Enzymatic resolution of ethyl α-hydroxyphosphinates in a modified reaction environment

Article abstract of DOI:10.1080/10426500903365595

The enzymatic resolutions of two racemic ethyl hydroxyalkane(P-phenyl) phosphinates were performed by both esterification and hydrolysis approaches. The first reaction was performed in anhydrous diisopropyl ether with triethylamine or pyridine as additive

Full text of DOI:10.1080/10426500903365595

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Phosphorus, Sulfur, and Silicon and the
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Enzymatic Resolution of Ethyl α-
Hydroxyphosphinates in a Modified
Reaction Environment
Paulina Majewska a , Barbara Lejczak a & Paweł Kafarski a
a Department of Bioorganic Chemistry, Faculty of Chemistry ,
Wrocław University of Technology , Wrocław, Poland
Published online: 25 Aug 2010.
To cite this article: Paulina Majewska , Barbara Lejczak & Paweł Kafarski (2010) Enzymatic
Resolution of Ethyl α-Hydroxyphosphinates in a Modified Reaction Environment, Phosphorus, Sulfur,
and Silicon and the Related Elements, 185:9, 1915-1920, DOI: 10.1080/10426500903365595
To link to this article: http://dx.doi.org/10.1080/10426500903365595
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Phosphorus, Sulfur, and Silicon, 185:1915–1920, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500903365595
ENZYMATIC RESOLUTION OF ETHYL
α-HYDROXYPHOSPHINATES IN A MODIFIED
REACTION ENVIRONMENT
Paulina Majewska, Barbara Lejczak, and Paweł Kafarski
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University
of Technology, Wrocław, Poland
The enzymatic resolutions of two racemic ethyl hydroxyalkane(P-phenyl)phosphinates
were performed by both esterification and hydrolysis approaches. The first reaction was
performed in anhydrous diisopropyl ether with triethylamine or pyridine as additives
by using lipases from three different sources (Candida cylindracea, Aspergillus niger,
and Mucor javanicus). The increase in enantioselectivity was observed when NEt3 was
applied. The second reaction—lipase-catalysed hydrolysis of ethyl butyryloxyalkane(P-
phenyl)phosphinates—was carried out by Candida cylindracea lipase in diisopropyl ether
saturated with water or in aqueous solutions containing MgCl2, LiCl, or Triton X-100. The
usefulness of biphasic systems consisting of diisopropyl ether and water or aqueous solution
of MgCl2, LiCl, or Triton X-100 also were tested. The use of biphasic system in the presence
of Triton X-100 resulted in the higher conversion of the substrates.
Keywords Biocatalysis; enantioselectivity; hydroxyphosphinates; hydroxyphosphonates;
lipases
INTRODUCTION
Chiral hydroxyphosphonates are considered as useful precursors of structurally
variable organophosphonic compounds.1–4 Hydroxyalkanephosphonic acids also exhibit
promising biological activity. For example, some of these compounds are interesting an-
tibacterial, antiviral, or anticancer agents acting as inhibitors of enzymes important for
development of these pathogens.5 It is well acknowledged that the biological activity of
organic compounds is strongly dependent on their three-dimensional structure, and thus
configuration and optical purity. That is one of the reasons why studies on the procedures
leading to chiral hydroxyphosphonates are so intensive.1
A range of methods is available for the preparation of pure enantiomers of these
compounds based on lipase-catalyzed reactions. It is well acknowledged that some ad-
ditives have an influence on the environment of the enzymes and hence on their selec-
tivity and/or activity.6 For example, Okamoto and Ueji7 improved the enantioselectivity
Received 16 July 2009; accepted 24 September 2009.
Address correspondence to Paulina Majewska, Department of Bioorganic Chemistry, Faculty of Chem-
istry, Wrocław University of Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wrocław, Poland. E-mail:
paulina.majewska@pwr.wroc.pl
1915

