Enzymatic resolution of α-hydroxyphosphinates with two stereogenic centres and determination of absolute configuration of stereoisomers obtained

Article abstract of DOI:10.1016/j.tetasy.2009.06.013

The use of quinine as a chiral solvating agent allows us to determine a tentative absolute configuration at the phosphorus atom of hydroxyphosphinates with two stereogenic centres (at the phosphorus and α-carbon atoms). Two ethyl butyryloxyalkane(P-phenyl

Full text of DOI:10.1016/j.tetasy.2009.06.013

Tetrahedron: Asymmetry 20 (2009) 1568–1574
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enzymatic resolution of
a-hydroxyphosphinates with two stereogenic
centres and determination of absolute configuration of stereoisomers obtained
b
a
a
Paulina Majewska a, , Marek Doskocz , Barbara Lejczak , Paweł Kafarski
*
a
_
´
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
b Chair and Department of Toxicology, Silesian Piasts University of Medicine in Wrocław, ul. Traugutta 57/59, 50-417 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2009
Accepted 18 June 2009
The use of quinine as a chiral solvating agent allows us to determine a tentative absolute configuration at
the phosphorus atom of hydroxyphosphinates with two stereogenic centres (at the phosphorus and
a
-carbon atoms). Two ethyl butyryloxyalkane(P-phenyl)phosphinates were hydrolysed using various
lipases. In all cases isomers possessing -carbon atom with an (S)-configuration were hydrolysed
preferentially. The absolute configuration of both chiral centres of obtained -hydroxyphosphinates
was determined by using (S)-(+)-MTPA-Cl and quinine. The mode of chiral discrimination of -hydrox-
a
a
a
yphosphinates by quinine was studied by means of computational chemistry, which confirmed the
experimental findings that the signals in 31P NMR spectra of compounds with an (RP)-configuration are
situated upfield when compared with the respective (SP) isomers.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Chiral hydroxyphosphonates are an interesting group of com-
pounds because of their well-recognised biological activity. For
example, some of these compounds act as inhibitors of chosen en-
zymes and exhibit interesting antibacterial, antiviral or anticancer
activities.1 In some cases, their activity depends not only on the
configuration of the stereogenic centre at the carbon atom but
also on the spatial structure of the phosphorus atom.2 The simple
and effective methods for the determination of the absolute con-
figuration of both centres are still challenging and need to be
developed. So far, the application of quinine, a well-acknowledged
chiral discriminator,3 seems to be the method of choice when con-
sidering the determination of the enantiomeric excess of hydrox-
yphosphonates. Thus, quinine has been widely applied as a chiral
2.1. Enzymatic hydrolysis
Racemic hydroxyphosphinates 1a and 1b, obtained by the addi-
tion of ethyl phenylphosphinite to the appropriate aldehyde,5 were
converted into butyryloxyphosphinates (compounds 2) by simple
acylation with butyryl chloride. Esters 2 were then hydrolysed
using various lipases (Scheme 1).
R
R
R
O
O
H
H
lipase
H
+
P
O
P
O
O
O
P
O
EtO
EtO
OH
EtO
2
1:
2:
(RP,R) and (SP,R)
(RP,S) and (SP,S)
solvating agent to determine the absolute configuration at the
a-
or b-carbon atoms of hydroxyphosphonates with one stereogenic
centre.4
H2SO4/CH3OH
Herein we report preliminary studies on the use of quinine for
the determination of the absolute configuration at the phospho-
rus atom of 1-hydroxy-2-methylpropane(P-phenyl)phosphinate
1a and 1-hydroxy-3,4-methoxyphenylmethane(P-phenyl)phosph-
inate 1b, compounds possessing two stereogenic centres, one lo-
a
R = -iPr
b R = -Ph(mOCH3)(pOCH3)
R
H
OH
P
O
EtO
cated at the
atom.
a-carbon atom and the other at the phosphorus
1
: (RP,R) and (SP,R)
Scheme 1. Hydrolysis of butyryloxyphosphinates 2 by lipases.
The hydrolysis was stopped when the chemical yield reached a
value close to 50% (which is typical for kinetic resolution) or before
* Corresponding author. Tel.: +48 71 320 2597.
E-mail address: paulina.majewska@pwr.wroc.pl (P. Majewska).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.06.013

P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
1569
the seventh day of reaction. Hydrolysis of 1-butyryloxy-2-methyl-
propane(P-phenyl)phosphinate 2a with lipase from porcine pan-
creas gave the pair of diastereoisomers (SP,S) and (RP,S) of
compound 1a as a major product with excellent enantiomeric
excess >98% (Table 1). In the case of compound 1b, the best results
were obtained when Aspergillus niger lipase was applied. As can be
seen from Table 1, the reaction was stereoselective in most cases;
(CH3–O)00 moiety of isomers (SP,S,R)—3.52 ppm; (RP,R,R)—
3.69 ppm; (RP,S,R)—3.53 ppm and (SP,R,R)—3.57 ppm.
