The enzyme-catalysed stereoselective transesterification of phenylalanine derivatives in supercritical carbon dioxide

Article abstract of DOI:10.1071/CH01018

The subtilisin Carlsberg catalysed transesterification of N-acetyl phenylalanine methyl ester (1), N-acetyl phenylalanine ethyl ester (2), N-trifluoroacetyl phenylalanine methyl ester (3) and N-trifluoroacetyl phenylalanine ethyl ester (4) was studied in supercritical carbon dioxide. The water content of the reaction affects the reactivity of the system; for the transesterification of the methyl esters with ethanol the optimum concentration of water was determined to be about 0.74 M, while for the transesterification of the ethyl esters with methanol the optimum concentration of water was about 1.3 M. The conversion is also dependent upon the concentration of alcohol; for ethanol, 2% v/v gives the maximum conversion, whilst for methanol, only 0.8-1. 2% v/v is required. This is probably due to a difference in the solubility of the substrates in the two alcohol/supercritical carbon dioxide mixtures. The reaction is highly stereoselective, in all cases no evidence for reaction of the D-isomer could be detected by chiral gas chromatography.

Full text of DOI:10.1071/CH01018

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Aust. J. Chem. 2002, 55, 259–262
The Enzyme-Catalysed Stereoselective Transesterification
of Phenylalanine Derivatives in Supercritical Carbon Dioxide
Andrew J. Smallridge,A,B Maurie A. TrewhellaA and Z. WangC
A School of Life Sciences and Technology (F008), Victoria University of Technology,
P.O. Box 14428, Melbourne City MC 8001, Australia.
B Author to whom correspondence should be addressed (e-mail: Andrew.Smallridge@vu.edu.au).
C Deceased.
The subtilisin Carlsberg catalysed transesterification of N-acetyl phenylalanine methyl ester (1), N-acetyl
phenylalanine ethyl ester (2), N-trifluoroacetyl phenylalanine methyl ester (3) and N-trifluoroacetyl phenylalanine
ethyl ester (4) was studied in supercritical carbon dioxide. The water content of the reaction affects the reactivity of
the system; for the transesterification of the methyl esters with ethanol the optimum concentration of water was
determined to be about 0.74 M, while for the transesterification of the ethyl esters with methanol the optimum
concentration of water was about 1.3 M. The conversion is also dependent upon the concentration of alcohol; for
ethanol, 2% v/v gives the maximum conversion, whilst for methanol, only 0.8–1.2% v/v is required. This is probably
due to a difference in the solubility of the substrates in the two alcohol/supercritical carbon dioxide mixtures. The
reaction is highly stereoselective, in all cases no evidence for reaction of the D-isomer could be detected by chiral
gas chromatography.
Manuscript received: 9 March 2001.
Final version: 21 March 2002.
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Introduction
CO2CH2CH2Cl
subtilisin Carlsberg
CO2Et
Supercritical fluids and, in particular, supercritical carbon
dioxide (scCO2), are rapidly becoming ‘green’ alternatives
to organic solvents in chemical processes. The use of
supercritical fluids in place of organic solvents for extraction
purposes is now an established industrial procedure although
the use of supercritical fluids as a solvent for chemical
reactions dates back to the 19th century.[1] More recently,
supercritical fluids have been used for reactions such as
asymmetric hydrogenation,[2] Diels–Alder addition[3] and
the Fischer–Tropsch synthesis of wax.[4] The use of
supercritical fluids for biocatalytic processes is a relatively
new development and was first reported in 1985.[5–7] The
solvent properties of supercritical fluids can be readily
changed by altering either the temperature or pressure of the
system. This ability to tune the solvent makes supercritical
fluids attractive media in which to conduct biocatalytic
reactions and this is now a rapidly expanding area of
research.[8,9] The enzyme-catalysed transesterification
reaction is probably the most widely studied enzymatic
reaction in supercritical fluids.[1] The selective
transesterification of triglycerides in scCO2 is particularly
appealing as it can provide a commercially viable pathway
for the modification of fats and oils.