1916
P. MAJEWSKA ET AL.
of lipase-catalysed hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates by
using diisopropyl ether saturated with an aqueous solution of MgCl2 and LiCl instead of
using the same solvent saturated with water. The same system, with the use of MgCl2 as an
additive, was used for lipase-catalysed hydrolysis of hydroxyphosphonates.8 Bashkar Rao
et al.9 used 2% solution of nonionic surfactant Triton X-100, which might be considered
as an open-chain crown ether analogue, as an additive to increase enantioselectivity of the
Candida cylindracea lipase. It is also known that basic additives such as triethylamine
have influenced enantioselectivity and/or activity of these enzymes in transesterification
reactions.6
In this article, we report a method to engineer the environment of the lipase-catalysed
reactions in order to improve enantioselectivity and conversion of the applied substrates
containing two centers of chirality—one located at the phosphorus atom and the sec-
ond at α-carbon atom of the molecules. The goal was reached by an application of
two approaches. The first approach was the esterification in diisopropyl ether contain-
ing NEt3 or pyridine as basic additives. The second approach was hydrolysis of ethyl
butyryloxyalkane(P-phenyl)phosphinates either in diisopropyl ether saturated with water
or with aqueous solutions of MgCl2, LiCl, or Triton X-100 or using biphasic diisopropyl
ether/aqueous system.
RESULTS AND DISCUSSION
Enzymatic Transesterifications
Racemic hydroxyphosphinate (compound 1a) was obtained by addition of ethyl
phenylphosphinite to the appropriate aldehyde.10 The kinetic resolution of this compound
by transesterification with lipases from Candida cylindracea, Aspergillus niger, and Mucor
javanicus in organic solvents has been already described.11 The observed enantioselectivity
and degree of conversion was quite good but not excellent. In this report, we have studied
how the application of additives such as pyridine and triethylamine affect the course of this
reaction (Scheme 1A). As seen in Table I, addition of triethylamine results in enhancement
of enantioselectivity of the reaction catalyzed by all three lipases, with the addition of
pyridine having a far less pronounced effect.
Enzymatic Hydrolysis
In order to study the hydrolytic procedure, racemic hydroxyphosphinates 1 were
converted into their O-butyryl derivatives 2 by simple acylation with butyryl chloride.11 The
esters 2 were then hydrolyzed using Candida cylindracea lipase. The kinetic resolutions of
compounds 2 (Scheme 1B) were carried out in two solvent systems—either in diisopropyl
ether saturated with water or aqueous solutions of MgCl2, LiCl, or Triton X-100 or in
analogous biphasic system. The hydrolysis was stopped when chemical yield reached a
value close to 50% (which is typical for kinetic resolution). As shown in Table II, all
the reactions carried out in a homogenous system resulted in low enatioselectivity of the
reaction, which disqualifies this procedure.
Therefore we decided to use a biphasic system composed of the same organic and
water fractions, namely water or aqueous solution of MgCl2, LiCl, or Triton X-100 and
diisopropyl ether in a volume ratio of 1:2. As seen from Table III, this approach resulted in
increasing enantioselectivities of the hydrolysis of compound 2b and did not affect either