As can be seen from these data, the signals of the
a-hydrox-
yphosphinate R groups with an (S)-configuration are situated up-
field when compared with the respective (R)-isomers (difference
of about 0.05–0.15 ppm for compound 3a and of 0.007–0.17 ppm
for compound 3b). This derives from the anisotropic effect exerted
by the phenyl group (Fig. 2) and thus conforms directly from the
Mosher model.
isomers bearing an
a-carbon atom of (S)-configuration were
hydrolysed preferentially, unfortunately with a lack of the stere-
oselectivity towards the phosphorus atom.
2.3. Determination of the absolute configuration at the
phosphorus atom
2.2. Determination of the absolute configuration at the
a
-carbon atom
Spectroscopic data for the two stereoisomers of compound 1a
(Fig. 3) have been described in the literature.7 The authors therein
defined the pair of isomers as (RP,R)- and (SP,S)-, but our studies
indicate that this configurational assignment was not correct. We
showed this in the case of 1-hydroxyethyl(P-phenyl)phosphinate
1c8 where the correct configuration of the discussed isomers has
to be set as (SP,R) and (RP,S). Moreover, Shioji used (R)-MTPA-Cl,7
which resulted in a change of configuration during derivatisation
to an (S)-MTPA-ester. Since our spectra for (SP,R)- and (RP,S)-iso-
mers (Fig. 3) are identical with those described by Shioji, this addi-
tionally supports our assignment.
After the purification of compounds 2, we unsuccessfully tried
to resolve the diastereoisomers using column chromatography
via the application of various eluents. Only when the silica gel col-
umn and a mixture of methylene chloride/n-hexane/ethyl acetate
(1:1:0.5 v/v) as eluent were used was some enrichment of isomers
obtained. These mixtures were hydrolysed using Candida cylindra-
cea lipase (compound 2a; time of hydrolysis—168 h) and A. niger li-
pase (compound 2b; time of hydrolysis—48 h) leading to a mixture
of compounds in molar ratios of (RP,S):(SP,R):(RP,R):(SP,S) =
2.36:0.44:0.39:0.99 (compound 1a) and (RP,S):(SP,R):(RP,R):(SP,S) =
1.20:0.48:0.57:1.10 (compound 1b) as determined by 31P NMR
using quinine as a chiral discriminator (Fig. 1). The remaining,
non-hydrolysed esters 2 were then chemically hydrolysed (Scheme
1) and purified to yield hydroxyphosphinates 1a and 1b with molar
ratios of (RP,S):(SP,R):(RP,R):(SP,S) = 0.18:2.45:1.00:0.35 in the case
of compound 1a and (RP,S):(SP,R):(RP,R):(SP,S) = 1.00:3.35:2.30:0.85
for compound 1b. These mixtures of isomers, after purification,
were acylated using (S)-(+)-MTPA-Cl. After the reactions were
complete, the mixtures of four isomers of (R)-MTPA esters 3 were
Comparison of the 31P NMR spectra of 1-hydroxy-2-methylpro-
pane(P-phenyl)phosphinate 1a obtained after enzymatic hydroly-
sis, recorded in the presence of quinine (Fig. 1—spectrum A) with
the spectra of two other compounds described previously, namely,
1-hydroxyethyl(P-phenyl)phosphinate 1c9,10 and 1-hydroxy-1-
phenylmethane(P-phenyl)phosphinate 1d8 (Figs. 4 and 5) indicates
that the signals of enantiomers with (RP) configuration are situated
upfield versus those of (SP) configuration. This finding allowed to
determine the absolute configuration at the phosphorus atom of
compound 1b (Fig. 1).
obtained and the absolute configuration of their
a-carbon atom
In conclusion, the use of a Mosher reagent enables us to estab-
additionally analysed by the method proposed by Mosher.6
lish configuration of the
a-carbon atom in the hydroxyphospho-
The 1H NMR chemical shifts of the phosphinate ester methyl
groups of 3a and 3b (CH3–CH2–O–P) were assigned as follows:
3a (SP,S,R)—1.29 ppm; (RP,R,R)—1.248 ppm; (RP,S,R)—1.27 ppm;
(SP,R,R)—1.245 ppm; 3b (SP,S,R)—1.34 ppm; (RP,R,R)—1.31 ppm;
(RP,S,R)—1.29 ppm and (SP,R,R) – 1.27 ppm. The signals derived
from the (RP,R,R)—and (SP,R,R)-isomers of compound 3a are very
close to each other; for this reason we described the chemical
shifts with an accuracy to the third place. The signals resulting
from a methyl group of (S)-configuration are downfield if com-
pared with the (R)-isomers (difference of about 0.02–0.04 ppm).
The 1H NMR chemical shifts of methyl groups ((CH3)2CH–CH–P)
of compound 3a were assigned as follows: (CH3)0 moiety of isomers
(SP,S,R)—1.00 ppm; (RP,R,R)—1.05 ppm; (RP,S,R)—0.892 ppm; (SP,R,R)—
1.04 ppm, whereas for (CH3)00 moiety of isomers (SP,S,R)—0.75 ppm;
(RP,R,R)—0.85 ppm; (RP,S,R)—0.889 ppm and (SP,S,R)—0.99 ppm.