NHR
NHCOCH3
scCO2/EtOH
Scheme 1
the supercritical system was superior to similar systems
utilizing organic solvents. The effect of temperature and the
carbon dioxide/ethanol ratio upon the reaction was
investigated, but no comment appeared concerning the
stereoselectivity of the reaction in scCO2 or the importance
of water to the reaction. The stereospecificity of the
subtilisin Carlsberg catalysed transesterification of N-
acetyl-D/L-phenylalanine ethyl ester with methanol has been
investigated in supercritical fluoroform[11] but no reports
have appeared concerning the stereoselectivity of the
reaction in scCO2.
Results and Discussion
In order to examine the stereoselectivity of the subtilisin
Carlsberg catalysed transesterification of phenylalanine
esters in scCO2, four phenylalanine derivatives were
prepared: N-acetyl phenylalanine methyl ester (1), N-acetyl
phenylalanine
ethyl
ester
(2),
N-trifluoroacetyl
phenylalanine methyl ester (3) and N-trifluoroacetyl
phenylalanine ethyl ester (4). The N-acetyl and N-
trifluoroacetyl derivatives were prepared to determine
whether the N-protecting group affected either the reactivity
Pasta et al. reported the subtilisin Carlsberg catalysed
transesterification reaction between N-acetyl-L-phenyl-
alanine chloroethyl ester and ethanol in supercritical carbon
dioxide (Scheme 1).[10] The report clearly demonstrated that
© CSIRO 2002
10.1071/CH01018
0004-9425/02/040259

260
A. J. Smallridge, M. A. Trewhella and Z. Wang
CO2Me
100
80
60
40
20
0
CO2Et
(1)
NHCOCH3
NHCOCH3
(1)
(3)
(2)
(4)
(2)
CO2Me
CO2Et
(3)
NHCOCF3
NHCOCF3
(4)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
or the stereoselectivity of the reaction. The D, L and D/L
derivatives of all four compounds were prepared in moderate
to good yields with no loss of chirality as evidenced by chiral
gas chromatography.
Water (M)
Fig. 1. The effect of water concentration upon the subtilisin
Carlsberg catalysed transesterification of phenylalanine derivatives
in scCO2.
Two reactions were studied: the transesterification of a
phenylalanine methyl ester to the corresponding ethyl ester
using ethanol, and the transesterification of a phenylalanine
ethyl ester to the corresponding methyl ester using methanol
(Scheme 2). The transesterification reactions were carried
out using subtilisin Carlsberg that had been buffered at pH
7.8 using a phosphate buffer. Reactions involving enzyme
preparations, which had been buffered at pH values above or
below this, resulted in substantially lower conversions. The
enzyme reactions were performed using a batch process in a
15 mL stainless steel reaction vessel containing a magnetic
stirring bar. The vessel was pressurized to 1650 psi with dry
carbon dioxide and immersed in a water bath at 47°C for 1 h.
Longer reaction times had little effect upon the conversion.
Gas chromatography was used to measure the extent of
reaction and the conversions quoted are an average of at least
two repetitions.
and water content was evident in the same reaction carried out
in supercritical fluoroform.[14] The optimal amount of water
is dependent upon the alcohol present in the reaction. For the
transesterification of the methyl esters with ethanol the
optimum concentration of water is about 0.74 M, while for
the transesterification of the ethyl esters with methanol the
optimum concentration of water is about 1.3 M. This
difference is probably due to differences in the solubility of
water in scCO2/ethanol and scCO2/methanol mixtures.
Hydrophilic solvents tend to strip the essential water layer
from the enzyme, consequently more water is required to
maintain the appropriate amount of water around the enzyme
in a hydrophilic solvent. The maximum water solubility in
scCO2 containing 450 mM ethanol at 40°C and 13 MPa is
2.9 g/L.[15] Whilst the maximum water solubility in scCO2/
methanolmixtureshasnotbeenreported, itwouldbeexpected
to be higher than for the corresponding ethanol/scCO2 mix,
due to the increased hydrophilicity of methanol compared
with ethanol. Hence, the optimal water content is higher for
the reactions involving the methanol/scCO2 system.