ENZYMATIC RESOLUTION OF ETHYL α-HYDROXYPHOSPHINATES
1917
A
B
R
R
R
lipase / vinyl butyrate
additive
O
H
H
H
+
P
O
O
P
O
P
O
OH
OH
EtO
EtO
EtO
1a
2a: (Rp,S) and (Sp,S)
1a: (Rp,R) and (Sp,S)
R
R
R
O
H
O
H
lipase
H
+
P
O
P
O
O
O
P
O
EtO
EtO
OH
additive
EtO
2
1
: (Rp,S) and (Sp,S)
2: (Rp,R) and (Sp,S)
a
b
R = -CH3
R = -C6H5
Scheme 1
the rate of hydrolysis or enantioselectivity of hydrolysis of 2a. The application of Triton X-
100 also resulted in significant increase of the conversion of compound 2b without harmful
effect on the enantioselectivity of the process, which still is low.
Solvent engineering belongs to the classic tools for the improvement of the course
of biocatalytic processes. Esterification of 1-hydroxyethane(P-phenyl)phosphinate 1a in
diisopropyl ether under molecular sieves (3Å mesh) proceeds with satisfactory enantios-
electivity.11 Using the same system enriched with triethylamine resulted in improved
enatioselectivity of this reaction. It is worth mentioning that this biocatalytic process
Table I Enzymatic esterification of ethyl 1-hydroxyethane(P-phenyl)phosphinate 1a by lipases from different
sources
Conversion (%)
ee of ester (%)
(RP,S) (SP,S)
91 79
Enzyme
amount (mg)
Time
(h)
Lipase
CCL
Additive
(SP,R) (RP,S)
(RP,R) (SP,S)
∗
—
200
200
200
200
200
200
20
72
72
72
168
72
72
96
72
72
44
40
39
44
43
53
48
45
43
42
46
41
44
43
50
46
50
43
NEt3
pyr∗idine
—
NEt3
pyr∗idine
—
95
79
91
93
89
38
79
37
90
85
>98
>98
>98
51
ANL
MJL
NEt3
pyridine
20
20
>98
47
Reactions were carried out in diisopropyl ether with addition of triethylamine or pyridine (0.023 mmol of
substrate, 0.023 mmol of additive) and compared with esterification carried out without additive as described
previously.11
∗Data described previously in publication.11

1918
P. MAJEWSKA ET AL.
Table II Enzymatic hydrolysis of ethyl 1-butyryloxyethane(P-phenyl)phosphinate 2a and 1-butyryloxyphenyl
methane(P-phenyl)phosphinate 2b in organic solvent
Conversion (%)
ee of alcohol (%)
Substrate
Additive
Time (h)
(RP,R); (SP,S)
(RP,S); (SP,R)
(SP,S)
(RP,S)
2a
—
MgCl2
LiCl
Triton X-100
—
MgCl2
LiCl
2
2
2
37
37
33
37
48
24
21
29
63
65
61
63
34
20
20
23
18
21
16
15
28
11
18
25
50
46
48
45
18
11
11
14
2
2b
168
168
168
168
Triton X-100
Reactions were carried out in diisopropyl ether saturated with water or aqueous solution of LiCl, MgCl2, or
Triton X-100 (100 mg of Candida cylindracea lipase, 0.036 mmol of substrate, 15 µL water or aqueous solution,
3 mL diisopropyl ether).
did not recognize chirality at the phosphorus atom. The esterification of the second
substrate—hydroxy(phenyl)methane(P-phenyl)phosphinate 1b—did not proceed despite
of the solvent used. Hydrolysis of its acylated derivative is an alternative approach.
Hydrolysis of butyryloxyphosphinates 2 was carried out using Candida cylindracea
lipase, and two solvent systems indicated that the biphasic system, rather than the organic
solvent saturated with water, gives more reliable results. The biphasic system supplemented
with surfactant such as Triton X-100 appeared to be the most effective. Quite interestingly,
in this case better resolutions were obtained for compound 2b, which shows that the two
reactions are complementary. However, also in this case, chirality of the phosphorus atom
has no influence on the discrimination by the enzyme.
Table III Enzymatic hydrolysis of ethyl 1-butyryloxyethane(P-phenyl)phosphinate 2a and 1-butyryloxyphenyl
methane(P-phenyl)phosphinate 2a in biphasic system
Conversion (%)
ee of alcohol (%)
Substrate
Additive
Time (h)
(RP,R); (SP,S)
(RP,S); (SP,R)
(SP,S)
(RP,S)
2a
—
MgCl2
LiCl
Triton X-100
—
MgCl2
LiCl
Triton X-100
1
1
1
44
39
40
47
59
35
40
71
60
67
64
61
59
30
37
67
13
16
20
29
64
67
70
30
44
46
28
58
49
38
46
34
1
2b
48
48
48
48
Reactions were carried out in a modified biphasic system consisting of diisopropyl ether and water or aqueous
solutions of LiCl, MgCl2, or Triton X-100 (20 mg of Candida cylindracea lipase, 0.036 mmol of substrate, 1 mL
water or aqueous solution, 2 mL diisopropyl ether).

