Whole-cell biotransformation of diethyl 1-hydroxy-1-phenylmethanephosphonate in a different reaction environment

Article abstract of DOI:10.1080/10426507.2016.1247088

The whole-cell biocatalytic resolutions of enantiomers of racemic 1-butyryloxy-1-phenylmethane-phosphonate was performed by Bacillus subtilis strain in different media. For this purpose, the well-known medium, which induce lipolytic properties, was tested

Full text of DOI:10.1080/10426507.2016.1247088

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Whole-cell biotransformation of diethyl 1-
hydroxy-1-phenylmethanephosphonate in a
different reaction environment
Paulina Majewska
To cite this article: Paulina Majewska (2016): Whole-cell biotransformation of diethyl 1-
hydroxy-1-phenylmethanephosphonate in a different reaction environment, Phosphorus,
Sulfur, and Silicon and the Related Elements, DOI: 10.1080/10426507.2016.1247088
To link to this article: http://dx.doi.org/10.1080/10426507.2016.1247088
Accepted author version posted online: 13
Oct 2016.
Published online: 13 Oct 2016.
Submit your article to this journal
Article views: 17
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gpss20
Download by: [Fudan University]
Date: 12 January 2017, At: 14:01

PHOSPHORUS, SULFUR, AND SILICON
http://dx.doi.org/./..
Whole-cell biotransformation of diethyl -hydroxy--phenylmethanephosphonate in a
different reaction environment
Paulina Majewska
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland
ABSTRACT
ARTICLE HISTORY
Received  July 
Accepted  October 
The whole-cell biocatalytic resolutions of enantiomers of racemic 1-butyryloxy-1-phenylmethane-
phosphonate was performed by Bacillus subtilis strain in different media. For this purpose, the well-known
medium, which induce lipolytic properties, was tested as well as nutrient broth medium. The enantios-
electivity of the hydrolysis reactions was at the same level (50% conversion, ∼80% ee) regardless of the
used medium, but the time of reaction varied significantly, depending on the medium in which the
microorganism was grown and on the reaction medium. The addition of 0.01% of tributyrin shortened the
response time in the range from 30 min to 1 h. In addition, when the nutrient broth with tributyrin was
used as a medium for grown and for biotransformation reactions, enantioselectivity also increased from 23
to 35.
KEYWORDS
Biocatalysis;
enantioselectivity;
hydroxyphosphonates
GRAPHICAL ABSTRACT
extracellular lipases (LipA and LipB).10 The biotransformations
of hydroxyphosphonates conducted by this strain are highly
enantioselective.7–9
Introduction
Many research teams are interested in chiral hydroxyphospho-
nates and obtaining them in an optically pure form because they
are a class of organophosphorus compounds applied as building
blocks for the synthesis of medicinally important molecules.1,2
Moreover, substituted hydroxyphosphonic acids constitute a
class of mimics of natural hydroxycarboxylic acids3 and conse-
quently are considered as those that may exhibit promising bio-
logical activity. This is indeed the fact and some of these com-
pounds exhibit interesting antibacterial, antiviral or anticancer
agents acting as inhibitors of enzymes important for pathogens
development.3,4
The whole-cell biotransformation is becoming an increas-
ingly popular method, because stereoselective biocatalytic
processes constitute a useful alternative to asymmetric syn-
thesis of chiral compounds.5 Until now, there are only few
publications about whole-cell biotransformation of hydrox-
yphosphonates by microorganisms with lipolytic activity. The
lipases produced by bacteria strains can catalyse the hydroly-
sis of esters of hydroxyphosphonates by means of kinetic res-
olution providing high enantiomeric excess of both hydrox-
yphosphonates and their unreacted esters.6–9 There are many
bacterial strains which exhibit lipolytic activity. One of them
is the common bacteria Bacillus subtilis, which produces two
The activities of most of bacterial lipases are usually induced
in a medium that contains fatty acids and oils,11–13 but also other
triglycerides like tributyrin or surfactants can induce lipoly-
tic activity.14,15 The well-known growth medium for bacteria
strain used in biotransformation reactions of hydroxyphospho-
nates (medium A.2, see paragraph 2.1.1) contained: 10 g of
soluble starch, 1 g of yeast extract, 5 g of (NH4)2SO4, 2 g of
K2HPO, and 100 μL of tributyrin.7–9 After 24 h of incuba-
tion, the microorganism was centrifuged and transferred to
the buffer, wherein the biotransformation reactions were per-
formed. There are reports that the nutrient broth was applied
as both medium for growing of bacteria and as a medium for
biotransformations.16 The use of the medium in which the
microorganism is growing as the reaction medium appears to be
appropriate, because of the simplification of biotransformation
procedures. In this work it is studied how the composition of
the reaction medium, and the culture medium has an influence
on the reaction enantioselectivity. For this purpose, the follow-
ing media were tested: popular A.2 medium, a similar medium
lacking tributyrin, and a classical nutrient broth with and with-
out tributyrin.
CONTACT Paulina Majewska
paulina.majewska@pwr.edu.pl
Wybrzez˙e Wyspian´skiego , - Wrocław, Poland.
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology,
©  Taylor & Francis Group, LLC

