Biocatalyzed kinetic resolution of racemic mixtures of chiral α-aminophosphonic acids

Article abstract of DOI:10.1016/j.molcatb.2013.02.004

Several fungal species namely: Aspergillus niger, Aspergillus parasiticus, Penicillium funiculosum, Trigonopsis variabilis and two different strains of Fusarium oxysporum were tested toward racemic mixtures of following phosphonic acids: 1-amino-2-methylp

Full text of DOI:10.1016/j.molcatb.2013.02.004

Journal of Molecular Catalysis B: Enzymatic 91 (2013) 32–36
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalyzed kinetic resolution of racemic mixtures of
chiral ␣-aminophosphonic acids
∗
˙
Kinga Kozyra , Małgorzata Brzezin´ ska-Rodak, Magdalena Klimek-Ochab, Ewa Zyman´ czyk-Duda
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzez˙ e Wyspian´skiego 27, 50-370 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Several fungal species namely: Aspergillus niger, Aspergillus parasiticus, Penicillium funiculosum, Trigonopsis
variabilis and two different strains of Fusarium oxysporum were tested toward racemic mixtures of follow-
ing phosphonic acids: 1-amino-2-methylpropanephosphonic acid, 1-aminophenylmethanephosphonic
acid and 1-amino-2-phenylethanephosphonic acid. Application of F. oxysporum strain (UW1) allowed
obtaining optically pure S-isomer of 1-aminophenylmethanephosphonic acid and R-isomer of
1-amino-2-phenylethanephosphonic acid, whereas biotransformation of racemic mixture of 1-amino-2-
methylpropanephosphonic acid with F. oxysporum strain (DSM 12646) cells resulted in obtaining pure
enantiomer of R-configuration.
Received 18 December 2012
Received in revised form 18 February 2013
Accepted 18 February 2013
Available online 27 February 2013
Keywords:
␣-Aminophosphonic acid
Deracemization
Fungi
© 2013 Elsevier B.V. All rights reserved.
Whole-cell biocatalyst
1. Introduction
not surprising, because in the most cases, compounds exerting
biological activity are chiral molecules with defined absolute
Diversified biological activity of aminophosphonic acids
and their derivatives causes, that they have found wide
configuration. It must be considered especially during design
and preparation of new drugs. That is why the efficient methods
of synthesis of the optically pure aminophosphonates are being
intensively investigated.
Simultaneously, in recent years scientist are focused on
application of environmentally-friendly technologies. Dynamic
development of green chemistry is observed and biocataly-
sis is becoming a standard technology for the production of
chemicals on an industrial scale [14–17]. However biocatalytic
methods leading to optically pure aminophosphonic products
are rather rare, so it was an inspiration for presented work.
a
range of application [1–10]. First compound of this class, 1-
aminomethanephosphonic acid, was synthesized by Pikl during the
Second World War, in 1943, but more extensive studies, which
shown their noteworthy properties arising from structural anal-
ogy between ␣-aminophosphonic acids and natural amino acids,
were undertaken notably later [11]. These compounds are valuable
for medicine, because of their mammalian toxicity, which has been
reported to be negligible. Phosphonopeptide alafosfalin (Fig. 1)
shows broad antibacterial activity against both Gram-positive and
Gram-negative microorganisms. Similar to ␤-lactam antibiotics,
alafosfalin inhibits the biosynthesis of bacterial cell walls, but the
mechanism of its action is different. Specific dipeptide permeases
transport this molecule into the cells. There, by means of intra-
cellular hydrolysis, alanine and ␣-aminoethanephosphonic acid,
which is the inhibitor of alanine racemase involved in the biosyn-
thesis of peptidoglycan, are produced. What is important, also some
strains resistant to commonly used antibacterial agents, such as
kanamycin, gentamicin or tetracycline, are susceptible to alafos-
falin [12].
Undertaken efforts allowed developing
a simple, economic
and efficient method of resolution of racemic mixtures of ␣-
aminophosphonic acids and as a result, desired enantiomers were
obtained as pure enantiomers or meaningly enriched racemic
mixtures.
