Preparative scale application of Mucor circinelloides ene–reductase and alcohol dehydrogenase activity for the asymmetric bioreduction of α,β-unsaturated γ-ketophosphonates

Article abstract of DOI:10.1016/j.bioorg.2019.103548

The fungus Mucor circinelloides exhibits high potential for green chemistry and technological applications. Recently M. circinelloides, which so far was considered mainly as a platform for biodiesel production, was found to exhibit high ene-reductase acti

Full text of DOI:10.1016/j.bioorg.2019.103548

Journal Pre-proofs
Preparative scale application of Mucor circinelloides ene–reductase and alco-
hol dehydrogenase activity for the asymmetric bioreduction of α,β-unsatura-
ted γ-ketophosphonates
Ignacy Janicki, Piotr Kiełbasiński, Jakub Szeląg, Adrian Gł ębski, Mirosława
Szczęsna-Antczak
PII:
S0045-2068(19)31830-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103548
YBIOO 103548
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To appear in:
Bioorganic Chemistry
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30 October 2019
17 December 2019
21 December 2019
Please cite this article as: I. Janicki, P. Kiełbasiński, J. Szeląg, A. Gł ębski, M. Szczęsna-Antczak, Preparative
scale application of Mucor circinelloides ene–reductase and alcohol dehydrogenase activity for the asymmetric
bioreduction of α,β-unsaturated γ-ketophosphonates, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103548
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Preparative scale application of Mucor circinelloides
ene-reductase and alcohol dehydrogenase activity for the
asymmetric bioreduction of α,β-unsaturated
γ-ketophosphonates.
Ignacy Janicki,a* Piotr Kiełbasiński,a Jakub Szeląg,b Adrian Głębski,b Mirosława
Szczęsna-Antczakb
a
Division of Organic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of
Sciences, Sienkiewicza 112, 90-363 Łódź, Poland
e-mail address: ijanicki@cbmm.lodz.pl
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Science, Technical University of
b
Łódź, Stefanowskiego 4/10, 90-924 Łódź, Poland
Abstract. The fungus Mucor circinelloides exhibits high potential for green chemistry and technological
applications. Recently M. circinelloides, which so far was considered mainly as a platform for biodiesel
production, was found to exhibit high ene-reductase activity. In our current research we applied this promising
microorganism to the biotransformation of a series of α,β-unsaturated γ-ketophosphonates. The biotransformations
were conducted using cheap corn steep liquor or minimal media. The products were obtained with excellent
enantiomeric purity (>99% ee in most cases) and in good isolated yields, highlighting the great potential of this
microorganism for asymmetric synthesis. Moreover, the products obtained may be further applied as chiral
building blocks for the synthesis of biologically active compounds, such as glutamic acid or fosmidomycin
derivatives.
Keywords: biotransformation; biocatalysis; chiral phosphonates; asymmetric catalysis; enzymes
Introduction
Phosphonates and phosphonic acids have gained a lot of interest as biologically active compounds
because of their unique ability to mimic natural compounds such as carboxylic acids, amino acids,
phosphate esters or anhydrides [1]. Because of that property, phosphonates represent a very broad group
of potential inhibitors of numerous enzymes that can be targets for treatment of various diseases.
Starting from the first acclaimed organophosphorus chemotherapeutic agent cyclophosphamide, which
was explored in our institute,[2] phosphorus containing drugs continue to be of interest in cancer
research, for example as glycolysis inhibitors [3]. Moreover, phosphonates have found attention as
potential therapeutics for treatment of malaria (fosmidomycin and its derivatives) [4-8], treatment of
nervous system diseases (glutamic acid derivatives) [9-11] and many others [1, 12-13]. Due to the fact
that biomedical applications are one of the essential areas of phosphonate chemistry, a great effort has
been made to develop new stereoselective methodologies for the synthesis of these molecules, as
chirality is a crucial aspect of medicinal chemistry [12-16]. One of the most useful approaches for the
synthesis of enantiomerically pure compounds is the asymmetric reduction of carbon-carbon double
bonds [17]. There is a number of successful examples of application of this concept in the synthesis of
chiral phosphonates by reductions of the corresponding unsaturated substrates catalyzed by copper
hydride species with chiral ligands [18-19], or catalytic hydrogenations using iridium [20] or rhodium
[21-24] complexes with chiral ligands. However, most successful hydrogenations require application of
hydrogen under high pressure (up to 100 bar) which may be problematic. An interesting alternative to
traditional chemical methods is biocatalysis, because it does not require harsh conditions and minimizes
the environmental impact. Hence, it is generally considered as a „green methodology.” In this context,
the biocatalytic asymmetric hydrogenation of alkenes using enzymes from the Old Yellow Enzyme
family and its homologues - ene-reductases, deserves special attention (Scheme 1).

