Enzymatic reactions in ionic liquids: Lipase-catalysed kinetic resolution of racemic, P-chiral hydroxymethanephosphinates and hydroxymethylphosphine oxides

Article abstract of DOI:10.1016/S0957-4166(02)00167-2

Lipase-mediated acetylation of racemic P-chiral hydroxymethanephosphinates and hydroxymethylphosphine oxides was performed in ionic liquids under kinetic resolution conditions. Lipase AK (Amano) and lipase from Pseudomonas fluorescens (Fluka) were up to s

Full text of DOI:10.1016/S0957-4166(02)00167-2

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 735–738
Enzymatic reactions in ionic liquids: lipase-catalysed kinetic
resolution of racemic, P-chiral hydroxymethanephosphinates and
hydroxymethylphosphine oxides
Piotr Kiełbasin´ski,* Małgorzata Albrycht, Jerzy Łuczak and Marian Mikołajczyk
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Ło´dz, Poland
Received 19 March 2002; accepted 8 April 2002
Abstract—Lipase-mediated acetylation of racemic P-chiral hydroxymethanephosphinates and hydroxymethylphosphine oxides
was performed in ionic liquids under kinetic resolution conditions. Lipase AK (Amano) and lipase from Pseudomonas fluorescens
(Fluka) were up to six times more enantioselective in BMIM·PF6 solutions than in common organic solvents. On the contrary,
the analogous reactions performed in BMIM·BF4 were practically non-stereoselective. © 2002 Published by Elsevier Science Ltd.
1. Introduction
synthesis of biologically active compounds,9 among
them herbicides.10 As the enantiomeric excess values of
the products obtained in the above reaction were mod-
erate (only in one case reached 90%), we have decided
to check whether the use of ionic liquids will improve
stereoselectivity of the reaction. This paper describes a
successful lipase-mediated kinetic resolution of racemic
P-chiral hydroxymethanephosphinates and a hydroxy-
methylphosphine oxide achieved in ionic liquids as
solvents. It is noteworthy that this is the first example
of the application of ionic liquids in an enzyme-
catalysed resolution of primary alcohols bearing a
remote stereogenic heteroatom centre.
Ionic liquids are becoming more and more popular as
effective solvents for a great variety of organic reac-
tions.1 Very recently some reports appeared on their
application as reaction media for enzyme-promoted
transformations, in which several advantages of this
approach were presented. The first example concerned
a thermolysin-catalysed synthesis of Z-aspartame, per-
formed in BMIM·PF6 containing 5% water, which
proved to enhance the stability of the enzyme and the
reaction rate.2 In turn, the first example of the use of an
enzyme in water-free ionic liquids was a series of lipase-
catalysed transformations of carboxylic acid deriva-
tives.3 Those two papers were quickly followed by two
reports on the successful use of ionic liquids to enhance
the enantioselectivity of the lipase-promoted kinetic
resolution of secondary alcohols.4,5 Finally, the most
recent paper by Park and Kazlauskas described modifi-
cations in the preparation of ionic liquids and their
influence on the reactivity of lipases and stereoselectiv-
ity of the enzymatic reactions.6
2. Results and discussion
2.1. Synthesis of racemic hydroxymethanephosphinates
The required racemic hydroxymethanephosphinates 2
were synthesised in the reaction of the corresponding
H-phosphinates 1 with paraformaldehyde in the pres-
ence of triethylamine (Eq. (1)). The compounds 2d, 3d
and the hydroxymethylphosphine oxide 2e were
obtained previously.10,11
In the course of our investigations on the enzyme-pro-
moted syntheses of chiral heteroatom compounds,7 we
have recently applied a lipase-catalysed acetylation
under kinetic resolution conditions for the synthesis of
optically active P-chiral hydroxymethanephosphinates
and phosphonates.8 The latter are gaining increasing
attention due to their applicability as substrates for the
(1)
* Corresponding author. Fax: (+48-42) 6847126; e-mail: piokiel@
bilbo.cbmm.lodz.pl
0957-4166/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0957-4166(02)00167-2