2. Experimental
2.1. General: substrates synthesis and characterization
It should be emphasized, that only the (S,R) diastereoisomer
of alafosfalin shows significant antibacterial activity [13]. It is
All chemicals were commercially available. Substrates for bio-
transformation were synthesized according to standard procedure
described in literature [18,19] and (if necessary) purified by column
chromatography (Dowex 50WX8, 200–400 mesh) or by recrystal-
lization from ethyl alcohol:water mixture (3:1, v/v).
∗
Corresponding author. Tel.: +48 71 320 4614; fax: +48 71 320 2427.
E-mail address: kinga.kozyra@pwr.wroc.pl (K. Kozyra).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.02.004

K. Kozyra et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 32–36
33
Table 1
Data for the NMR samples preparation.
␣-Aminophosphonic
acid (abbreviation)
The amount
of sample
The amount of
␣-cyclodextrin
pH value
ValP
PhgP
PheP
27 mg
20 mg
20 mg
39 mg
58 mg
58 mg
10
2
10
Fig. 1. Alafosfalin.
2.1.1. 1-Amino-2-methylpropanephosphonic acid (ValP)
31P NMR ı (ppm): 13.44.
After 3 days of cultivation, biomass was separated by filtra-
tion (P. funiculosum, A. parasiticus and A. niger) or centrifugation
(T. variabilis: 4500 rpm/15 min; F. oxysporum: 6000 rpm/15 min).
Then the pellet was washed with distilled water and centrifuged
again and the cells were applied for particular biotransformation
procedure.
1H NMR ı (ppm): 0.95 (dd, J = 6.9 Hz, 6H, (CH3)2CH), 2.00–2.15
(m, 1H, (CH3)2CH), 2.94 (dd, J = 6.3 Hz, 1H, CHP).
2.1.2. 1-Aminophenylmethanephosphonic acid (PhgP)
31P NMR ı (ppm): 10.60.
1H NMR ı (ppm): 4.19 (d, J = 15.5 Hz, 1H, CHP), 7.29 (s, 5H, Ph).
2.4. Biotransformation procedures
2.1.3. 1-Amino-2-phenylethanephosphonic acid (PheP)
31P NMR ı (ppm): 11.51.
(I) P. funiculosum. Wet fungal biomass (10 g) was transferred into
100-mL flask containing 50 mL of inducer solution (d-Val, l-
Val, d-Phe or l-Phe, respectively) in final concentration of
30 mM. Induction was carried out for 3 days in room tempera-
ture with shaking at 130 rpm. Then the biomass was separated
by filtration, washed with distilled water and added to 100-mL
flask containing 50 mL of aqueous solution of substrate (ValP in
case of cells induced with valine; for cells induced with phen-
ylalanine PhgP or PheP were applied) in final concentration of
5 mM. The reaction mixtures were incubated on a rotary shaker
(130 rpm) at room temperature for 4 days.
(II) A. niger. Wet fungal biomass (10 g) was used as biocatalyst or
preincubated for 24 h under starvation conditions (in 50 mL of
distilled water at 130 rpm and room temperature). Biotrans-
formation was carried out according to procedure above (I)
with the addition of 0.5 g of glycerol. Biotransformation was
carried out at 130 rpm and at room temperature for 3 days.
(III) A. parasiticus, T. variabilis and F. oxysporum. Wet fungal biomass
(10 g) was used directly after cultivation as biocatalyst or was
preincubated for 24 h under starvation conditions (in 50 mL of
distilled water at 130 rpm and room temperature). Biotrans-
formation was carried out for 3 days as described above (I).
1H NMR ı (ppm): 2.58–2.77 (m, 1H, CHP), 3.34–3.80 (m, 2H,
CH2), 7.14–7.36 (m, 5H, Ph).
The 1H and 31P NMR spectra were recorded on Bruker AvanceTM
600 spectrometer or Bruker Avance DRX 300 spectrometer at 298 K.
The optical rotation was measured using the polarimeter PolAAr 31.
