A. Varga et al. / Tetrahedron: Asymmetry 24 (2013) 1389–1394
1393
ꢀ
1
redissolved in n-hexane–isopropanol (1:1, v/v), and analyzed by
HPLC.
temp.: 130 °C (100 min), 130 °C to 140 °C (1 °C min , 140 °C
ꢀ1
(20 min); 140 °C to 150 °C (1 °C min ), 150 °C (20 min); FID], t
R
(
min): 141.0 [(R)-3], 147.2 [(S)-3].
4
.4.2. Fermenting biotransformations on an analytical scale
A fresh wet cake of yeast (2.5 g) and sucrose (1 g) was added
4
2
6
.7. Conversion of ethyl (R)- and (S)-3-hydroxypropanoates (R)-
and (S)-2 into enantiomers of (S)- and (R)-b-phenylalanine (S)-
and (R)-6
into water (25 mL). The resulting suspension was stirred for
0 min, followed by the addition of ethyl 3-oxo-3-phenylpropano-
3
ate 1 (24 mg) dissolved in DMSO (0.5 mL). Further experiments
were performed as described in the previous section.
4
.7.1. Ethyl (S)- and (R)-3-amino-3-phenylpropanoates (S)-5 and
(
R)-5
Into a cooled (0 °C) solution of optically active (S)- or (R)-ethyl 3-
hydroxy-3-phenylpropanoate (R)- or (S)-2 (136 mg, 0.7 mmol) and
diphenyl phosphorazide (214 L, 1 mmol) in dry THF (2 mL), neat
DBU (150 L, 1 mmol) was added under argon. The reaction mixture
was stirred for 3 h at 0 °C, then slowly warmed to rt and stirred for
6 h. The resulting two-phase mixture was washed with water
4
.4.3. Biotransformations under fermenting conditions in the
presence of additives
l
Experiments were conducted as previously described. The addi-
tives (25 mg) were introduced into the suspension together with
the sucrose.
l
1
(
2 ꢁ 10 mL) and 5% HCl (10 mL). The organic layer was concentrated
4
.5. Preparative synthesis of (S)-3-hydroxy-3-phenylpropanoate
in vacuum. The crude product was purified by vacuum-chromatogra-
phy using petrol ether-ethyl acetate (9.5:0.5, v/v) as eluent to yield
(S)-2 with S. cerevisiae
(
S)- or (R)-ethyl 3-azido-3-phenylpropanoate (S)-4 or (R)-4 in 92%
yield as a colorless semisolid which was used in the next step as such.
Into a solution of (S)- or (R)-4 in THF (2 mL), water (18 L,
mmol) and PPh (155 mg, 0.6 mmol) were added. The mixture
was refluxed overnight, cooled to rt, and then chromatographed
without any work-up procedure using a mixture of CH Cl –EtOH
7:3, v/v) as eluent to yield (S)- or (R)-ethyl 3-amino-3-phenylpro-
Into a suspension of cells (15 g) in water (150 mL), sucrose was
added (3 g) and the mixture was stirred for 30 min. A solution of
ethyl 3-phenyl-3-oxopropanoate 1 (144 mg, 0.75 mmol) dissolved
in DMSO (2 mL) was added into the fermenting suspension. The
reaction mixture was stirred at room temperature until the trans-
l
1
3
formation of the substrate was completed (samples of 100 lL were
2
2
(
taken and extracted by ethyl acetate, then analyzed with TLC).
After completion of the reaction, the mixture was extracted with
EtOAc (3 ꢁ 200 mL). The combined organic layer was dried over
1
panoate (S)- or (R)-5 as a colorless semisolid (97 mg, 72%). H NMR
spectra of the optically active products were identical to those re-
3
5
ported in the literature for (S)-5.
2 4
anhydrous Na SO , the solvent was removed in vacuo and the crude
2
5
), ee = 96% by HPLC {lit.35
3
), ee = 99.8%}; HPLC [on Chiracel OD-H col-
(
S)-5: ½
a
ꢂ
¼ ꢀ21:8 (c 1, CHCl
3
:
product obtained was purified by column chromatography on silica
D
[a
]
D
= ꢀ22.9 (c 1, CHCl
gel using dichloromethane to give (S)-2 as a clear oil (75 mg, 86%).