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P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 13 (2002) 735–738
2.2. Kinetic resolution of racemic substrates 2
To perform the title reaction we chose 1-butyl-3-
methylimidazolium hexafluorophosphate (BMIM·PF6)
and 1-butyl-3-methylimidazolium tetrafluoroborate
(BMIM·BF4) as our model ionic solvents. The former
one is stable and immiscible with water and selected
organic solvents, whereas the latter can be mixed with
water in various proportions. The racemic hydroxy-
methanephosphinates were dissolved in an ionic liquid
and acetylated using vinyl acetate in the presence of
selected lipases, which were added directly to the reac-
tion mixture (Eq. (2)). The reaction was carried out
under kinetic control conditions, i.e. it was stopped at
ca. 50% conversion, which was determined by 31P
NMR of the aliquots taken directly from the reaction
mixture. It should be added that in the case of
BMIM·PF6 the 31P NMR signal of the PF6 anion of the
solvent appears at l=−145 ppm and does not affect the
observation of the signals of 2 and 3, which appear in
the region of 30–40 ppm. The product and the uncon-
sumed substrate were then extracted from the ionic
liquid solution (see Section 4) and separated by column
chromatography or TLC. The enantiomeric excess (ee)
values were determined both by 1H NMR, using (−)-(S)
or (+)-(R)-tert-butylphenylphosphinothioic acid as a
chiral solvating agent12 and by HPLC using a chiral
column. The results are summarised in Table 1.13
(2)
no case was the E value lower than that seen in the
analogous reactions performed in common organic sol-
vents. The improvement in the E values was particu-
larly marked (three to six times) for substrates having
larger organic substituents. In contrast to BMIM·PF6,
its tetrafluoroborate analogue, BMIM·BF4, surprisingly
showed an opposite effect. In spite of the fact that the
enzymatic resolution of secondary alcohols was found
to proceed in BMIM·BF4 with high stereoselectivity for
the lipases from Pseudomonas cepacia and Candida
antarctica,4–6 in our experiments practically no enan-
tioselectivity was observed, although the reactions pro-
ceeded with rates comparable to those seen for
reactions in BMIM·PF6. At the moment no satisfactory
explanation for this phenomenon can be provided due
to the lack of systematic studies on the influence of
ionic liquids on enzymes. However, it would be reason-
able to assume that the lack of stereoselectivity might
be attributed to the miscibility of BMIM·BF4 with
water. It is known that relatively hydrophilic solvents
are capable of stripping off the essential water from the
Of the two ionic liquids applied, only BMIM·PF6
proved to meet the expectations for enhancing the
stereoselectivity of the enzymatic kinetic resolution. In
Table 1. Kinetic resolution of 2 in ionic liquids
Entry
Substr.
Lipase
Ionic
Recovered alcohol 2*
Acetate 3*
E14
Eb
liquid
Yielda
(%)
[h]D
(CHCl3)
E.e. (%) Abs.
Yielda
(%)
[h]D
(CHCl3)
E.e. (%) Abs.
conf.
conf.
1
2
3
4
5
6
7
8
2a
2a
2a
2a
2b
2b
2b
2c
2c
2c
2d
2e
2e
AK
PFL
AK
PFL
AK
PFL
AK
AK
PFL
AK
PFL
AK
AK
PF
PF
BF
BF
PF
PF
BF
PF
PF
BF
PF
PF
BF
33.3
40.0
44.0
14.0
36.0
44.0
24.0
36.0
39.0
56.0
32.0
33.0
43.0
−21.5
−20.0
+0.4
−0.83
−12.1
−9.5
89
75
R
R
S
R
R
R
R
R
R
S
37.7
42.0
10.0
37.0
36.5
30.0
33.0
48.5
49.0
38.0
55.0
42.0
42.0
+49.8
+41.0
−1.2
89
78
4.8
0.3
83
76
1
80
68
S
S
S
S
S
S
S
S
51
18
1.1
1.0
26
14
1.0
32
15
1.3
12
5
45c
1.2
1.4
79
63
0.4
95
86
4.0
+0.5
+39.6
+37.3
+0.2
+31.0
+26.0
−0.3
+10.4
+7.0f
−1.4f
5c
5c
−1.1
−21.3
−16.1
+0.4
−12.1
−18.7f
+0.1f
9
S
10
11
12
13
4.5
R
N.d.
R
S
95
43
1
N.d.
S
R
50
53
2
12d
4e
1.1
Lipases: AK: Lipase AK (AMANO); PFL: Lipase from Pseudomonas fluorescens (FLUKA).
Ionic liquids: PF: BMIM·PF6; BF: BMIM·BF4.
N.d.=not determined.
a Yields after separation.
b The highest E values calculated for the same reactions performed in diisopropyl ether with the same enzymes and under comparable conditions,
taken from:
c Ref. 8.
d Ref. 10.
e Ref. 15.
f In C6H6.