2.2. Fungal strains
Fusarium oxysporum (DSM 12646) and Trigonopsis variabilis
(DSM 70714) were purchased from DSMZ, Germany. Aspergillus
niger (OP1) was a kind gift from the Opole University, Poland. F.
oxysporum (UW1) was a kind gift from the University of Wroclaw,
Poland. Aspergillus parasiticus (NRRLY 2999) was a kind gift from
the Anadolu University, Turkey. Penicillium funiculosum (Thom S3)
was isolated from soil sample and identified by Klimek-Ochab [20].
2.3. Cultivation of microorganisms
All cultures were grown in 250-mL flasks containing 100 mL of
appropriate medium on a rotary shaker (130 rpm) at room temper-
ature for 3 days, until the mid-log growth phase was reached.
P. funiculosum (Thom S3) and A. parasiticus (NRRLY 2999) were
cultivated on commercially available Potato Dextrose Broth.
A. niger (OP1) was cultivated on special medium for optimal
d-amino acid oxidase production, this medium was consisted of:
4 g/L casamino acids, 1 g/L KH2PO4, 0.1 g/L MgSO4•7H2O, 0.5 g/L
yeast extract, 10 g/L glycerol (as an effective carbon source for
enzyme production) and enzyme synthesis inducer (d-valine or
d-phenylalanine) in final concentration of 30 mM [21].
T. variabilis (DSM 70714) was cultivated on Universal Medium
For Yeasts, which was consisted of 3 g/L yeast extract, 3 g/L malt
extract, 5 g/L peptone from soybeans and 10 g/L glucose.
F. oxysporum (UW1) was cultivated on three different media:
(a) Czapek-Dox Medium, which was consisted of 30 g/L sucrose,
0.5 g/L MgSO4•7H2O, 0.5 g/L KCl, 2.64 g/L (NH4)2SO4, 0.01 g/L FeSO4
and 0.5 g/L KH2PO4 [22]; (b) special medium containing inducer:
18 g/L glucose, 4 g/L K2HPO4, 4 g (NH4)2SO4, 4 g/L yeast extract,
1 g/L MgSO4•7H2O, 0.5 g/L CaCl2•2H2O, 0.1 g/L H3BO3, 0.04 g/L
NaMoO4, 0.04 g/L ZnSO4•7H2O, 0.045 g/L CuSO4•7H2O, 0.025 g/L
FeSO4•7H2O and inducer (d-valine or d-phenylalanine) in final
concentration of 15 mM [23]; (c) commercially available Potato
Dextrose Broth.
Subsequently, after bioconversion was completed, biocatalyst
was removed by filtration (P. funiculosum, A. parasiticus and A. niger)
or centrifugation (T. variabilis: 4500 rpm/15 min and F. oxysporum:
6000 rpm/15 min) and the filtrate/supernatant was evaporated.
2.5. Enantiomeric excess assignment
The enantiomeric excess was determined by means of 31P
NMR spectroscopy with the addition of ␣-cyclodextrin as a chi-
ral discrimination agent [24]. NMR samples were prepared as
follows: appropriate amounts of biotransformation products and
␣-cyclodextrin were dissolved in 600 ␮L of D2O (specific data in
Table 1). Then the solutions were adjusted to proper pH (Table 1).
Thereafter the measurement was done.
2.6. Absolute configuration assignment
The absolute configuration was assigned by measurements of
20
the optical rotation [˛]
in 1 M NaOH. The results were eval-
578
20
uated and confirmed according to literature data [25]: [˛]
−1.0 for (R)-1-amino-2-methylpropanephosphonic acid; [˛]
+18.0 for (S)-1-aminophenylmethanephosphonic acid; [˛]
+37.0 for (R)-1-amino-2-phenylethanephosphonic acid.
=
=
=
578
20
578
20
F. oxysporum (DSM 12646) was cultivated on Malt Extract
Peptone Broth containing 30 g/L malt extract and 3 g/L soya
peptone.
578

34
K. Kozyra et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 32–36
Fig. 2. Biotransformed ␣-aminophosphonic acids.
3. Results and discussion
for the effectiveness of resolution of racemic mixtures of chiral
xenobiotics with P C bond, however in earlier research, only one
model compound was examined and obtained optical purity was
moderate – 50% of e.e. [40]. Therefore, current work extends these
experimental field in order to improve the results, to find efficient
microbial biocatalysts capable to resolve racemic mixtures of series
of structurally different chiral ␣-aminophosphonates (Fig. 2), and
finally to optimize the process conditions.