The 1H NMR chemical shifts of the methoxy groups ((CH3–O)2–
Ph–CH–P) of compound 3b were assigned as follows: for the
(CH3–O)0 moiety of isomers (SP,S,R)—3.854 ppm; (RP,R,R)—3.87
ppm; (RP,S,R)—3.847 ppm and (SP,R,R)—3.854 ppm., whereas for
nates. The knowledge of this configuration and the use of quinine
enable us to determine, tentatively, the configuration at the phos-
phorus atom in a-hydroxyphosphinates.
2.4. Determination of the absolute configuration at the
phosphorus atom using a theoretical method
The theoretical investigation of the change of the chemical shift
in 31P NMR spectra as a result of complexation of hydroxyphosph-
inates by quinine was carried out using 1c as a model compound
by means of DFT methods. Compound 1c (as well as all hydrox-
yphosphinates 1a–d) appears as a mixture of four isomers because
of the presence of two stereogenic centres, on phosphorus and
a–carbon atoms. Additionally all these isomers may have different
conformations (synclinals and antiperiplanar). The synclinal
conformation is stabilised by the strong intramolecular hydrogen
bonds between the hydroxyl group and the P@O group
(O–Hꢀ ꢀ ꢀO@P) whereas the antiperiplanar conformation forms the
Table 1
Hydrolysis of butyryloxyphosphinates 2 by lipases via Scheme 1.
Lipase
Substrate
Time (h)
Conversion of hydrolysis (%)
Enantiomeric excess of hydroxyphosphinates (%)
(RP,R); (SP,S)
(SP,R); (RP,S)
(SP,S)
(RP,S)
Candida cylindracea lipase
Aspergillus niger lipase
2a
2b
2a
2b
2a
2b
166
168
166
22
166
168
39
44
21
46
32
25
53
41
41
36
52
9
>98
42
>98
54
>98
82
62
52
90
82
>98
<5
Lipase from porcine pancreas

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P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
Figure 1. 31P NMR spectra of mixtures of stereoisomers of hydroxyphosphinates 1a (spectrum A; Candida cylindracea lipase—168 h) and 1b (spectrum B; Aspergillus niger
lipase—48 h) resolved with quinine.
downfield relative to
O
R
R
H
H
O
H
H
EtO
EtO
P
O
P
O
CF3
CF3
OH
OH
1
: (RP,S)
P
P
EtO
EtO
O
O
R
R
O
OCH3
O
OCH3
1
: (SP,R)
a
R = -iPr
3
3
(RP,R,R)
(SP,S,R)
c R = -CH3
upfield relative to
Figure 3. Configuration of the two isomers of ethyl 1-hydroxy-2-methylpropane(P-
phenyl)phosphinate 1a and of 1-hydroxyethyl(P-phenyl)phosphinate 1c as
obtained by Shioji.7
downfield relative to
H
O
H
O
EtO
EtO
CF3
CF3
P
P
O
O
R
O
R
OCH3
O
OCH3
CH3
H
H
P
O
P
O
OH
OH
EtO
EtO
3
3
(RP,S,R)
(SP,R,R)
1c
1d
upfield relative to
Figure 4. Structures of the two ethyl hydroxyalkane(P-phenyl)phosphinates
1
Figure 2. Determination of absolute configuration on the a-carbon atom.
described previously.8–10
O–Hꢀ ꢀ ꢀO–P hydrogen bond. The difference in energy (
D
G) between
quinine molecule and hydroxyphosphonate of a synclinal confor-
mation.12 The mode of interaction between quinine molecule and
one of the 1c isomers calculated in this work is showed in Figure 6.
Quinine, when interacting with 1c, forms two strong hydrogen
bonds of the type of (QUININE)–O–Hꢀ ꢀ ꢀO@P and (1c)O–Hꢀ ꢀ ꢀO(QUI-
NINE)–H. The energy of interactions in this complex is in the range
of 10–13 kcal/mol with the base superposition (BSSE) taken into
consideration. The investigation of complexes formed with differ-
ent synclinal conformations of 1c showed that the energy of these
conformations is similar and that quinine forms complexes with all
synclinal and antiperiplanar conformations amount to 0.2–
0.4 kcal/mol with synclinal conformations being dominant in
solution.
Quinine is a good chiral solvating agent for hydroxyphospho-
nates and hydroxyphosphites because it forms diastereoizomeric
complexes and causes the possibility to distinguish these isomers
by NMR technology. Previous theoretical studies on the structure
of the complexes of hydroxyphosphonates with quinine11 showed
that the most probable complexes in solution are formed between

P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
1571
Figure 5. 31P NMR spectra of hydroxyphosphinates 1c (spectrum A; Rhizopus sp. Lipase—24 h) and 1d (spectrum B; Candida cylindracea lipase—46 h) in the presence of
quinine.
1c, +sc, ꢁsc, ap were taken into consideration for each enantiomer.
The measured difference between the chemical shifts for the phos-
phorus atom of diastereoisomers (SPR,RPS) and (RPR,SPS) for 1c is
equal to 0.66 ppm. The calculated difference when taking into con-
sideration the Boltzmann distribution for the three conformers is
1.59 ppm. Calculations were performed for the population of con-
formers +sc, ꢁsc, ap for enantiomer (SPR) being 46.8%, 32.7% and
20.5%, respectively, and 51.0%, 17.5% and 31.5% for the (SPS)-iso-
mer. The calculated chemical shifts for the phosphorus atom of
the (SPR)-isomer have higher values than those for (SPS)-isomer
which is in agreement with the experimental assignment.