CO2Et
CO2Me
NHR
subtilisin Carlsberg
methanol
ethanol
NHR
R = COCH3 or COCF3
The observation that in some cases the conversion begins
to decrease as the water concentration increases is consistent
with hydrolysis of the ester to the corresponding carboxylic
acid. Miller et al.[16] reported that the rates of inter-
esterification of trilaurin and myristic acid, catalysed by a
1,3-specific lipase in scCO2, declined with increasing water
content due to hydrolysis of the product to the corresponding
acid. For the transesterification of the phenylalanine
derivatives in scCO2, no trace of free acid could be detected
by gas chromatography after the reaction, indicating that at
the level of water used in this study, hydrolysis was not
occurring. Once the optimal water level has been reached,
increasing amounts of water may precipitate from the solvent
leading to a build up of water droplets in the enzyme
particles, which form a mass-transfer barrier, and a
consequent decrease in reaction rate.[17,18] The precipitation
of water from scCO2 is the most likely cause of the drop in
reaction rates in the present study.
Scheme 2
Water Concentration
It has been shown that for enzymatic reactions carried out in
non-aqueous media a small amount of water is required for
enzyme activity.[12] In the earlier paper by Pasta et al. no
comment was made regarding the addition of water to the
reaction mixture[10] and we were interested to determine
whether water played a role in the activity of this scCO2
system. In order to control the water content of the reaction,
the carbon dioxide was passed through a drying tube (4 Å
molecular sieves) and the alcohols were dried using
established procedures.[13] The optimal water content for the
transesterification of the L-phenylalanine derivatives was
determined by conducting reactions with varying amounts of
added water (Fig. 1). These reactions all contained 2% v/v of
the appropriate alcohol.
Alcohol Concentration
As expected, little transesterification occurred in the
absence of water. As the water content was increased, the
conversion increased, reached a maximum and in some cases
decreased. A similar relationship between enzyme activity
Although scCO2 is a non-polar fluid, the solubility of polar
substrates in scCO2 can be increased by the addition of a polar
organic co-solvent.[19,20] The addition of a co-solvent can

Enzyme-Catalysed Stereoselective Transesterification in scCO2
261
lead to increases in reaction rate, although not always due to
increased substrate solubility.[21] In the present study, the
alcohol can be considered to be not only the reactant but also
a co-solvent to aid in the solubilization of the phenylalanine
derivatives in the scCO2. The effect of alcohol content upon
the transesterification of the L-phenylalanine derivatives was
studied by varying the alcohol concentration from 0.3–2.7%
v/v (Fig. 2). The optimum concentration of water as
determined in the previous section was used in each of the
reactions; i.e. 0.74 or 1.3 M as appropriate. The conversion
appears to be dependent upon the concentration of the
particular alcohol; for ethanol 2% v/v gives the maximum
conversion, whilst for methanol only 0.8–1.2% v/v is
required. This suggests that the different degrees of
conversion may be due to differing solubilities of the
phenylalanine derivatives in the two solvent mixtures.
Table 1. Stereoselectivity of the subtilisin Carlsberg catalysed
transesterification of phenylalanine derivatives in scCO2
CO2R1
CO2R3
NHR2
NHR2
+ R3OH
subtilisin Carlsberg
+ R
1OH
R1, R2, R3
Configuration Conversion Configuration
of starting
material
(%)
of product
(> 99% ee)
R1 = Me, R2 = COCH3,
R3 = Et
L
92
36
0
40
15
0
67
24
0
25
13
0
L
L
D/L
D
—
L
R1 = Me, R2 = COCF3,
R3 = Et
L
D/L
D
L
—
L
R1 = Et, R2 = COCH3,
R3 = Me
L
100
D/L
D
L
(1)
—
L
80
R1 = Et, R2 = COCF3,
R3 = Me
L
D/L
D
L
60
—
40
(2)
(3)
20
(4)
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
stereoselective process. The D-isomer does not react at all.
When racemic material is added to the reaction only the
L-isomer reacts and the extent of conversion is about half of
that when pure L-isomer is added.
Alcohol (% v/v)
Fig. 2. The effect of alcohol concentration (% v/v) upon the
subtilisin Carlsberg catalysed transesterification of phenylalanine
derivatives in scCO2.
Conclusion
The subtilisin Carlsberg catalysed transesterification of a
number of phenylalanine derivatives has been shown to
proceed in a highly stereoselective manner in scCO2; no
evidence for reaction of the D-isomer could be detected. The
concentration of both water and alcohol in the system is
important in determining the optimum reaction conditions.
This transesterification reaction in scCO2 complements the
previously reported reaction in supercritical fluoroform and
highlights the high reactivity and selectivity which can be
obtained in supercritical fluids.