P. Kiełbasin´ski et al. / Tetrahedron: Asymmetry 13 (2002) 735–738
737
enzyme surface, leading to insufficient hydration of the
4.4. Ethyl hydroxymethanephenylphosphinate 2b
enzyme which may in some cases exert a strong influ-
ence on the enzyme and decrease its activity.16
BMIM·BF4, which can mix with water in any propor-
tions, may thus be considered responsible for such an
interaction with certain kinds of enzymes.
Purified by a bulb-to-bulb distillation (ca. 140°C/0.2
mmHg), yield 80%. 31P NMR (C6D6): l=39.4. 1H
NMR (C6D6): l=0.96 (t, J=7.05, 3H), 3.6–4.0 (m,
2H), 4.05–4.30 (m, 2H), 6.4 (br. s, 1H), 6.9–7.1 and
7.7–7.9 (m, 5H). Anal. calcd for C9H13O3P: C, 54.00; H,
6.55; P, 15.47. Found: C, 53.60; H, 6.57; P, 15.29%.
3. Conclusion
4.5. i-Propyl hydroxymethanephenylphosphinate 2c
The results described above clearly demonstrate that
ionic liquids may be a promising medium for enzymatic
transformations involving chiral heteroatom substrates.
However, a lot of detailed investigations will be neces-
sary to gain sufficient knowledge about the influence of
ionic liquids on enzymes in general, and in particular,
the interactions of various types of ionic liquids with
different kinds of enzymes.
Purified by column chromatography, followed by crys-
tallisation from benzene; yield 60%, mp 70–72°C. 31P
NMR (C6D6): l =38.1. H NMR (C6D6): l=0.94 (d,
J=6.2, 3H), 1.22 (d, J=6.2, 3H), 4.0–4.35 (m, 2H),
4.5–4.7 (m, 1H), 6.1 (br. s, 1H), 6.95–7.1 and 7.75–7.9
(m, 5H). Anal. calcd for C10H15O3P: C, 56.05; H, 7.05;
P, 14.45. Found: C, 56.16; H, 6.99; P, 14.25%.
1
4.6. Kinetic resolution of 2—general procedure
4. Experimental
4.1. General
A mixture of a racemic 2 (1 mmol), the enzyme (10–20
mg) and vinyl acetate (1 mL) was stirred in the ionic
liquid (3 mL) at room temperature. The reaction was
monitored by 31P NMR and stopped at ca. 50% conver-
sion. The unconsumed substrate and the acetate formed
3 were extracted from the ionic liquid solution with
ether. To ensure full extraction of the products, the
ionic liquid layer was treated with water. In the case of
BMIM·PF6, which is immiscible with water, the layers
were separated and the aqueous layer was extracted
with chloroform. In the case of BMIM·BF4, the homo-
geneous solution formed after water addition was also
extracted with chloroform. In both cases the organic
layers were combined, dried over MgSO4 and the sol-
vents evaporated. The products were separated by
column chromatography using chloroform–methanol
(in gradient 100:1 to 15:1) as solvent or by preparative