R1
R1
Ene-reductase
EWG
*
EWG
*
R2
R2
R3
R3
NAD+ or NADP+
NADH or NADPH
Cofactor recycling system
Scheme 1. General scheme of ene-reductase-catalyzed asymmetric reduction of α,β-unsaturated compounds.
During the last two decades ene-reductases were identified as highly valuable biocatalysts for green
organic transformations, especially for asymmetric synthesis [25-33] with possible industrial
applications [34-36]. These nicotinamide-dependent enzymes can catalyze reduction of “activated”
carbon-carbon double bonds, being excellent catalysts for asymmetric reductions of various α,β-
unsaturated ketones, aldehydes, esters, nitriles, etc. In our recent research, we demonstrated
stereoselective reduction of a series of α,β-unsaturated phosphonates bearing additional electron
withdrawing group using isolated ene-reductases (Scheme 2) [37]. The methodology was found to be
perfectly stereoselective offering access to enantiomerically pure products. In our procedure we applied
a cofactor recycling system allowing application of only catalytic amounts of NAD and ammonium
formate as a cheap hydrogen source, which made the whole process more economical.
R
O
R
O
R
O
R
O
O
P
O
P
Ene-reductase
*
EWG
EWG
NADH
NAD+
NH3 + CO2
HCOONH4
Formate dehydrogenase
Scheme 2. Asymmetric reduction of α,β-unsaturated phosphonates using isolated ene-reductases [37].
Recent screening research towards identification of fungal ene-reductase activity by Romagnolo et.
al. revealed Mucor circinelloides to exhibit a high potential in this area [38-39]. Among a broad scope
of tested fungi, Mucor circinelloides was found to be one of the most versatile and effective strains
allowing conversion of model substrates in high yields. Moreover it was proven that it can efficiently
catalyze the hydrogenation of a series of more demanding ene-reductase substrates like α,β-unsaturated
nitriles or esters [40]. Its putative ene-reductases were recently identified and expressed, providing an
insight into mechanistic aspects of Mucor circinelloides reduction of the carbon-carbon double bonds
[41-42]. Interestingly, stereoselectivity was not investigated in the transformations performed in the
presence of whole cell fungi. Noteworthy, M. circinelloides strains exhibit high potential in context of
biorefineries, especially for biodiesel production [43-51].
The aim of our current research was to make the bioreduction process more applicable to a larger,
preparative scale using a whole-cell system. Although the use of isolated enzymes allows conducting
the desired reaction with simple workup, apparatus and limiting side reaction, its main drawbacks are a
necessity for using stoichiometric amounts of a cofactor or its recycling system and a time consuming,
expensive enzyme purification with often limited enzyme stability. On the other hand, alternative whole-
cell processes do not require cofactor recycling or enzyme purification steps [25]. Of course these types
of processes have their cons such as side reactions, a more difficult workup due to a large volume or a
large biomass production. However, some of these issues may be faced by the development of a more
sustainable process in which the biomass and side products are also valuable. For this reason, the latter

procedure undoubtedly fills the need for green processes and as such can be considered valuable in the
attempts to develop new methods for biocatalyzed alkene reductions.
Results and Discussion
General remarks
Based on the previous observations [37] and literature reports [38-48], we decided to test the
possibility of applying M. circinelloides whole cells to the asymmetric bioreduction of a series of α,β-
unsaturated phosphonates (Figure 1). The following phosphonates were selected as the most promising
substrates and were synthesized according to the literature procedures [37, 52-54].