The chemical shift in the 31P NMR spectra of solutions with qui-
nine is the average of three different conformers bonded with a
quinine molecule or three non-bonded. When quinine is used in
excess it was observed that the calculated chemical shifts of each
isomer are as follows: 42.32 ppm for (SPR); 42.27 ppm for (RPS);
42.08 ppm for (SPS) and 41.80 ppm for (RPR). Calculated chemical
shifts obtained from theoretical experiments of + and ꢁ synclinal
conformers 1c are as follows: 43.25 ppm for (SPR); 42.16 ppm for
(RPS); 42.02 ppm for (SPS) and 41.71 ppm for (RPR). The results of
our theoretical calculation are similar to the results obtained
experimentally and the signals from the isomers follow the same
order. The high difference between chemical shifts of different con-
formers of the same complex (e.g., +scRPR:Quinine 41.10 ppm,
ꢁscRPR:Quinine 44.55 ppm) can induce errors in interpretation
when only theoretical experiments are taken into consideration.
The theoretical experiments can only be used when they are per-
formed for more conformers with the appropriate calculated
method.
The theoretical studies confirmed the experimental determina-
tion of the absolute configuration of phosphorus atom in the
hydroxyphospinates and the experimental assignment of the sig-
nals in 31P NMR for certain isomers. They additionally showed
the mode of interaction of the quinine with the hydroxyphosphi-
nate molecules.
Figure 6. The structures of complex ethyl 1-hydroxyethyl(P-phenyl)phosphinate
with quinine (+scRPR:Quinine). B3LYP/6-31G(d,p).
the conformers forming species being in equilibrium in solution.
The calculated Boltzmann selection for +sc, ꢁsc conformations of
1c complexed with quinine is as follows: SPR (98.0, 2.0%); RPS
(88.6, 11.4%); SPS (69.3, 30.7%); RPR (17.6, 82.4%).
The observed chemical shifts for each atom represent the aver-
age conformation of the compound in solution. One method for the
precise determination of the chemical shifts is the analysis based
on the representative population of conformers.13 In the calcula-
tions of the NMR parameters for three conformers of compound
3. Experimental
3.1. General
All materials were purchased from commercial suppliers:
Sigma, Aldrich, Fluka, POCh and Serva, and were used without

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P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
purification. The sources of lipases were C. cylindracea (Sigma), A.
niger (Fluka) and porcine pancreas (Sigma). NMR spectra were
measured on a Bruker AvanceTM 600 at 600.58 MHz for 1H;
243.12 MHz for 31P and 151.02 MHz for 13C in CDCl3 or on a Bruker
Avance DRX 300 instrument operating at 300.13 MHz for 1H and
121.50 MHz for 31P in CDCl3. Chemical shifts (d) are reported in
ppm and coupling constants (J) are given in Hz. 1H NMR are refer-
enced to internal standard TMS (d = 0.00), the centre line of CHCl3
of the 13C NMR spectra (d = 77.23) and 85% phosphoric acid in H2O
for 31P NMR spectra were used as an external reference. All com-
pounds were purified by gradient column chromatography using
Merck Silica Gel 60 (63–230 mesh) or by HPLC (Varian, Dynamax
HPLC Column 250 ꢂ 21.4 mm; MICROSORB 300-10 C18).
16.39 (CH2CH3), 55.79 (OCH3), 56.04 (OCH3), 61.94 (OCH2), 73.43
(d, J = 112.4, CHP), 110.56 (d, J = 4.2 Hz), 110.76 (d, J = 1.7 Hz),
120.17 (d, J = 6.3 Hz), 128.06 (d, J = 110.1 Hz), 128.24, 128.42,
129.00, 132.62 (d, J = 2.1 Hz), 132.94, 133.19, 148.71 (d,
J = 2.9 Hz, COCH3), 148.94 (d, J = 2.5 Hz, COCH3) (Ph).
3.3. Synthesis of ethyl butyryloxyalkane(P-phenyl)phosphinates 2
Compounds 1a and 1b were converted to 2a and 2b with butyr-
yl chloride by the following procedure: Compound 1 (10 mmol)
was added to 100 ml of the reaction media containing chloroform
and triethylamine (10:1 v/v), followed by addition of 11 mmol of
butyryl chloride. The resulting solution was stirred for 24 h at
room temperature. After this time, the mixture was washed suc-
cessively by 100 ml of 5% hydrochloric acid, 100 ml distilled water
and dried over anhydrous magnesium sulfate. The product was
purified by means of silica gel column chromatography using
dichloromethane/ethyl acetate (5:3 v/v) as eluent.
3.2. Synthesis of ethyl hydroxyalkane-(P-phenyl)phosphinates 1
Compounds 1a and 1b were synthesised according to the Tex-
ier–Boullet method: Aluminium oxide (5 g) was mixed with 5 g
of potassium fluoride and powdered in a grinder. Then 20 mmol
of ethyl phenylphosphinate and 20 mmol of the required aldehyde
were added into this mixture and left at room temperature for
48 h. After this time the mixture was eluted by dichloromethane.