It is also clear that each derivative shows different
reactivity in this system, with N-acetyl-L-phenylalanine
methyl ester (1) giving the best conversion (82%) with
0.74 M water. For the transesterification of the corresponding
ethyl ester (2) the conversion is lower (52%) and more water
is required (1.3 M). The nature of the protecting group on the
nitrogen also influences the rate of transesterification. The
N-acetyl derivatives are significantly more reactive than the
corresponding N-trifluoroacetyl compounds.
Stereoselectivity
Experimental
Enzymes are generally highly stereoselective and they have
been shown to retain this property in scCO2.[4] The
stereoselectivity of subtilisin Carlsberg transesterification
reactions has not been studied in scCO2 although it has been
shown to be stereoselective in supercritical fluoroform. In
order to study the stereoselectivity of the transesterification
of phenylalanine derivatives catalysed by subtilisin Carlsberg
in scCO2, a series of reactions, utilizing the optimum
conditions previously determined for each derivative, were
carried out using the L, D/L and D phenylalanine derivatives
(Table 1). The conversion was measured using conventional
gas chromatography whilst the enantiomeric composition of
the product was determined using chiral gas chromatography.
The subtilisin Carlsberg catalysed transesterification of
the phenylalanine derivatives in scCO2 is a highly
General
Gas chromatography was performed on a Shimadzu GC-17A with FID;
the column was a BP-10 (15 m by 0.25 mm) with a phase thickness of
0.25 µm. For chiral gas chromatography a Chiraldex G-TA column (30
m × 0.25 mm) with a phase thickness of 0.125 µm was used. 1H nuclear
magnetic resonance (NMR) spectra were recorded at 300 MHz on a
Bruker DPX300 and refer to solutions with tetramethylsilane as the
internal reference (δ 0.0 ppm).
Enzyme Preparation
Crude protease (subtilisin Carlsberg Type VIII: Bacterial with 14 units/
mg solid, purchased from SIGMA–Aldrich) (215.1 mg) was dissolved
in pH 7.8 phosphate buffer (2 mL) with gentle agitation. The pH of the
solution was readjusted to pH 7.8 by titration with 0.1 M disodium
hydrogen phosphate under slow agitation. The solution was frozen in
liquid nitrogen and freeze-dried over 4 days. The modified protease
(345.0 mg) was stored below 4°C in a sealed vial.

262
A. J. Smallridge, M. A. Trewhella and Z. Wang
Preparation of Phenylalanine Derivatives
Transesterification Reactions
Using a procedure similar to that described by Yamada et al.,[22]
borontrifluoride/diethyl ether complex (15 mL) was added to a solution
of L-phenylalanine (1.47 g, 8.91 mmol) in methanol (30 mL) and the
mixture refluxed for 24 h with an anhydrous calcium carbonate drying
tube to exclude moisture. The solvent was then removed under reduced
pressure. Dichloromethane (300 mL) was added and saturated
potassium carbonate solution was added dropwise under strong
agitation until the solution was approximately pH 8 (at that time the
colour of the solution changed from dark pink to light yellow). The
resultant solid was filtered off and discarded and the dichloromethane
layer was separated and dried with magnesium sulfate. Removal of the
dichloromethane gave crude L-phenylalanine methyl ester.
The following is the general procedure that was used for all of the
reactions using scCO2.
N-Acetyl-L-phenylalanine methyl ester (15 mg) and modified
protease (10 mg) were placed in a 15 mL stainless steel vessel, which
contained a magnetic stirring bar, and dried ethanol (300 µL) and
distilled water (200 µL) were added. Liquid carbon dioxide was
pumped into the vessel to 1650 psi and the reaction stirred at 47°C for
1 h. The reaction vessel was then cooled in a water–ice mixture (to
convert the supercritical carbon dioxide into liquid carbon dioxide), the
pressure was carefully released and the gas bubbled through
dichloromethane. Dichloromethane was used to wash all parts of the
reaction vessel and then filtered. The dichloromethane solutions were
combined and subjected to gas chromatography, which showed a
mixture of N-Acetyl-L-phenylalanine methyl ester and N-Acetyl-L-
phenylalanine ethyl ester in an 8 : 92 ratio; the two components were
identified by comparison with authentic samples. Chiral gas
chromatography showed no evidence of the D-enantiomer.