O
P
O
P
O
P
O
O
O
O
O
O
O
O
O
1c
1a
1b
O
P
O
P
O
P
O
P
O
O
O
O
O
O
O
O
O
O
O
O
1d
1e
1f
1g
O
O
P
O
O
O
O
P
CN
O
1h
1i
Figure 1. Series of substrates tested for the Mucor circinelloides catalyzed asymmetric bioreduction.
Biotransformations using Mucor circinelloides whole cells (Scheme 3) were run using two different
culture mediums – defined minimal medium or corn steep liquor (CSL), the latter being a cheap by-
product of corn processing. The substrates were added to the reaction medium at the beginning of the
cultivation. Biotransformations using CSL were run for 3 days, however, the biocatalytic procedures
using defined media were run for 7 days because of a slower cell growth and lower reaction rate. All the
observations of the influence of substrate structure and media applied was supported by ANOVA
analysis. Among the substrates tested, 1a-1g were transformed into saturated products 2 in moderate to
good yields and with excellent stereoselectivity of 90% to 99% enantiomeric excess. These results
broaden the capability of the transformation compared to our previous report (R2 = methyl only) [37] as
more difficult, bulky substrates (R2 = Et or Ph) were successfully transformed. In case of substrates 1c-
1g, we have observed also the formation of additional products, which turned out to be unsaturated
alcohols 3 formed by the carbonyl group reduction as a result of the alcohol dehydrogenase activity.
Careful analysis of products 3 using a chiral shift reagent and NMR techniques [55-57] or HPLC

equipped with chiral columns revealed that they were produced with excellent enantiomeric excesses
(99% in all cases). Surprisingly, we did not observe saturated phosphono-γ-alcohols which could be
formed during the carbon-carbon double bond reduction and subsequent carbonyl group reduction. It is
worth mentioning that the inverse process, namely the initial carbonyl group reduction followed by the
carbon-carbon double bond reduction is unlikely due to an insufficient activation of the carbon-carbon
double bond by the phosphonate group [37]. Substrates 1h and 1i were not converted during cultivation.
Moreover, an interesting observation was made for substrate 1h which was found to inhibit the growth
of the fungus. This observation suggests that compound 1h may exhibit antifungal activity, also reported
for other α,β-unsaturated phosphonates [58].
R1
O
R1
O
R1
O
R1
O
R1
O
R1
O
O
P
O
P
O
P
Mucor circinelloides
+
*
R2
R2
R2
*
O
O
HO
1
2
3
Scheme 3. Mucor circinelloides catalyzed biotransformation of α,β-unsaturated phosphonates.
Table 1. Results of Mucor circinelloides catalyzed biotransformations.
Entr
y
Substrate
R1
Culture
Medium
Product 2
Product 3
Total
biomass
yield
Symb
ol
R2
Isolated
Yield [%]
Enantiomeric
excess [%]
Isolated
Yield [%]
Enantiomer
ic excess
[%]
[g/l]
1
2
3
4
5
6
7
8
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
1f
Me
Me
Et
Me Minimal 58
Me CSL 59
Me Minimal 59
Me CSL 61
99 (+)
99 (+)
97 (+)
93 (+)
90 (+)
95 (+)
96 (-)
96 (-)
94 (-)
93 (-)
99 (+)
n.d.a
-
-
-
-
-
-
-
-
11.9
35.6
5.2
18.1
12.7
18.5
13.1
25.0
3.2
12.5
13.6
33.7
2.7
Et
i-Pr Me Minimal 53
i-Pr Me CSL 30
Minimal 66
CSL 26
Minimal 21
CSL 18
Minimal 47
9
99 (-)
99 (-)
n.d.
38
traces
5
38
41
26
71
25
52
Me
Me
Et
Et
Et
Et
Et
Ph
Ph
Ph
Ph
99
9
99 (-)
99 (-)
99 (+)
99 (+)
99 (-)
99 (-)
10
11
12
13
14
Et
Me
Me
Et
1f
CSL
Minimal 24
CSL
traces
1g
1g
99 (+)
n.d.