TLC (chloroform–methanol 20:1).
BMIM·PF6 was prepared according to the procedure
described by Rogers et al.17 BMIM·BF4 was prepared
by a straightforward adaptation of the procedure
described by Carlin et al18 for EMIM·BF4 and purified
according to the procedures described by Park and
Kazlauskas.6
The enzymes were purchased from AMANO or
FLUKA. NMR spectra were recorded on Bruker
1
instruments at 200 MHz for H and 81 MHz for 31P,
with C6D6 or CDCl3 as solvents. Optical rotations were
measured on a Perkin–Elmer 241 MC polarimeter.
Column chromatography was carried out using Merck
60 silica gel. TLC was performed on Merck 60 F254
silica gel plates. The HPLC analyses were performed on
an LKB instrument, using CHIRALCEL OD column;
flow 0.25 mL/min; hexane:i-PrOH 98:2; u=254 nm.
4.7. Methyl acetoxymethanephenylphosphinate 3a
1
31P NMR (C6D6): l=34.1. H NMR (C6D6): l=1.49
(s, 3H), 3.30 (d, J=10.9, 3H), 4.27–4.56 (2×AB, 2H),
6.9–7.1 and 7.7–7.9 (m, 5H).
4.2. Synthesis of racemic 2—general procedure
Equimolar amounts of 1 and paraformaldehyde and a
few drops of triethylamine were heated under argon at
80°C until the precipitate dissolved. The crude reaction
mixture was then evacuated under vacuum at ca. 60°C
to remove triethylamine and volatile by-products and
finally purified either by column chromatography using
chloroform–methanol (in gradient 100:1 to 15:1) as
eluent, or by bulb-to-bulb distillation, or crystallisation.
4.8. Ethyl acetoxymethanephenylphosphinate 3b
1
31P NMR (C6D6): l=32.4. H NMR (C6D6): l=0.97
(t, J=7.05, 3H), 1.50 (s, 3H), 3.65–4.0 (m, 2H), 4.25–
4.60 (2×AB, 2H), 7.0–7.1 and 7.8–7.95 (m, 5H).
4.9. Isopropyl acetoxymethanephenylphosphinate 3c
31P NMR (C6D6): l=31.2. H NMR (C6D6): l=0.98
(d, J=6.2, 3H), 1.17 (d, J=6.2, 3H), 1.49 (s, 3H),
4.2–4.65 (2×m, 4H), 7.0–7.1 and 7.85–7.95 (m, 5H).
1
4.3. Methyl hydroxymethanephenylphosphinate 2a
After purification by column chromatography yield
60%. 31P NMR (C6D6): l=41.9. 1H NMR (C6D6):
l=3.31 (d, J =10.5, 3H), 4.0–4.28 (m, 2H), 6.0 (br. s,
1H), 6.9–7.15 and 7.7–7.9 (m, 5H). Anal. calcd for
C8H11O3P: C, 51.62; H, 5.96; P, 16.64. Found: C, 51.40;
H, 5.88; P, 16.30%.
4.10. Acetoxymethyl-tert-butylphenylphosphine oxide 3e
1
31P NMR (C6D6): l=40.4. H NMR (C6D6): l=0.94
(d, J=14.5, 9H), 1.52 (s, 3H), 4.63 and 4.65 (2×AB,
2H), 7.0–7.15 and 7.6–7.75 (2×m, 5H).

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Stowasser, B.; Budt, K.-H.; Jian-Qi, L.; Peyman, A.;
Acknowledgements
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