11.37; found: C 68.55, H 7.67,
N 11.10.
Strains and microbial growth
A collection of marine yeasts was created previously from deep
sub-seafloor sediments.[7] All isolates are available in the UBO Cul-
ture Collection (http://www.univ-brest.fr/ubocc).
The screening of growth was performed in Erlenmeyer flasks
(100 mL) that contained 10 mL of YPD liquid medium (2.0% glu-
cose, 2.0% polypeptone and 1.0% yeast extract, w/v) prepared
with freshwater or natural seawater[21] and incubated in an orbital
shaker at 180 rpm at 288C for 48 h. Samples were withdrawn at
appropriate intervals and used to monitor the cell growth by
measuring the optical density (OD) at 600 nm by using a spectro-
photometer after appropriate dilution. For dry weight determina-
tion, washed culture samples were filtered through a 0.45 mm glass
microfiber GF/A filter (Whatman) and dried for 24 h at 1108C. The
results were an average of five replicates.
(S)-(1-Thiophen-2-yl)ethanol (2c)
The reaction progress and ee were determined by using HPLC
using a Phenomenex Lux cellulose–3 column (n-hexane/iPrOH
90:10, 0.5 mLminÀ1
,
254 nm): tr (S)-2c 13.5 min, tr (R)-2c
15.90 min.[25] Oil. Rf =0.3 (hexane/EtOAc=8:2); [a]2D5 =À20.80
(c=0.2 chloroform) lit.[25] [a]2D0 =À23.2 (c=0.79 chloroform);
1H NMR (300 MHz, CDCl3): d=7.25–7.18 (m, 1H), 6.95–6.92 (m, 2H),
5.07 (q, J=6.5 Hz, 1H), 2.54 (br s, 1H, OH), 1.56 ppm (d, J=6.5 Hz,
3H); 13C NMR (75 MHz, CDCl3): d=150.0, 126.6, 124.4, 123.2, 66.2,
25.2 ppm; elemental analysis calcd (%) for C6H8OS (128.03):
C 56.22, H 6.29; found: C 56.04, H 6.58.
Bio-transformations
Reductions of all substrates were performed with freshly prepared
cells, grown in freshwater-YPD or in YPD-seawater. Cells grown in
freshwater-YPD medium (50 mgdry weight/mL) were suspended in
phosphate buffer (pH 7.5, 0.1m), whereas cells grown in seawater-
YPD medium (50 mgdry weight/mL) were suspended in natural micro-
filtered seawater. The bio-transformations of 1a–g were performed
in the presence of 5% glucose by adding the substrates as concen-
(S)-(1-Furan-2-yl)ethanol (2d)
The reaction progress and ee were determined by using HPLC
using a Phenomenex Lux cellulose–3 column (n-hexane/iPrOH
98:2, 0.5 mLminÀ1, 220 nm): tr (S)-2d 19.3 min, tr (R)-2d 21.3 min.[25]
Oil. Rf =0.3 (hexane/EtOAc=8:2); [a]2D5 =À20.5 (c=0.2 chloroform)
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1
lit.[26] [a]D20 =À13.6 (c=0.55 chloroform); H NMR (400 MHz, CDCl3):
17S)-2h, 24.61 min.[19] White solid. Rf =0.3 (hexane/EtOAc=1:1).
ee=84%. H NMR (300 MHz,C3D6O): d=0.82 (t, J=7.5 Hz, 3H), 1.53
1
d=7.36 (dd, J=2.0, 1.0 Hz, 1H), 6.32 (dd, J=3.0, 2.0 Hz, 1H), 6.22
(d, J=3.0 Hz, 1H), 4.86 (q, J=6.5 Hz, 1H), 2.35 (br s, 1H, OH),
1.52 ppm (d, J=6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=157.7, 141.8, 110.1, 105.1, 63.6, 21.2 ppm;[14] elemental analysis
calcd (%) for C6H8O2 (112.05): C 64.27, H 7.19; found: C 63.97,
H 7.34.
(non, J=7.5 Hz, 2H), 1.78 (qui, J=6.2 Hz, 2H), 1.95–2.07 (m, 1H),
2.23–2.31 (m, 3H;), 2.43–2.46 (m, 2H; CH2), 2.51 (t, J=6.2 Hz, 2H),
2.74 (t, J=6.2 Hz, 2H), 3.77 (s, 3H), 4.13 (d, J=4.2 Hz, 1H; OH),
4.32 (q, J=5.3 Hz, 1H), 6.0 (t, J=7.5 Hz, 1H), 6.64 (d, J=2.8 Hz,
1H), 6.72 (dd, J=2.8, 8.7 Hz, 1H), 7.50 ppm (d, J=8.7 Hz, 1H);
13C NMR (300 MHz,C3D6O): d=8.2, 23.6, 25.3, 26.6, 26.8, 28.4, 30.9,
34.8, 54.9, 57.2, 75.1, 112.8, 113.2, 118.1, 125.3, 129.6, 135.4, 138.7,
159.0, 219.1 ppm; elemental analysis calcd (%) for C20H26O3
(314.19): C 76.40, H 8.33; found: C 76.11, H 8.39.
(S)-3-Hydroxy-3-phenylpropanenitrile (2e)
The reaction progress and ee were determined by using HPLC
using
a
Chiralcel OJ-H column (n-hexane/iPrOH 90:10,
1.0 mLminÀ1, 220 nm), tr (S)-2e 24.5, tr (R)-2e 31.1 min.[13e] Oil.