Et
traces
5.0
a) n.d. – not determined
Influence of R1 and R2 substituents on substrate reactivity
Inspection of Table 1 shows that generally substrates bearing smaller substituents R1 and R2 are
better accepted by M. circinelloides ene-reductases. Thus, both 1a and 1b, in which R2 = Me, and R1 =
Me or Et, respectively, are effectively transformed into saturated products 2a and 2b in 58-61% yield
and no significant difference in their reactivity is observed. However, substrate 1c with R1 = i-Pr and R2
= Me gives lower yields and also a competitive carbonyl group reduction occurs. Compounds 1d and 1f
with R1 = Me and R2 = Et or Ph, respectively, are transformed to the corresponding saturated products
(2d and 2f) with higher yields than compounds 1e and 1g bearing R1 = Et. For example, using minimal
growth medium, biotransformation of 1d results in 66% isolated yield of 2d while biotransformation of
1e under the same conditions gives only 21% of 2e. A slight decrease of enantioselectivity may be
observed comparing enantiomeric purity of products with R1 = Et1 and R2 = Me or Et (2b and 2e,
respectively) to products bearing R1 = Me (2a and 2d). Influence of the size of R2 is also noticeable, for

example biotransformation of 1a, 1d and 1f bearing R1 = Me and R2 = Me, Et, and Ph, respectively, in
CSL medium results in a gradual decrease of product 2 formation giving 59% of 2a, 26% of 2d, and
only traces of 2f.
In case of substrates 1c-1g the competitive, alcohol dehydrogenase catalyzed reaction, leading to
the formation of carbonyl group reduction products 3c-3g was also observed. All these products were
obtained with excellent enantiomeric excess of 99%, showing a great potential of M. circinelloides for
biocatalytic, asymmetric carbonyl group reduction. Product 3d was formed only in 5% isolated yield
when using CSL growth medium, while products with bigger groups R1 and R2, 3c and 3e-3g, were the
main products of the biotransformations of compounds 1c and 1e-1g in the CSL medium.
Influence of growth media on biotransformations
Comparison of biotransformations using minimal or rich in nutrients (corn steep liquor) culture
media shows an interesting relation between the activities of alcohol dehydrogenase and ene-reductase.
In case of compounds 1a-1b, the results obtained using two different growth media are comparable and
the only products observed and isolated in 58-61% yield are 2a-2b (we have observed only a slightly
lower ee value of product 2b when using corn steep liquor). These results are similar to
biotransformations using isolated ene-reductase OYE3 which gave 61-63% isolated yield of 2a and 2b
[37]. Interestingly, in case of substrates 1c-1g we have also observed another products formation – 3c-
3g. Starting from 1c with isopropyl R1 group we isolated 53% of 2c but also 9% of 3c when using
minimal medium. Application of CSL medium resulted in reverse proportions of products – 30% of 2c
and 38% of 3c. In case of substrate 1d product 2d was isolated in very good 66% yield and only traces
of 3d were observed using minimal medium. However, biotransformation in CSL medium resulted in
significant decrease of the yield of 2d (26%) and gave only a small amount of 3d (5%). The main product
obtained after biotransformation of 1e was 3e, which was isolated in 38-41% yield along with 18-21%
of 2e. We did not observe significant difference between biotransformations of 1e using two growth
media – there were only slightly inversed proportions of 2e to 3e depending on the media applied.
The most interesting results were obtained for substrates 1f and 1g where a clear reactivity
preference can be observed using minimal and nutrient rich media. Application of minimal growth
medium for biotransformation of 1f allowed preferential carbon-carbon double bond reduction and
formation of 2f in 47% isolated yield along with 26% of 3f. On the other hand application of CSL
medium, resulted in a preferential carbonyl group reduction and formation of 3f in 71% isolated yield
with only traces of 2f. Similar observation was made for 1g where products 2g and 3g were formed in
almost equal yields (24% and 25%) using minimal growth medium, while the use of CSL medium led
to preferential formation of 3g in 52% isolated yield with only traces of 2g observed.
A plausible explanation of the above observations may be a higher alcohol dehydrogenase activity
during the stationary phase of cells growth than during the exponential phase. Substrates 1a-1b are
transformed by ene-reductases very quickly [37] while bulkier compounds 1c-1g are more demanding
substrates and therefore their conversion is slower. Because of the high ene-reductase activity towards
substrates 1a-1b, they may be fully converted into relevant products very fast, during the exponential
phase, while bulkier substrates 1c-1g are still not fully converted before the stationary phase, when
alcohol dehydrogenase activity is higher, which results in the competitive carbonyl group reduction.