2
P. MA JEWSKA
Figure . P NMR spectrum with quinine after hydrolysis of diethyl -butyryloxy--phenylmethanephosphonate  ( h, medium A., method ).
ucts were analyzed by 31P NMR spectroscopy using quinine as
CSA (Figure 1) and by analytical HPLC using a chiral column
(Figure 2). Because of the nature of the kinetic resolution only
the results when conversion was close to 50% are shown.
An increased reaction time resulted in a decrease of enan-
tiomeric excess of product up to 0% for a conversion being near
100% at 24–48 h depending on the medium and procedure. The
enantiomeric excess of unreacted substrate is increasing during
the time, but it is connected with a decrease of yield of obtained
unreacted ester.
As can be seen from Tables 1–2, the conversion near 50%
was obtained faster when the method 1 was used. This proce-
dure is far easier to perform than method 2 because, after incu-
bation, the cells remained in the medium, consequently cen-
trifugation and washing of bacterial cells was omitted. It can
therefore be concluded that the separation of cells of bacte-
ria followed by their suspension in the buffer is inefficient and
unnecessary. At the same time it can be deduced that the addi-
tion of tributyrin reduce time required to reach 50% of degree
conversion (in some cases up to 1 h), which suggest that addi-
tion of this component assists in producing a more amount of
Results and discussion
Racemic hydroxyphosphonate (compound 1) was obtained by
the addition of diethyl phosphite to the corresponding alde-
hyde. After purification, racemic hydroxyphosphonate 1 was
converted into O-butyryl derivative 2 by simple acylation with
butyryl chloride. The ester 2 was then hydrolysed using Bacil-
lus subtilis strain as a whole-cell biocatalyst. Such kinetic res-
olution of this compound by hydrolysis with Bacillus subtilis
was described previously.9 The reaction resulted in an enan-
tiomeric excess of 95% at 44% substrate conversion after 8 h.
Also Serrratia liquefaciens, Escherichia coli, Pseudomonas aerug-
inosa, and Pseudomonas fluorescens were tested following the
same procedures of growth of these strains (medium A.2)
and biotransformation procedures (phosphate buffer 0.017M
pH = 7.0).9
In this report the change in composition of the growing
medium and procedure of hydrolysis affecting the course of
this reaction was studied. Thus, biotransformation reactions
were carried out in four different media and additionally by
two different methods. Each time, the biotransformation prod-
Figure . Chromatogram after hydrolysis of diethyl -butyryloxy--phenylmethanephosphonate  ( h, medium A., method ).

PHOSPHORUS, SULFUR, AND SILICON
3
for 31P and 151.02 MHz for 13C in CDCl3 (99.8% of D, con-
taining 0.03% v/v TMS) or on a Bruker AvanceTM DRX 300
Table . Hydrolysis of diethyl -butyryloxymethanephenylphosphonate by Bacillus
subtilis in media A and A.
1
Time Conversion ee of ester ee of alcohol
instrument operating at 300.13 MHz for H; 121.50 MHz for
31P and 75.45 MHz for 13C in CDCl3 (99.8% D with 0.03% v/v
TMS). Chemical shifts (δ) are reported in ppm in relation to
standards. The biotransformations products were analysed by
31P NMR with quinine used as a Chiral Solvating Agent (CSA).
In the case where not all the signals from the enantiomers have
been separated, the sample was also analysed on HPLC using
LaChrome D-7000 system with chiral column Chiralpak AD
250 × 4.6 mm. Synthesized compounds were purified by gra-
dient column chromatography using Merck Silica Gel 60 (63–
230 mesh). Separation of unreacted substrate from the product
was done by Medium-pressure Liquid Chromatography system
Interchim PuriFlash 430evo on reversed phase column Puri-
Flash C18-HP with a grain size of 15 microns. Specific rotation
was measured on polAAr 31 polarimeter in chloroform (λ = 589
nm).
Medium Method [h]
[%]
[%]
[%]
E
A.