ENZYMATIC RESOLUTION OF ETHYL α-HYDROXYPHOSPHINATES
1919
EXPERIMENTAL
All materials were purchased from commercial suppliers: Sigma, Aldrich, Fluka,
POCh, and Serva, and were used without purification. Details for lipase purchases are
as follows: Candida cylindracea—CLA (Sigma), Aspergillus niger—ANL (Fluka), and
Mucor javanicus—MJL (Fluka). All compounds were purified by gradient column chro-
matography using Merck Silica Gel 60 (63–230 mesh) or by HPLC (Varian, Dynamax
HPLC Column 250 × 21.4 mm; MICROSORB 300–10 C18). The enantiomeric excess
(ee) was measured by 31P NMR with quinine used as a chiral solvating agent or by HPLC
with chiral column CHIRALPAK AD. Absolute configuration of hydroxyphosphinates 1
was determined and described previously.11,12
Synthesis of Ethyl Hydroxyalkane(P-phenyl)phosphinates 1 and Ethyl
Butyryloxy Alkane(P-phenyl)phosphinates 2
Compounds 1 and 2 were synthesized and characterized previously.11,12
Enzymatic Transesterification in Modified Organic Solvent
Enzymatic esterification of ethyl 1-hydroxyethane(P-phenyl)phosphinate 1a was car-
ried out in diisopropyl ether (2 mL) with the addition of powdered molecular sieves (20 mg,
3Å mesh). Triethylamine or pyridine (0.023 mmol), substrate (0.023 mmol), suitable lipase
(20 or 200 mg), and vinyl butyrate (0.165 mmol) were added into the reaction mixture (see
Table III). The reaction was carried out at 36◦C in a shaker (150 rpm) and stopped when
conversion was close to 50% by filtration of biocatalyst followed by evaporation of organic
layer. The resulting product was purified by HPLC (C-18 column, gradient: 40% acetoni-
trile in water to 70% acetonitrile in water, retention time of 2a: 11.2 min) and analyzed by
HPLC (CHIRALPAK AD, Diacel, 10% 2-propanol in n-hexane, retention time: (SP,S): 7.6
min; (RP,R): 8.2 min; (RP,S): 8.5 min, and (SP,R): 9.7 min).
Enzymatic Hydrolysis Reactions in Diisopropyl Ether Saturated
with Aqueous Solutions
Distilled water (15 µL) or aqueous solution (15 µL) of LiCl (2.4 M), MgCl2 (1.2 M),
or Triton X-100 (2%) was added to diisopropyl ether (2 mL) and mixed. After addition of
substrate 2 (0.2 mmol) and Candida cylindracea lipase (100 mg), the enzymatic reaction
was carried out at room temperature with shaking (150 rpm). The reaction was stopped when
the conversion was close to 50% or after 168 h by filtration of catalyst and addition of ethyl
acetate (15 mL). Drying of the organic solvent over anhydrous magnesium sulfate, filtration,
and removal of the solvent by evaporation yielded the desired hydroxyphosphinate, which
was analyzed with 31P NMR using quinine as a chiral discriminator.
Enzymatic Hydrolysis Reactions in Modified Biphasic Medium
The biphasic system (3 mL) was composed of diisopropyl ether (2 mL) and water
or aqueous solution (1 mL) of LiCl (2.4 M), MgCl2 (1.2 M), or Triton X-100 (2%). After
addition of substrate (0.2 mmol) and Candida cylindracea lipase (100 mg), reactions were
carried out at room temperature with shaking (150 rpm). The reaction was stopped after

1920
P. MAJEWSKA ET AL.
certain intervals of time, and the product was extracted twice with ethyl acetate (15 mL),
and the organic phase was dried over anhydrous magnesium sulfate. After filtration, the
organic solvent was removed by evaporation, and the obtained hydroxyphosphinate was
analyzed with 31P NMR using quinine as a chiral discriminator.
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