The crude L-phenylalanine methyl ester was immediately dissolved
in dichloromethane (30 mL). Acetic anhydride (15 mL) was added
dropwise under strong agitation and the mixture stirred at room
temperature for 24 h. Removal of the solvent under reduced pressure
followed by bulb-to-bulb distillation (220–230°C/1 mm) gave N-acetyl-
L-phenylalanine methyl ester as colourless crystals (1.38 g, 70%) (m.p.
References
1
60–61°C, lit.[23] 60–61°C). H NMR (300 MΗz): δ 2.03, s, 3H, CH3;
3.11, dd, J 6, 14 Hz, 1H, H3; 3.18, dd, J 6, 14 Hz, 1H, H3; 3.75, s, 3H,
CH3; 4.91, dt, J 6, 7.8 Hz, 1H, H2; 5.95, d, J 6 Hz, 1H, NH; 7.1, dd, J
1.5, 7.5 Hz, 2H, H2´, H6´; 7.3–7.4, m, 3H, H3´, H4´, H5´. Chiral gas
chromatography showed only one enantiomer (> 99% ee).
A similar procedure was used for the preparation of the other
derivatives; ethanol or trifluoroacetic anhydride was used in place of
methanol or acetic anhydride as appropriate.
N-Acetyl-D/L-phenylalanine methyl ester. Yield 54%. Chiral gas
chromatography showed two peaks in a 1 : 1 ratio indicating that the
product was racemic.
[1] P. G. Jessop, W. Leitner (Eds), Chemical Synthesis using
Supercritical Fluids 1999 (Wiley–VCH: Weinheim).
[2] M. J. Burk, S. Feng, M. F. Goss, W. Turnas, J. Am. Chem. Soc.
1995, 117, 8277.
[3] A. A. Clifford, K. Pople, W. J. Gaskill, K. D. Bartle, C. M.
Raynor, Chem. Commun. 1997, 595.
[4] L. Fan, S. Yan, K. Fujimoto, K. Yoshii, J. Chem. Eng. Jpn 1997,
30, 923.
[5] T. W. Randolph, H. W. Blanch, J. M. Prausnitz, C. R. Wilke,
Biotechnol. Lett. 1985, 7, 325.
N-Acetyl-D-phenylalanine methyl ester. Yield 60%. Chiral gas
chromatography indicated a single enantiomer (> 99% ee).
N-Acetyl-L-phenylalanine ethyl ester. Yield 41% (m.p. 91–92°C,
[6] D. A. Hammond, M. Karel, A. M. Klibanov, Appl. Biochem.
Biotechnol. 1985, 11, 393.
[7] K. Nakamura, Y. M. Chi, Y. Yamada, T. Yano, Chem. Eng.
Commun. 1985, 45, 405.
1
lit.[24] 92–94°C). H NMR (300 MΗz): δ 1.25, t, J 7.2 Hz, 3H, CH3;
2.17, s, 3H, CH3; 3.09, dd, J 5.7, 14.1 Hz, 1H, H3; 3.16, dd, J 5.7, 14.1
Hz, 1H, H3; 4.17, q, J 7.2 Hz, 2H, CH2; 4.86, dt, J 5.7, 7.8 Hz, 1H, H2;
5.89, d, J 6.3 Hz, 1H, NH; 7.1, dd, J 1.5, 7.2 Hz, 2H, H2´, H6´; 7.24–
7.28, m, 3H, H3´, H4´, H5´. Chiral gas chromatography showed one
peak only (> 99% ee).
N-Acetyl-D/L-phenylalanine ethyl ester. Yield 69%. Chiral gas
chromatography showed two peaks in a 1 : 1 ratio indicating that the
product was racemic.
[8] S. V. Kamat, E. J. Beckman, A. J. Russell, Crit. Rev. Biotechnol.
1995, 15, 41.
[9] A. J. Mesiano, E. J. Beckman, A. J. Russell, Chem. Rev. 1999, 99,
623.
[10] P. Pasta, G. Mazzola, G. Carrea, S. Riva, Biotechnol. Lett. 1989,
11, 643.
[11] S. V. Kamat, E. J. Beckman, A. J. Russell, J. Am. Chem. Soc.
1993, 115, 8845.
N-Acetyl-D-phenylalanine ethyl ester. Yield 64%. Chiral gas
chromatography indicated a single enantiomer (> 99% ee).