.





































A.





.













lipases. Only in one case, when the medium B.2 was used, the
time of reaction time increased significantly. Unfortunately, the
addition of tributyrin does not affect the enantioselectivity of
biotransformation.
Moreover, it can be noticed that the change of the method
from medium-based to buffer-based does not increase the time
required to achieve 50% of degree conversion and does not
improve the enantioselectivity of the reaction.
Synthesis of diethyl
1-hydroxy-1-phenylmethanephosphonate and diethyl
1-butyryloxy-1-phenylmethanephosphonate
Finally it can be perceived that the direct use of nutrient broth
significantly reduced the time of biotransformation reactions,
and that the combination of the use of cell culture media and
medium of reaction in one (medium B.1 and method 1) gave
the best results.
Compounds 1 and 2 were synthesized according to the method
described previously.9
Microorganism: growth and biotransformations conditions
Bacillus subtilis strain was stored in nutrient agar at a temper-
ature of 4 °C. In order to obtain good conditions for microor-
ganism growth and ensuring satisfied results of biotransfor-
mation reactions, several media were tested. They were as
follows:
Experimental
Medium A.1 composed of: 10 g of starch soluble, 1 g of yeast
extract, 5 g of (NH4)2SO4, 2 g of K2HPO4 and 1000 mL of dis-
tilled water;
Materials and methods
All chemicals were purchased from Sigma Aldrich (St. Louis,
Missouri, United States), POCh (Gliwice, Poland) or BIO-
CORP (Warszawa, Poland) and were used without further
purification.
Medium A.2:7 10 g of starch soluble, 1 g of yeast extract, 5 g of
(NH4)2SO4, 2 g of K2HPO4, 100 μL of tributyrin and 1000 mL
of distilled water;
B. subtilis strain was from our own collection (isolated
from soil) and was identified by the Deutsche Sammlung für
Mikroorganismen und Zellkulturen (DSMZ), Braunschweig,
Germany.
Medium B.1: 8 g of nutrient broth (BIOCORP PS 90) and
1000 mL of distilled water;
Medium B.2: 8 g of nutrient broth (BIOCORP PS 90), 100 μL
of tributyrin and 1000 mL of distilled water.
Microorganisms were incubated at 26 °C with shaking at
150 rpm for 24 h. Then biotransformation reactions were pre-
pared in two ways:
NMR (Nucleic Magnetic Resonance) spectra were measured
on a Bruker AvanceTM 600 at 600.58 MHz for 1H; 243.12 MHz
Method 1: After incubation cells remained in the medium
and the 50 μL of substrate was added.
Table . Hydrolysis of diethyl -butyryloxymethanephenylphosphonate by Bacillus
subtilis in media B and B.
Method 2: After incubation cells were centrifuged at
3000 rpm for 10 min and washed twice in 0.017 M phos-
phate buffer, pH 7.0. The washed cells were suspended in
100 mL of 0.017 M phosphate buffer and 50 μL of substrate was
added.
Time Conversion ee of ester ee of alcohol
Medium Method [h]
[%]
[%]
[%]
E
B.

.

.












Biotransformation reactions were carried out at 26 °C with
shaking at 150 rpm. After an appropriate time depending on
conversion (close to 50%, see Tables 1 and 2), reactions were
stopped by biomass centrifugation followed by extraction of bio-
transformation products with ethyl acetate (50 mL). Organic
layer was dried over anhydrous magnesium sulfate and, after fil-
tration, the organic solvent was evaporated.


.


.




















B.



.