Thus, for bulkier substrates, longer exponential growth phase using minimal growth medium should
result in higher yields of carbon-carbon double bond reduction while using nutrient rich CSL medium,
where stationary phase starts earlier, may lead to preferential carbonyl group reduction. This is in
accordance with the experimental data obtained, especially in case of 1c-1d and 1f-1g. This creates an
interesting possibility of controlling the chemical outcome of the biotransformation by the appropriate
selection of its conditions and growth medium. We believe that the process may be further optimized to
achieve the desired results.
Influence of biotransformation conditions on the growth of the fungal biomass
A high yield of manufactured products is a main parameter determining the profitability of using
the strain of M. circinelloides. The substances which can be obtained by the bioreduction of α,β-
unsaturated phosphonates are the first products. The second one is a biomass containing intracellular
lipase and chitosanase, from which it is also possible to obtain lipids that are accumulated as reserve
substances in mycelium grown in both media used in the experiment [44].
The yield of lipids was dependent on culture conditions (type of the medium and substrate) and
reached even more than 60% of the dry biomass. Generally, the percentage of fungal oils was higher

when the biomass yield was high. It was found that the accumulation of lipids in the mycelium growing
in the mineral medium was reduced in the presence of substrates containing substituents R1 and R2 that
are larger than Me. This phenomenon was not observed in CSL medium in which the strain accumulated
more than 50% of the lipids regardless of the substrate being converted (with two exceptions). Only in
the presence of 1e and 1g, the amount of oils was reduced to 30% and 11%, respectively.
When no substrate 1 was added, the total yield of the biomass was 33.7 g/l for CSL medium and
15.1 g/l for defined minimal medium with glucose. These results are similar to those described in
previous works performed in the Institute of Technical Biochemistry [44]. In almost all
biotransformations performed, the yield of the M. circinelloides biomass was lower than in the control
samples. There were two exceptions from this tendency, namely in the biotransformation experiments
with substrates 1a and 1f carried out in CSL medium, the yield of M. circinelloides biomass were, in
comparison to control, slightly higher (35.6 g/l) or the same. Taking into consideration the influence of
substituents R1 and R2 on the fungal growth, it was observed that for R1 = Me the biomass obtained was
more than twice higher than for substituents R1 = Et and R1 = i-Pr. On the other hand, in case of
substituents R2, a very low effect on the biomass yield was observed. The ANOVA analysis shows that
this parameter has an impact on the variability lower than 3,5%, with p-value higher than 0.45, thus this
effect is negligible. Interestingly, the isolated yield of products 2 and 3 was not dependent on the amount
of the biomass obtained after the culture. For example: the yields of the biotransformation products 2a
and 2b were similar although the total biomass yield for substrate 1b was twice lower. This reduction
in biomass yield in the case of 1b was observed for both media used. To conclude, no clear correlation
between the yield of products 2 and 3 and the M. circinelloides biomass could be observed.
Conclusion
We have demonstrated a possibility of a preparative scale application of Mucor circinelloides in
the asymmetric reduction of carbon-carbon double bonds, as well as in the asymmetric carbonyl group
reduction of α,β-unsaturated γ-ketophosphonates. The obtained products of an excellent enantiomeric
purity may be highly valuable building blocks for further transformations into biologically relevant
products. Based on previous reports [38-42], we are also convinced that the developed procedure may
be applied to other different substrates to obtain valuable products without negative effect on the
biomass growth. Presented biotransformations show great potential of Mucor circinelloides for the
application in asymmetric synthesis.