Compounds 1a and 1b were purified by a gradient column chroma-
tography on silica gel using dichloromethane/ethyl acetate (5:3 v/
v) as eluent.
3.3.1. Ethyl 1-butyryloxy-2-methylpropane(P-phenyl)phosphinate
2a
Rf = 0.68, molar ratio 1:1, yield 54%. Mixture of (RP,R)- and
(SP,S)-isomers: 31P NMR d (ppm): 37.24; 1H NMR: d (ppm): 0.81
(t, J = 7.4 Hz, 3H CH2CH2CH3), 0.93 (d, J = 6.7, 3H, CHCH3), 1.10 (d,
J = 6.8, 3H, CHCH3), 1.29 (t, J = 7.0 Hz, 3H, CH2CH3), 1.56–1.62 (m,
2H, CH2CH2CH3), 2.03–2.32 (m, 3H, CH2CH2CH3, CHCH3), 3.91–
3.98 (m, 2H, OCH2), 5.28 (dd, J = 7.2, 7.2 Hz, 1H, CHP), 7.45–7.49
(m, 2H, Ph), 7.54–7.57 (m, 1H, Ph), 7.78–7.82 (m, 2H, Ph); 13C
NMR d (ppm): 13.71 (CH2CH2CH3), 16.61 (d, J = 6.5 Hz, CHCH3),
18.38 (OCH2CH3), 18.79 (d, J = 8.2 Hz, CHCH3), 20.03 (d, J = 6.1 Hz,
CH(CH3)2), 28.99 (CH2CH2CH3), 35.92 (CH2CH2CH3), 61.34 (d,
J = 6.3 Hz, OCH2), 74.11 (d, J = 121.3 Hz, CHP), 128.54, 128.73,
129.83 (d, J = 77.7 Hz), 132.52, 132.66 (Ph), 133.83 (d, J = 2.9 Hz,
Ph), 172.67 (d, J = 3.9 Hz, CO).
3.2.1. Ethyl 1-hydroxy-2-methylpropane(P-phenyl)phosphinate
1a
Rf = 0.30, molar ratio 1:1, yield 42%. Mixture of (RP,R) and (SP,S)
isomers: 31P NMR d (ppm): 40.11; 1H NMR: d (ppm): 1.00 (d,
J = 6.6 Hz, 3H, CHCHCH3), 1.01 (d, J = 6.7 Hz, 3H, CHCHCH3), 1.32
(t, J = 7.0 Hz, 3H, CH2CH3), 3.69–3.77 (m, 1H, CHP), 3.89–3.95 (m,
1H, PCHCH), 4.10–4.19 (m, 2H, OCH2), 7.45–7.50 (m, 2H, Ph),
7.55–7.58 (m, 1H, Ph), 7.80–7.86 (m, 2H, Ph); 13C NMR d (ppm):
16.70 (d, J = 5.3 Hz, CHCHCH3), 18.15 (d, J = 7.6 Hz, CHCHCH3),
20.32 (d, J = 7.8 Hz, OCH2CH3), 29.77 (d, J = 2.9 Hz, PCHCH), 61.33
(d, J = 7.4 Hz, OCH2), 75.29 (d, J = 113.4 Hz, CHP), 128.67, 128.75,
129.51 (d, J = 118.7 Hz) 132.63 (2C), 132.57 (Ph).
Mixture of (RP,S)- and (SP,R)-isomers: 31P NMR d (ppm): 36.72;
1H NMR: d (ppm): 0.90 (t, J = 7.4 Hz, 3H CH2CH2CH3), 0.99 (d,
J = 6.8, 3H, CHCH3), 1.01 (d, J = 6.7, 3H, CHCH3), 1.33 (t, J = 7.0 Hz,
3H, CH2CH3), 1.43–1.48 (m, 2H, CH2CH2CH3), 2.03–2.32 (m, 3H,
CH2CH2CH3, CHCH3), 4.08–4.16 (m, 2H, OCH2), 5.18 (dd, J = 2.4,
6.4 Hz, 1H, CHP), 7.45–7.49 (m, 2H, Ph), 7.54–7.57 (m, 1H, Ph),
7.78–7.82 (m, 2H, Ph); 13C NMR d (ppm): 13.83 (CH2CH2CH3),
16.70 (d, J = 5.8 Hz, CHCH3), 18.49 (OCH2CH3), 18.59 (d, J = 6.8 Hz,
CHCH3), 20.27 (d, J = 7.7 Hz, CH(CH3)2), 28.83 (CH2CH2CH3), 36.08
(CH2CH2CH3), 61.30 (d, J = 5.9 Hz, OCH2), 74.63 (d, J = 117.3 Hz,
CHP), 128.62, 128.81, 129.00 (d, J = 80.1 Hz), 132.45, 132.60 (Ph),
132.75 (d, J = 2.2 Hz, Ph), 172.51 (d, J = 4.5 Hz, CO).