Rf =0.4 (hexane/EtOAc=7:3); [a]2D0 =À58.5 (c=1.0, EtOH) lit.[15]
(S)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothiazole
(2i)
[a]2D0 =À57.7 (c=2.6, EtOH)]; H NMR (300 MHz,CDCl3): d=2.74 (d,
1
J=6.1 Hz, 2H), 5.01 (t, J=6.1 Hz, 1H), 7.36–7.39 ppm (m, 5H);
13C NMR (75 MHz,CDCl3): d=27.9, 70.0, 117.4, 125.5, 128.8, 128.9,
141.1 ppm; elemental analysis calcd (%) for C9H9ON (147.07):
C 73.45, H 6.16, N 9.52; found: C 73.01, H 6.31, 9.32.
The reaction progress and ee were determined by using HPLC
using a Chiralpak IA column (n-hexane/iPrOH 8:2 as eluent, flow
rate: 0.7 mLminÀ1, 254 nm), tr (R)-2i 10.9 min, tr (S)-2i 14.8 min.[19]
Yellowish solid. Rf =0.25 (CH2Cl2/MeOH, 95:5). ee=64%. 1H NMR
(300 MHz,CD3OD): d=1.91 (dddd, J=14.2, 8.6, 7.7, 5.9 Hz, 1H), 2.04
(dddd, J=14.2, 6.2, 5.9, 2.9 Hz, 1H), 2.26 (s, CH3, 3H), 2.61–2.71 (m,
2 h), 2.79 (dddd, J=16.5, 5.9, 5.9, 1.8 Hz, 1H), 2.99 (ddd, J=15.7,
4.7, 1.8 Hz, 1H), 4.15 ppm (dddd, J=8.6, 6.9, 4.7, 2.9 Hz, 1H);
13C NMR (300 MHz, CD3OD): d=21.2, 23.2, 30.5, 30.7, 66.2, 120.9,
143.2, 156.4, 169.1 ppm; elemental analysis calcd (%) for
C9H12O2N2S (212.06): C 50.92, H 5.70, N 13.20; found: C 50.96,
H 5.89 N 12.85.
(S)-3-(2-Thienyl)-3-hydroxypropanenitrile (2 f)
The reaction progress and ee were determined by using HPLC
using
a
Chiralcel OJ-H column (n-hexane/iPrOH=90:10,
1.0 mLminÀ1, 220 nm), tr (S)-2 f 27.0, tr (R)-2 f 32.3 min.[13e] Oil.
Rf =0.3 (hexane/EtOAc=7:3); [a]2D8 =À19.4 (c=1.0, CHCl3) lit.[26]
[a]2D8 =À16.71 (c=1.01, CHCl3); 1H NMR (300 MHz,CDCl3): d=2.85
(d, J=6.2 Hz, 2H), 3.04 (s, br, OH), 5.26 (t, J=6.2 Hz, 1H), 6.98–7.00
(m, 2H), 7.06–7.07 (d, J=3.5 Hz, 1H), 7.29–7.32 ppm (m, 1H);
13C NMR (75 MHz, CDCl3): d=28.2, 66.2, 117.1, 124.7, 125.7, 127.1,
144.5 ppm; elemental analysis calcd (%) for C7H7ONS (153.02):
C 54.88, H 4.61, N 9.14; found: C 54.81, H 4.81, 8.92.
Acknowledgements
This work was supported by the European Community FP7-
KBBE.2012.3.2–02 project MACUMBA, grant agreement no.
311975.
(S)-3-(2-Furyl)-3-hydroxypropanenitrile (2g)
The ee was determined by using HPLC using a Chiralcel OJ-H
column (n-hexane/iPrOH=90:10, 1.0 mLminÀ1, 220 nm), tr (S)-2g
21.9, tr (R)-2g 25.6 min.[18] Oil. Rf =0.3 (hexane/EtOAc=7:3);
[a]2D0 =À40.2 (c=1.0, EtOH) lit.[27] [a]2D0 =À38.6 (c=1.3, CHCl3);
1H NMR (300 MHz,CDCl3): d=2.57 (s, br, OH), 2.88 (d, J=6.2 Hz,
2H), 5.05 (t, J=6.2 Hz, 1H), 6.37–6.41 (m, 2H), 7.41–7.42 ppm (m,
1H); 13C NMR (75 MHz,CDCl3): d=24.9, 63.8, 107.5, 110.6, 116.9,
142.9, 152.8 ppm; elemental analysis calcd (%) for C7H7O2N
(137.05): C 61.31, H 5.14, N 10.21; found: C 61.06, H 5.36, 9.94.
Keywords: biocatalysis · ketones · enzymes · reduction ·
stereoselectivity
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Published online on && &&, 0000
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These are not the final page numbers! ÞÞ

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I. Serra, B. Guidi, G. Burgaud,
M. L. Contente, P. Ferraboschi, A. Pinto,*
C. Compagno, F. Molinari, D. Romano*
&& – &&
Seawater-Based Biocatalytic Strategy:
Stereoselective Reductions of Ketones
with Marine Yeasts
All at sea: Marine yeasts from deep-
sub-seafloor sediments were grown in
seawater and used as biocatalysts to
perform the stereoselective reduction of
different ketones in seawater as well.
Strains of Meyerozyma guilliermondii and
Rhodotorula mucilaginosa were able to
reduce different aromatic ketones with
high molar conversions and moderate-
to-high enantioselectivity.
&
ChemCatChem 2016, 8, 1 – 8
www.chemcatchem.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!