Experimental Section
Fungal strain and general biotransformation procedure
The strain of Mucor circinelloides IBT-83 from the culture collection of Institute of Technical Biochemistry (TUL)
was used in all biotransformations [44, 46-48, 59-60]. All the cultures were performed in 1000 ml flasks containing
200 ml of culture medium, at 30°C and with shaking 180 rpm. Culture media were inoculated with a suspension
of M. circinelloides spores (obtained from 3-days culture slants containing 8.5°Blg wort-medium, pH 5.8),
supplemented with 2 mmols of the given substrate per flask (final concentration 10 mM) and incubated in a
reciprocal shaker for 3 days in CSL medium (rich in C and N-sources) and for 7 days in defined medium
(containing glucose and minerals). Both media had initial pH of 4.7
Defined minimal medium was composed of glucose (5%) and minerals (concentration in g/dm3): NH3NO3(0.25),
KH2PO4 (8.0), NaCl (0.1), MgSO4·7H2O (0.5), CaCl2 (0.1) as well as microelements (mg/dm3): MnSO4 (0.5),
H3BO3 (0.5), CuSO4 (0.040), KJ (0.1), ZnSO4 (0.4), Na2MoO4 (0.2) and Fe2(SO4)3 (2.46).
CSL medium consisted of 3.66% (w/v) of corn steep liquor (spray-dried, Solulys 095E, from Roquette, France)
and 2.7% (v/v) of rape oil.
The biomass after the culture was filtered off and washed with water. All the collected liquid was condensed to
approximately 100 ml and acetone was added to precipitate the residual proteins. The precipitate was filtered off
through a pad of Celite and the liquid was evaporated under reduced pressure to remove acetone and extracted 3
times using ethyl acetate. The organic phase was dried using MgSO4, concentrated under reduced pressure and the
residue was subjected to column chromatography using ethyl acetate and acetone (gradient) as eluent. All the
products were obtained as dense liquids. Experimental data of the products are provided in the supplementary
material.
The yield of oils was calculated after gravimetric determination of the mass of lipids extracted with acetone (3-
times) from water-rinsed biomass (extracts were separated from defatted biomass using filter funnel, subsequently
collected and dried to solvents evaporation). The yield of defatted biomass was calculated after determination of

dry weight of lipid-free biomass from each flask. Total yield of oleaginous biomass (YB) was obtained by summing
up the above values.
Experimental data of the products obtained
1
All the new, previously not reported products were identified by H-NMR, 13C-NMR, 31P-NMR and elemental
analysis.
2a
1H NMR (200 MHz, CDCl3) δ 3.68 (dd, J = 10.5 Hz, J = 0.8 Hz , 6H), 2.79 – 2.94 (m, 1H), 2.40 – 2.61 (m, 2H),
2.15 (s, 3H), 1.14 (dd, J = 18.4 Hz, J = 6.8 Hz 3H). 31P NMR (CDCl3) δ 36.40 (s).
2b
1H NMR (200 MHz, CDCl3) δ 4.01-4.16 (m, 4H), 2.78 – 2.95 (m, 1H), 2.36 – 2.56 (m, 2H), 2.16 (s, 3H), 1.30 (td,
J = 7.1 Hz, J = 0.8 Hz, 6H) 1.13 (dd, J = 18.2 Hz, J = 6.8 Hz, 3H). 31P NMR (CDCl3) δ 33.80 (s).