Mixture of (RP,S)- and (SP,R)-isomers: 31P NMR d (ppm): 40.03;
1H NMR: d (ppm): 1.01 (d, J = 6.7 Hz, 6H, CHCHCH3), 1.06 (d,
J = 6.8 Hz, 6H, CHCHCH3), 1.34 (t, J = 7.0 Hz, 3H, CH2CH3), 3.69–
3.77 (m, 1H, CHP), 3.95–4.02 (m, 1H, PCHCH), 4.10–4.19 (m, 2H,
OCH2), 7.45–7.50 (m, 2H, Ph), 7.55–7.58 (m, 1H, Ph), 7.80–7.86
(m, 2H, Ph); 13C NMR d (ppm): 16.74 (d, J = 5.3 Hz, CHCHCH3),
17.47 (d, J = 5.9 Hz, CHCHCH3), 20.45 (d, J = 10.1 Hz, OCH2CH3),
29.59 (d, J = 4.0 Hz, PCHCH), 61.46 (d, J = 7.3 Hz, OCH2), 75.40 (d,
J = 109.9 Hz, CHP), 128.72, 128.80, 129.62 (d, J = 119.0 Hz) 132.41,
132.47, 132.73 (d, J = 2.7 Hz) (Ph).
3.3.2. Ethyl 1-butyryloxy-1-(3,4-methoxyphenyl)methane(P-
phenyl)phosphinate 2b
3.2.2. Ethyl 1-hydroxy-1-(3,4-methoxyphenyl)methane(P-
phenyl)phosphinate 1b
Rf = 0.47, molar ratio 1:1, yield 69%. Mixture of (RP,R) and (SP,S)
isomers: 31P NMR d (ppm): 34.38; 1H NMR: d (ppm): 0.90 (t,
J = 7.4 Hz, 3H CH2CH2CH3), 1.32 (t, J = 7.0 Hz, 3H, CH2CH3), 1.59–
1.64 (m, 2H, CH2CH2CH3), 2.35 (td, J = 2.0, 7.2 Hz, 2H,
C(O)CH2CH2CH3), 3.76 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.77 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 4.01–4.14 (m, 2H, OCH2), 6.16 (d,
J = 7.9 Hz, 1H, CHP), 6.74–6.99 (m, 3H, Ph), 7.40–7.74 (m, 5H, Ph);
Rf = 0.17, molar ratio 1:1, yield 38%. Mixture of (RP,R) and (SP,S)
isomers: 31P NMR d (ppm): 38.98; 1H NMR: d (ppm): 1.32 (t,
J = 7.0 Hz, 3H, CH2CH3), 3.64 (s, 3H, OCH3), 3.85 (s, 3H, OCH3),
3.94–4.18 (m, 2H, OCH2), 5.12 (d, J = 9.8 Hz, 1H, CHP), 6.73–6.82
(m, 3H, Ph), 7.32–7.35 (m, 2H, Ph), 7.44–7.49 (m, 3H, Ph); 13C
NMR d (ppm): 16.43 (CH2CH3), 55.67 (OCH3), 56.04 (OCH3), 61.99
(OCH2), 73.09 (d, J = 113.1, CHP), 110.35 (d, J = 4.2 Hz), 110.65 (d,
J = 1.9 Hz), 119.79 (d, J = 6.3 Hz), 127.29 (d, J = 122.3 Hz), 128.16,
128.34, 128.66, 132.68 (d, J = 2.1 Hz), 132.88, 133.13, 148.61 (d,
J = 2.7 Hz, COCH3), 148.71 (d, J = 2.9 Hz, COCH3) (Ph).
13C NMR
d (ppm): 13.70 (CH2CH2CH3), 16.73 (d, J = 5.6 Hz,
OCH2CH3), 18.46 (CH2CH2CH3), 36.20 (C(O)CH2CH2CH3), 55.91
(OCH3), 56.00 (OCH3), 62.04 (d, J = 7.1 Hz, OCH2), 72.81 (d,
J = 118.9 Hz, CHP), 111.04 (d, J = 1.8 Hz), 111.29 (d, J = 4.3 Hz),
120.86 (d, J = 5.7 Hz), 125.78, 128.43, 128.52, 128.47 (d,
J = 129.8 Hz), 132.66, 132.72, 132.85 (d, J = 3.2 Hz), 148.87 (d,
J = 2.1 Hz, COCH3), 149.40 (d, J = 2.9 Hz, COCH3) (Ph) 172.09 (d,
J = 7.4 Hz, CO).