The purpose of this research was to obtain optically pure enan-
tiomers of ␣-aminophosphonic acids via microbial transformation
of racemic mixture of chiral substrates, which is considered as
kinetically controlled bioconversion. Several asymmetric syn-
thesis methods for ␣-aminophosphonic acids derivatives have
been developed so far [13,26–28]. The main strategies involve
enantioselective and diastereoselective hydrophosphonylation of
imines; enantioselective and diastereoselective reaction of alde-
hyde/ketone with amine and dialkyl phosphite; diastereoselective
alkylation of phosphoglycinates derivatives; diastereoselective
amination of phosphonates; chiral pool processes; catalytic
asymmetric hydrogenation of dehydroaminophosphonates and
resolution methodologies. Procedures developed until 2012 for
the stereoselective synthesis of ␣-aminophosponic acids analogs
of valine and phenylalanine were reviewed by Ordón˜ez et al. [29],
whereas asymmetric synthesis methods concerning the analog of
phenylglycine were reported by Joly and Jacobsen [30], Chen and
Yuan [31], Abell and Yamamoto [32], Metlushka et al. [33], Zhao
et al. [34] and Ohara et al. [35].
Relevant disadvantages of asymmetric synthesis (toxic wastes,
organic solvents, catalysts, economy) make biocatalysis an inter-
esting alternative. However, obtaining ␣-aminophosphonic acids
via enzymatic resolution is still only partly explored field of
biotechnology. Chenault et al. reported the application of Acyl-
ase I for the kinetic resolution of amino acids and their analogs
with high enantiomeric excesses. However, that method was not
applicable for ␣-aminophosphonic acids [36]. Solodenko et al.
applied penicillin acylase for stereoselective hydrolysis of racemic
1-(N-phenylacetylamino)alkylphosphonic acids. Subsequently, the
corresponding l-aminophosphonic acid was separated by column
chromatography and acid hydrolysis of unreacted d-enantiomer
allowed obtaining d-aminophosphonic acid [37].
Especially noteworthy is applying of whole-cell biocatalysts,
because this strategy eliminates the necessity of using additional
cofactors required by many enzymes. Microorganisms have the
ability to perform diversified chemical reactions, most of them
are very selective. Fungi are important source of enzymes of var-
ied activities, some of them are commercially useful. Moreover,
among these microorganisms, there are also strains able to biode-
grade organophosphorus compounds by cleaving C P bond. Some
fungi utilize phosphonates as sole source of phosphorus for growth
[20,38,39], therefore they are not susceptible to potential toxicity of
these compounds. That is why chosen fungal strains were applied
as biocatalysts, which implied in obtaining pure enantiomers of
␣-aminophosphonic acids – bioconversion substrates.
Among whole-cell biocatalysts tested, the most potent were two
different strains of F. oxysporum (Table 2).
It should be pointed out, that the influence of cultivation con-
ditions on bioconversion results, was significant. Application of
Czapek-Dox Medium or special, rich medium containing inducer
(compound mediating the amino acids transport cross plasma
membrane and/or synthesis of enzymes of desired activity; here:
amino acid oxidases) for growing F. oxysporum strain (UW1),
implied in unsatisfactory results (enantiomeric excess up to 20%).
In contradiction for the same fungal strain, direct addition of the
substrate into the bioreaction medium, after the biomass culti-
vation on Potato Dextrose Broth for 3 days, fruited in obtaining
of (S)-1-aminophenylmethanephosphonic acid and (R)-1-amino-
2-phenylethanephosphonic acid as pure enantiomers (Fig. 3).
Furthermore, applying F. oxysporum strain (DSM 12646) culti-
vated on Malt Extract Peptone Broth resulted in obtaining pure
(R)-1-amino-2-methylpropanephosphonic acid. What is impor-
tant, the preincubation of that biocatalyst for 24 h under starvation
conditions, before biotransformation, was crucial. It could arise
from the fact, that this compound is not a physiological substrate
Table 2
Resolution of racemic mixtures of chiral ␣-aminophosphonic acids.