2c
1H NMR (500 MHz, CDCl3) δ 4.74 – 4.60 (m, 2H), 2.95 – 2.78 (m, 1H), 2.50 – 2.29 (m, 3H), 2.16 (s, 3H), 1.30
(dd, J = 6.1, 4.0 Hz, 12H), 1.18 – 1.06 (m, 3H). 13C NMR (126 MHz, CDCl3) δ 206.07 (d, J = 16.1 Hz), 70.19 (d,
J = 6.9 Hz), 44.11 (s), 30.36 (s), 26.96 (d, J = 145.9 Hz), 24.05 (d, J = 5.0 Hz), 13.84 (d, J = 5.4 Hz). 31P NMR
(202 MHz, CDCl3) δ 31.93 (s). Anal. calcd. for C11H23O4P1: C, 52.7897; H, 9.2630. Found: C, 51.95; H, 9.42
2d
1H NMR (500 MHz, CDCl3) δ 3.71 (d, J = 10.6 Hz, 6H), 2.87 – 2.76 (m, 1H), 2.56 – 2.41 (m, 2H), 2.17 (s, 3H),
1.64 (dm, 2H), 0.96 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 205.77 (s), 52.50 (dd, J = 32.5, 6.8 Hz),
42.05 (s), 31.68 (d, J = 141.1 Hz), 30.26 (s), 22.17 (d, J = 3.9 Hz), 12.16 (d, J = 9.7 Hz). 31P NMR (202 MHz,
CDCl3) δ 36.30 (s). Anal. calcd. for C8H17O4P1: C, 46.1524; H, 8.2304. Found: C, 46.11; H, 7.94
2e
1H NMR (500 MHz, CDCl3) δ 4.15 – 4.00 (m, 4H), 2.88 – 2.78 (m, 1H), 2.54 – 2.37 (m, 2H), 2.18 (s, 3H), 1.83 –
1.69 (m, 1H), 1.53 – 1.39 (m, 1H), 1.29 (dt, J = 7.1, 4.0 Hz, 6H), 0.97 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 206.03 (d, J = 11.2 Hz), 61.68 (dd, J = 24.7, 6.7 Hz), 42.18 (s), 32.28 (d, J = 141.5 Hz), 30.30 (s), 22.22
(d, J = 3.8 Hz), 16.47 (d, J = 5.9 Hz), 12.20 (d, J = 9.7 Hz). 31P NMR (202 MHz, CDCl3) δ 33.68 (s). Anal. calcd.
for C10H21O4P1: C, 50.8400; H, 8.9597. Found: C, 50.26; H, 9.15.
2f
1H NMR (500 MHz, CDCl3) δ 7.43 – 7.17 (m, 5H), 3.83 – 3.72 (m, 1H), 3.58 (dd, J = 108.4, 10.6 Hz, 6H), 3.22 –
3.04 (m, 2H), 2.09 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 204.69 (d, J = 14.2 Hz), 135.57 (d, J = 7.1 Hz), 129.09
(d, J = 6.6 Hz), 128.70 (d, J = 2.3 Hz), 127.48 (d, J = 3.0 Hz), 53.23 (dd, J = 108.9, 7.1 Hz), 43.73 (s), 38.37 (d, J
= 140.0 Hz), 30.40 (s). 31P NMR (202 MHz, CDCl3) δ 30.78 (s).
2g
1H NMR (500 MHz, CDCl3) δ 7.43 – 7.11 (m, 5H), 4.09 – 3.98 (m, 2H), 3.93 – 3.83 (m, 1H), 3.77 – 3.66 (m, 2H),
3.20 – 3.06 (m, 2H), 2.08 (s, 3H), 1.17 (dt, J = 97.6, 7.1 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 204.94 (d, J =
14.6 Hz), 135.83 (d, J = 6.9 Hz), 129.17 (d, J = 6.5 Hz), 128.57 (d, J = 2.3 Hz), 127.34 (d, J = 3.0 Hz), 62.47 (dd,
J = 108.0, 7.1 Hz), 43.83 (s), 38.95 (d, J = 140.2 Hz), 30.45 (s), 16.31 (dd, J = 22.4, 5.9 Hz). 31P NMR (202 MHz,
CDCl3) δ 28.35 (s). Anal. calcd. for C14H21O4P1: C, 59.1477; H, 7.4455. Found: C, 59.01; H, 7.46.
3c
1H NMR (500 MHz, CDCl3) δ 6.52 (ddd, J = 24.1, 8.0, 1.5 Hz, 1H), 4.74 – 4.55 (m, 3H), 3.12 (s, 1H), 1.80 (dd, J
= 14.8, 1.4 Hz, 3H), 1.38 – 1.19 (m, 15H). 13C NMR (126 MHz, CDCl3) δ 148.56 (d, J = 8.5 Hz), 125.58 (d, J =
179.4 Hz), 70.46 (d, J = 5.4 Hz), 64.47 (d, J = 21.5 Hz), 24.44 – 23.45 (m), 22.48 (s), 12.76 (d, J = 9.7 Hz). 31P
NMR (202 MHz, CDCl3) δ 18.77 (s). Anal. calcd. for C11H23O4P1: C, 52.7897; H, 9.2630. Found: C, 51.86; H,
9.64.