Mixture of (RP,S)- and (SP,R)-isomers: 31P NMR d (ppm): 37.53;
1H NMR: d (ppm): 1.29 (t, J = 7.0 Hz, 3H, CH2CH3), 3.68 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 3.94–4.18 (m, 2H, OCH2), 5.05 (d,
J = 7.3 Hz, 1H, CHP), 6.73–6.82 (m, 3H, Ph), 7.38–7.42 (m, 2H, Ph),
7.52–7.55 (m, 1H, Ph), 7.63–7.66 (m, 2H, Ph); 13C NMR d (ppm):
Mixture of (RP,S)- and (SP,R)-isomers: 31P NMR d (ppm): 33.83;
1H NMR: d (ppm): 0.84 (t, J = 7.4 Hz, 3H CH2CH2CH3), 1.27 (t,

P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
1573
J = 7.1 Hz, 3H, CH2CH3), 1.52–1.58 (m, 2H, CH2CH2CH3), 2.28 (td,
J = 2.9, 7.4 Hz, 2H, C(O)CH2CH2CH3), 3.77 (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 4.01–4.14 (m, 2H, OCH2), 6.26 (d, J = 10.3 Hz, 1H,
CHP), 6.74–6.99 (m, 3H, Ph), 7.40–7.74 (m, 5H, Ph); 13C NMR d
(ppm): 13.61 (CH2CH2CH3), 16.60 (d, J = 5.9 Hz, OCH2CH3), 18.49
(CH2CH2CH3), 36.22 (C(O)CH2CH2CH3), 55.93 (OCH3), 56.00
(OCH3), 61.88 (d, J = 6.6 Hz, OCH2), 72.15 (d, J = 121.0 Hz, CHP),
111.08, 111.40 (d, J = 4.4 Hz), 121.11 (d, J = 5.7 Hz), 126.93,
128.43, 128.51, 128.62 (d, J = 128.9 Hz), 132.72, 132.78, 132.83
(d, J = 3.1 Hz), 148.89 (d, J = 1.8 Hz, COCH3), 149.51 (d, J = 2.8 Hz,
COCH3) (Ph), 171.95 (d, J = 7.6 Hz, CO).
J = 12.1 Hz, 1H, CHP), 6.73–6.85 (m, 3H, Ph), 7.30–7.64 (m, 10H,
Ph).
(SP,S,R): 31P NMR d (ppm): 33.26; 1H NMR d (ppm): 1.33 (t,
J = 7.1 Hz, 3H, CH2CH3), 3.44 (s, 3H, OCH3), 3.52 (s, 3H, PhOCH3),
3.855 (s, 3H, PhOCH3), 3.97–4.18 (m, 2H, OCH2), 6.31 (d,
J = 10.0 Hz, 1H, CHP), 6.73–6.85 (m, 3H, Ph), 7.30–7.64 (m, 10H,
Ph).
3.5. Enzymatic reactions—general procedures
Enzymatic hydrolysis of ethyl 1-butyryloxyphosphinate 2 was
carried out in a biphasic system (3.8 ml) consisting of 0.05 M phos-
phate buffer, pH 7.0 (3.0 ml), and mixture of diisopropyl ether
(0.2 ml) with n-hexane (0.6 ml). After the addition of 0.2 mmol of
substrate and 100 mg of suitable lipase (see Table 1), the reaction
was carried out at room temperature with shaking (150 rpm).
The reaction was stopped after certain periods of time, and the
product was extracted twice with 15 ml of ethyl acetate and organ-
ic phase was dried over anhydrous magnesium sulfate. After filtra-
tion, the organic solvent was removed by evaporation and the
obtained hydroxyphosphinate was analysed with 31P NMR using
quinine as a chiral discriminator.
3.4. Synthesis of Mosher esters 3
A mixture of stereoisomers of compound 1 was derivatised
according to a literature method: At first, 0.10 mmol of 1 was dis-
solved in the mixture composed of dry dichloromethane (300
ll)
and dry pyridine (300 l), followed by the addition of 0.14 mmol
l
of (S)-(+)MTPA-Cl. The mixture was left for 3 days at room temper-
ature. After this time 3-dimethylamino-1-propylamine (0.20
mmol) was added into the mixture. After 5 min the mixture was
diluted with diethyl ether (10 ml), washed with cold 5% hydrochlo-
ric acid (10 ml) and water (10 ml), and dried over anhydrous
magnesium sulfate. After filtration of drying agent ether was evap-
orated and compound 3 was purified by means of HPLC (gradient:
from 40% of acetonitrile in water to 100% of acetonitrile, death time
7.5 min).
3.6. Computational details
The structures of 1-hydroxyethyl(P-phenyl)phosphite 1c (Fig. 4)
and quinine complexes were optimised using the density func-
tional theory (DFT) with the B3LYP functional14 and the standard
6-31G(d,p) basis of atomic orbitals.15 All enantiomers of 1c (SPR,
RPS, SPS, RPR) were considered as conformers of three local minima,
namely, those with sinclinal+ (sc+), sinclinal- (scꢁ) and antiperipla-
nar (ap) position of the phosphonic moiety. The 1c:quinine com-
plexes were optimised for + and ꢁ sinclinal conformers of 1c.
Vibrational frequencies for 1c were calculated by applying the
ideal gas, rigid rotor and harmonic oscillator approximations.16
All the conformer energy minima of 1c were confirmed by fre-
quency calculations.
3.4.1. Mosher ester 3a
Retention time 15.0 min.
(RP,R,R): 31P NMR d (ppm): 34.80; 1H NMR d (ppm): 0.85 (d,
J = 6.8 Hz, 3H, CHCHCH3), 1.05 (d, J = 7.1 Hz, 3H, CHCHCH3), 1.25
(t, J = 7.0 Hz, 3H, CH2CH3), 2.12–2.37 (m, 1H, PCHCH), 3.45 (s, 3H,
OCH3), 3.91–4.19 (m, 2H, OCH2), 5.54 (dd, J = 5.2, 10.9 Hz, 1H,
CHP), 7.34–7.82 (m, 10H, Ph).