N-Trifluoroacetyl-L-phenylalanine methyl ester. Yield 43% (m.p.
51–52°C, lit.[25] 52–53°C). 1H NMR (300 MΗz): δ 3.17, dd, J 5.4, 14.1
Hz, 1H, H3; 3.24, dd, J 5.7, 14.1 Hz, 1H, H3; 3.78, s, 3H, CH3; 4.88, dt,
J 5.7, 7.5 Hz, 1H, H2; 6.62, s, 1H, NH; 7.06, dd, J 2, 7.5 Hz, 2H, H2´,
H6´; 7.25–7.31, m, 3H, H3´, H4´, H5´. Chiral gas chromatography
showed only one enantiomer (> 99% ee).
[12] A. Zaks, A. M. Klibanov, J. Biol. Chem. 1988, 263, 8017.
[13] W. L. F. Armareggo, D. D. Perrin, Purification of Laboratory
Chemicals 1996 (Butterworth–Heinemann: Oxford).
[14] A. K. Chaudhary, S. V. Kamat, E. J. Beckman, D. Nurok, R. M.
Kleye, P. Hajdu, A. J. Russel, J. Am. Chem. Soc. 1996, 118, 12891.
[15] A. Marty, W. Chulalaksananukul, R. M. Willemot, J. S. Condoret,
Biotechnol. Bioeng. 1992, 39, 273.
[16] D. A. Miller, H. W. Blanch, J. M. Prausnitz, Ind. Eng. Chem. Res.
1991, 30, 939.
[17] T. Dumont, D. Barth, M. Perrut, J. Supercrit. Fluids 1993, 6, 85.
[18] T. Dumont, D. Barth, C. Corbier, G. Branlant, M. Perru,
Biotechnol. Bioeng. 1992, 40, 329.
[19] J. M. Wong, K. P. Johnston, Biotechnol. Prog. 1986, 2, 29.
[20] G. Brunner, S. Peter, Sep. Sci. Technol. 1982, 17, 199.
[21] T. W. Randolph, D. S. Clark, H. W. Blanch, J. M. Prausnitz,
Science 1988, 239, 387.
[22] T. Yamada, N. Isono, A. Inui, T. Miyazawa, S. Kuwata, H.
Watanabe, Bull. Chem. Soc. Jpn 1978, 51, 1897.
[23] B. M. Iselin, H. T. Huang, R. V. MacAllister, C. Niemann, J. Am.
Chem. Soc. 1950, 72, 1729.
N-Trifluoroacetyl-D/L-phenylalanine methyl ester. Yield 57%.
Chiral gas chromatography showed two peaks in a 1 : 1 ratio indicating
that the product was racemic.
N-Trifluoroacetyl-D-phenylalanine methyl ester. Yield 60%.
Chiral gas chromatography indicated a single enantiomer (> 99% ee).
N-Trifluoroacetyl-L-phenylalanine ethyl ester. Yield 43% (m.p.
1
56–57°C, lit.[26] 57–58°C). H NMR (300 MΗz): δ 1.30, t, J 7.2 Hz,
3H, CH3; 3.19, dd, J 5.7, 14.1 Hz, 1H, H3; 3.26, dd, J 5.7, 14.1 Hz, 1H,
H3; 4.25, q, J 7.2 Hz, 2H, CH2; 4.87, dt, J 5.7, 7.8 Hz, 1H, H2; 6.78, br
s, 1H, NH; 7.1, dd, J 2.1, 7.2 Hz, 2H, H2´, H6´; 7.2–7.4, m, 3H, H3´,
H4´, H5´. Chiral gas chromatography showed one peak only
(> 99% ee).
N-Trifluoroacetyl-D/L-phenylalanine ethyl ester. Yield 64%.
Chiral gas chromatography showed two peaks in a 1 : 1 ratio indicating
that the product was racemic.
N-Trifluoroacetyl-D-phenylalanine ethyl ester. Yield 83%. Chiral
gas chromatography indicated a single enantiomer (> 99% ee).
[24] J.-M. Ricca, D. H. G. Crout, J. Chem. Soc., Perkin Trans. 1 1993,
11, 1225.
[25] A. Spisni, R. Corrandini, R. Marchelli, A. Dossena, J. Org.
Chem. 1989, 54, 684.
[26] D. Landini, M. Penso, J. Org. Chem. 1991, 56, 420.