4
P. MA JEWSKA
Enantioselectivity assignment
Acknowledgment
The mixtures of biotransformations products (alcohol and unre-
acted ester) were analyzed by 31P NMR spectroscopy using qui-
nine as a chiral solvating agent and by analytical HPLC with
chiral column (CHIRALPAK AD, Diacel, gradient method:
15 min of isocratic flow of 1% of 2-propanol in n-hexane, 5 min
from 1% to 4% of 2-propanol in n-hexane, 15 min of isocratic
flow of 4% of 2-propanol in n-hexane, products retention time:
ester: (S) –11.5 min, (R) – 13 min, alcohol: (S) – 25 min, (R) –
26.5 min.)
I would like to thank Agnieszka Tysler, student at the Department of Chem-
istry, who helped in the implementation of the initial research.
Funding
This study was financed from the Project: “Biotransformations for phar-
maceutical and cosmetics industry” No. POIG.01.03.01-00-158/09 part-
financed by the European Union within the European Regional Develop-
ment Fund for the Innovative Economy.
The degree of the enantiomeric excess was expressed as a per-
centage (%) and defined as:
References
1. Kiss, N. Z.; Rádai, Z.; Mucsi, Z.; Keglevich, G. Heteroat. Chem. 2016,
27, 1-9.
2. Demkowicz, S.; Rachon´, J.; Da´sko, M.; Kozak, W. RSC Adv., 2016, 6,
7101-7112.
3. Kolodiazhnyi, O. I. Tetrahedron: Asymm. 2005, 16, 3295-3340.
4. Collinsova, M.; Jiracek, J. Curr. Med. Chem. 2000, 7, 629-647.
5. Wachtmeister, J.; Rother, D. Curr. Opin. Biotechnol. 2016, 42, 169-177.
6. Skwarczyn´ski, M.; Lejczak, B.; Kafarski, P. Chirality 1999, 11, 109-114.
7. Majewska, P.; Kafarski, P.; Lejczak, B.; Bryndal, I.; Lis, T. Tetrahedron:
Asymmetry, 2006, 17, 2697-2701.
8. Majewska, P. Bioorg. Chem. 2015, 61, 28-35.
9. Malinowska, B.; Majewska, P.; Szatkowski, P.; Kafarski, P.; Lejczak, B.
Biocatal. Biotransform. 2011, 29, 271-277.
P1 − P2
ee =
× 100%
P1 + P2
where P1 and P2 are the values of the area under the signals
coming from the major and minor enantiomer of the product
respectively.
Enantiomeric ratio (E) was computed from the following for-
mula:
(1−ees )
ln[eep
ln[eep
]
]
(ees+eep)
E =
(1+ees )
(ees+eep)
10. Eggert, T.; Pouderoyen, G.; Dijkstra, B. W.; Jaeger, K.-E. FEBS Lett.
2001, 502, 89-92.
11. Joseph, B.; Ramteke, P. W.; Kumar, P. A. J. Gen. Appl. Microbiol. 2006,
52, 315-320.
Conclusions
The use of whole cells of Bacillus subtilis seems to be a method
of choice for obtaining α-hydroxyphosphonates with high enan-
tiomeric excess. The reaction conditions are environmentally
friendly and are compatible with the global trend of green chem-
istry. The use of culture medium as the reaction medium sub-
stantially accelerates the biotransformation, probably because
enzymes are already produced before introduction of substrate.
Additionally, the use of a substantially simpler and commercially
available cultivation medium is profitable, because its prepara-
tion is much easier and biotransformation reactions proceed
faster and more effective.
12. Immanuel, G.; Esakkiraj, P.; Jebadhas, A.; Iyapparaj, P.; Palavesam, A.
Food Technol. Biotechnol. 2008, 46, 60-65.
13. Kiran, G. S.; Shanmughapriya, S.; Jayalakshmi, J.; Selvin, J.; Gand-
himathi, R.; Sivaramakrishnan, S.; Arunkumar, M.; Thangavelu,
T.; Natarajaseenivasan, K. Bioprocess Biosyst. Eng. 2008, 31,
483-492.
14. Sharma, R.; Chisti, Y.; Banerjee, U. C. Biotechnol Adv. 2001, 19, 627-
662.
15. Prasanna Rajeshkumar, M.; Mahendran, V. S,; Balakrishnan, V. I.J.S.N.
2015, 6, 183-187.
16. Norul, A. S.; Nazira, M.; Tajuddin, S. N. Aust. J. Basic Appl. Sci 2015,
9, 155-159.