(SP,R,R): 31P NMR d (ppm): 34.36; 1H NMR d (ppm): 0.99 (d,
J = 6.8 Hz, 3H, CHCHCH3), 1.04 (d, J = 6.8 Hz, 3H, CHCHCH3), 1.25
(t, J = 7.0 Hz, 3H, CH2CH3), 2.12–2.37 (m, 1H, PCHCH), 3.46 (s, 3H,
OCH3), 3.91–4.19 (m, 2H, OCH2), 5.39 (dd, J = 3.5, 5.0 Hz, 1H,
CHP), 7.34–7.82 (m, 10H, Ph).
The NMR parameters were calculated using the coupled per-
turbed density functional theory (CP-DFT) method by including
the diamagnetic spin–orbit, paramagnetic spin–orbit, Fermi-con-
tact and spin-dipolar terms.17 The calculations were performed
within NMR dedicated IGLOII basis set for H, C, N and O atoms.18
Application of this procedure has usually been found to lead to rea-
sonable agreement with experimental data for chemical shifts of
diasterotopic protons,13 chemical shift effects of complexation aliz-
arin in metal19 and coupling constants across hydrogen bond.20
The chemical phosphorus shifts were defined in relation to TPP
(ꢁ17.8 ppm) as standard.
(RP,S,R): 31P NMR d (ppm): 34.31; 1H NMR d (ppm): 0.889 (d,
J = 6.8 Hz, 3H, CHCHCH3), 0.892 (d, J = 6.8 Hz, 3H, CHCHCH3), 1.27
(t, J = 7.2 Hz, 3H, CH2CH3), 2.12–2.37 (m, 1H, PCHCH), 3.63 (s, 3H,
OCH3), 3.91–4.19 (m, 2H, OCH2), 5.39 (dd, J = 3.5, 5.0 Hz, 1H,
CHP), 7.34–7.82 (m, 10H, Ph).
(SP,S,R): 31P NMR d (ppm): 34.73; 1H NMR d (ppm):0.73 (d,
J = 5.6 Hz, 3H, CHCHCH3), 1.00 (d, J = 8.6 Hz, 3H, CHCHCH3), 1.29
(t, J = 7.0 Hz, 3H, CH2CH3), 2.12–2.37 (m, 1H, PCHCH), 3.42 (s, 3H,
OCH3), 3.91–4.19 (m, 2H, OCH2), 5.56 (dd, J = 5.4, 10.8 Hz, 1H,
CHP), 7.34–7.82 (m, 10H, Ph).
The computations were carried out using the GAUSSIAN 03 suite of
22
codes21 and results were visualised by applying the GAUSSVIEW
package.
3.4.2. Mosher ester 3b
Figures with optimised complex ethyl 1-hydroxyethyl(P-phenyl)-
phosphinate:quinine and Cartesian coordinates for ꢁscRPR:Quinine
and ꢁscRPR:Quinine are available free of charge via the Internet at
http://www.molnet.eu/images/stories/Supp_Majewska_2009.pdf.
Retention time 13.9 min.
(RP,R,R): 31P NMR d (ppm): 32.72; 1H NMR d (ppm): 1.28 (t,
J = 7.1 Hz, 3H, CH2CH3), 3.42 (s, 3H, CHOCH3), 3.69 (s, 3H, PhOCH3),
3.87 (s, 3H, PhOCH3), 3.97–4.18 (m, 2H, OCH2), 6.33 (d, J = 8.7 Hz,
1H, CHP), 6.73–6.85 (m, 3H, Ph), 7.30–7.64 (m, 10H, Ph).
(SP,R,R): 31P NMR d (ppm): 32.92; 1H NMR d (ppm): 1.29 (t,
J = 7.1 Hz, 3H, CH2CH3), 3.38 (s, 3H, OCH3), 3.57 (s, 3H, PhOCH3),
3.855 (s, 3H, PhOCH3), 3.97–4.18 (m, 2H, OCH2), 6.45 (d,
J = 13.1 Hz, 1H, CHP), 6.73–6.85 (m, 3H, Ph), 7.30–7.64 (m, 10H,
Ph).
Acknowledgements
Authors greatly thank Professor Leszczynski group from the
Interdysciplinary Center for Nanotoxicity for the help in visualisa-
´
tion of data. Calculations were performed at Poznan Supercomput-
(RP,S,R): 31P NMR d (ppm): 32.87; 1H NMR d (ppm): 1.31 (t,
J = 7.1 Hz, 3H, CH2CH3), 3.44 (s, 3H, OCH3), 3.53 (s, 3H, PhOCH3),
3.848 (s, 3H, PhOCH3), 3.97–4.18 (m, 2H, OCH2), 6.35 (d,
ing and Networking Center (PCSS), Wroclaw Centre for Networking
and Supercomputing (WCSS) and Academic Computer Centre in
Gdansk (CI TASK).

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P. Majewska et al. / Tetrahedron: Asymmetry 20 (2009) 1568–1574
18. Kutzelnigg, W.; Fleischer, U.; Schindler, M.. In The IGLO-Method: Ab Initio
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