Substrate
ValP
The best results of biotransformation
(e.e./configuration)
Substrate
conversion [%]
40% R (Trigonopsis variabilis)
100% R (Fusarium oxysporum DSM
12646a)
36% S (Aspergillus niger)
27% S (Aspergillus nigera)
40% R (Fusarium oxysporum DSM
12646)
29
50
PhgP
26
21
27
54% S (Fusarium oxysporum DSM
33
12646a)
100% S (Fusarium oxysporum UW1)
21% S (Fusarium oxysporum UW1a)
19% S (Fusarium oxysporum DSM
12646)
50
16
16
PheP
15% R (Fusarium oxysporum DSM
12
12646a)
100% R (Fusarium oxysporum UW1)
50
Presented studies are continuation of previous ones, which
proved that, appropriate biotransformation conditions are crucial
Bold was applied to indicate, when the racemic mixtures were completely resolved.
a
Biotransformation procedure involved starvation step.

K. Kozyra et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 32–36
35
Fig. 3. 31P NMR spectra of biotransformation products recorded with ␣-cyclodextrin: biocatalyst: Fusarium oxysporum strain (UW1) cultivated on PDB; substrate:
1-aminophenylmethanephosphonic acid: (A) first day of biotransformation – 17% e.e. of S-enantiomer, (B) third day of biotransformation – only S-isomer of 1-aminophenyl-
methanephosphonic acid is present.
for fungi, therefore, it is utilized only if other substances are not
available. However, it is not a general phenomenon and conditions
should be established empirically.
– unreacted – remains in the supernatant solution, that allows
receiving optically pure ␣-aminophosphonic acid (Fig. 4). It could
suggest, that amino acid oxidase is involved in the reaction.
This enzyme under physiological conditions catalyzes the oxida-
tive deamination of amino acids into corresponding ␣-keto acids
[41–43]. There are l- and d-amino acid oxidases inside viable
cells, which are absolutely stereoselective and transform only one,
appropriate enantiomer. Thus, they can be used in processes lead-
ing to enantiomerically pure amino acids [44]. On the other hand,
these enzymes show broad substrate specificity and are capable
to deaminate several amino acids with hydrophobic side chains,
and even some other chemical compounds such as cephalosporin C
[43,45,46]. That is why, also ␣-aminophosphonic acids, as analogs
of amino acids are possible substrates for amino acids oxidases.
Other explanation of discussed lack of the signals deriving from
other organophosphorus molecules, is that, the particular enan-
tiomer – bioconversion substrate – is transported across plasma
membrane and converted inside the cell, therefore the possibility of
the transfer limits the process, living the optical purity of the prod-
ucts on moderate level. Finally it should be under consideration
On the other hand, in the case of F. oxysporum strain (DSM
12646) the influence of starvation period on the absolute config-
uration of product was evident. When fresh cells were used, the
enantiomeric excess after 3 days of biotransformation was 40% of
R-enatiomer of phenylglycine analog and 19% of S-enatiomer of the
phosphonic analog of phenylalanine. Applying cells preincubated in
distilled water (starvation step) resulted in enrichment of racemic
mixtures with the S-enatiomer of PhgP (54% e.e.) and R-enatiomer
of PheP (15% e.e.), respectively. Unfortunately, even when the bio-
transformation was carried out for 7 days, these mixtures were not
completely resolved.
The analysis of 31P NMR spectra of biotransformation mixture
suggests possible mechanism of presented microbial conversion.
Apart from signals corresponding to the products, there are no
signals of other organophosphorus compounds. Probably, one
enantiomer of ␣-aminophosphonic acid is transformed into ␣-
ketophosphonic acid, which is unstable in water. The second one
Fig. 4. Postulated mechanism of biotransformation.

36
K. Kozyra et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 32–36
that, despite other explanations, unstable oxoaminophosphonates
(deamination step products) split and served as inorganic source of
phosphorus atom for living cells and as a consequence any trace of
phosphorus was recorded.
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Assuming, this article reports for the first time protocols allowed
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Described procedure seems to be promising for scaling up and
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