3d
1H NMR (500 MHz, CDCl3) δ 6.49 (dd, J = 24.1, 8.5 Hz, 1H), 4.73 – 4.61 (m, 1H), 3.70 (dd, J = 10.9, 1.6 Hz,
6H), 2.55 (s, 1H), 2.37 – 2.18 (m, 2H), 1.31 (d, J = 6.4 Hz, 3H), 1.07 (t, J = 7.6 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 149.64 (d, J = 8.6 Hz), 129.75 (d, J = 174.0 Hz), 64.06 (d, J = 21.6 Hz), 52.44 (d, J = 5.3 Hz), 23.18 (s),
20.82 (d, J = 10.0 Hz), 14.41 (s). 31P NMR (202 MHz, CDCl3) δ 24.26 (s). Anal. calcd. for C8H17O4P1: C, 46.1524;
H, 8.2304. Found: C, 45.89; H, 8.27.

3e
1H NMR (500 MHz, CDCl3) δ 6.47 (dd, J = 24.0, 8.6 Hz, 1H), 4.70 – 4.62 (m, 1H), 4.16 – 3.98 (m, 4H), 2.47 (s,
1H), 2.37 – 2.20 (m, 2H), 1.36 – 1.28 (m, 9H), 1.09 (t, J = 7.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 148.66 (d,
J = 8.7 Hz), 131.00 (d, J = 174.1 Hz), 64.10 (d, J = 21.6 Hz), 61.81 (d, J = 5.4 Hz), 23.16 (s), 20.87 (d, J = 9.9
Hz), 16.37 (d, J = 6.4 Hz), 14.56 (s). 31P NMR (202 MHz, CDCl3) δ 21.42 (s). Anal. calcd. for C10H21O4P1: C,
50.8400; H, 8.9497. Found: C, 50.39; H, 8.89.
3f
1H NMR (500 MHz, CDCl3) δ 7.47 – 7.29 (m, 3H), 7.23 – 7.17 (m, 2H), 6.82 (dd, J = 23.2, 8.7 Hz, 1H), 4.42 –
4.23 (m, 1H), 3.70 (dd, 6H), 2.32 (s, 1H), 1.27 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 151.02 (d, J =
7.9 Hz), 134.09 (d, J = 9.1 Hz), 129.67 (s), 128.96 (d, J = 5.2 Hz), 128.54 (s), 128.07 (s), 64.89 (d, J = 19.7 Hz),
52.88 (s), 23.03 (s). 31P NMR (202 MHz, CDCl3) δ 19.75 (s).
3g
1H NMR (500 MHz, CDCl3) δ 7.40 – 7.29 (m, 3H), 7.25 – 7.20 (m, 2H), 6.78 (dd, J = 23.1, 8.7 Hz, 1H), 4.36 –
4.25 (m, 1H), 4.12 – 3.97 (m, 4H), 2.32 (s, 1H), 1.32 – 1.19 (m, 9H). 13C NMR (126 MHz, CDCl3) δ 149.99 (d, J
= 7.9 Hz), 134.31 (s), 132.31 (s), 129.06 (d, J = 5.1 Hz), 128.42 (s), 127.90 (s), 64.95 (d, J = 19.6 Hz), 62.33 (d, J
= 5.2 Hz), 23.02 (s), 16.30 (d, J = 6.1 Hz). 31P NMR (202 MHz, CDCl3) δ 16.91 (s). Anal. calcd. for C14H21O4P1:
C, 59.1477; H, 7.4455. Found: C, 59.51; H, 7.26.
Acknowledgements
The Authors thank the National Science Center (NCN), Poland, for financial support from grant PRELUDIUM UMO-
2016/21/N/ST5/03361 to I.J.
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Graphical abstract

Highlights:
•
•
Filamentous fungus Mucor circinelloides was applied for asymmetric biotransformation of α,β-
unsaturated-γ-ketophosphonates (1).
Products of carbon-carbon double bond and/or carbonyl group reduction (2 and 3 respectively)
were obtained in good yields.
•
•
•
All products were obtained with excellent enantiomeric excess – up to >99% ee.
Biotransformation outcome (2:3 product ratio) is dependent on the growth media applied.
Cheap growth medium (corn steep liquor) can be successfully applied for the biotransformation.

Conflict of Interest
Authors declare no conflict of interest.