1
13
umn (4.6 ꢁ 250 mm), hexane/i-PrOH = 98:2, 1.0 mL/min, 218 nm],
The H and C NMR spectra of the optically active (S)-2 were indis-
2
D
5
t
R
(min): 34.3 [(S)-5], 40.5 [(R)-5].
tinguishable from those of rac-2. ½
aꢂ
¼ ꢀ51:1 (c 1.5, CHCl
3
),
2
D
5
), ee = 96% by HPLC {lit.36
33
25
D
(
R)-5: ½
a
ꢂ
¼ þ21:7 (c 1, CHCl
3
:
ee = 96% by HPLC {lit. : ½
aꢂ
¼ ꢀ43:8 (c 1, CHCl
3
), Ee > 99%}; HPLC
[
a
]
D
= +21.1 (c 1, CHCl ), ee = 91%}; HPLC: see at (S)-5.
3
(
on Chiralpak IA/Chiralpack IB tandem, n-hexane/i-PrOH = 95:5,
1
R
.0 mL/min, 218 nm), t (min): 22.4 [(S)-2], 23.1 [(R)-2].
4
3
.7.2. Synthesis of enantiomerically enriched (S)- or (R)-3-amino-
-phenylpropanoic acid (S)-6 or (R)-6
A mixture of ethyl (S)- or (R)-3-amino-3-phenylpropanoate (S)-
4
3
.6. Analytical and preparative scale kinetic resolutions of ethyl
-acetoxy-3-phenylpropanoate rac-3
or (R)-5 (95 mg) in 6 M HCl solution (5 mL) was heated at reflux for
h. The solution was cooled to room temperature and the pH was
adjusted to give a basic solution with conc. NH solution. The crude
product was purified by recrystallization from water to give (S)- or
R)-3-amino-3-phenylpropanoic acid (S)-6 or (R)-6 in 75% yield.
1
The same yeast cell-based conditions applied for the microbial
3
reduction of 1 were used for the kinetic resolution of racemic ethyl
3
-acetoxy-3-phenylpropanoate rac-3.
(
Samples of yeast suspension (ca. 5 mL, each; obtained by mix-
1
13
The H NMR and C NMR data of the products were in accordance
with those reported in the literature.
ing 2.5 g of yeast cake in 20 mL of water) were transferred in free-
zer tubes and centrifuged for 5 min at 10,000g. The cell pellets
were flash-frozen in liquid nitrogen (ꢃ1 min) and freeze-dried
3
7
2
D
5
O), ee = 96% by HPLC {lit.38
2
O), Ee >99%}; HPLC [on Chiracel IA column
(
S)-6: ½
a
ꢂ
¼ ꢀ7:7 (c 0.25, H
2
:
2
5
½
a
ꢂ
¼ ꢀ8 (c 0.27, H
D
(
overnight at 0.2 mbar). The yeast preparation lyophilized in this
(
4.6 ꢁ 250 mm), hexane/i-PrOH = 1:1, 1.0 mL/min, 218 nm],
t
R
way was added to the solution of rac-3 in hexane (5 mL). The pro-
gress and enantioselectivity of the reactions were followed by ana-
lyzing the samples by chiral GC.
The preparative scale kinetic resolution of racemic ethyl 3-acet-
oxy-3-phenylpropanoate rac-3 (295 mg, 1.25 mmol) biotransfor-
mations was performed with 25 g of S. cerevisiae ATCC 9080 cells
under the same conditions which were applied for the microbial
reduction of 1 (see Section 4.5). The products were separated by
silica gel chromatography (hexane:EtOAc, 9.5:0.5, v/v), resulting
(
min): 27.8 [(S)-6], 33.1 [(R)-6]. t
R
(min): 27.8 [(S)-6], 33.1 [(R)-6].
2
O), ee = 96% by HPLC {lit. :
2
O}; HPLC: see at (S)-6.
2
D
5
39
(
R)-6: ½
a
ꢂ
¼ þ7:6 (c 0.25, H
2
5
½
a
ꢂ
¼ þ7 (c 0.27, H
D
Acknowledgements
L.P. thanks the financial support from Hungarian OTKA Founda-
tion (NN-103242). Support to AV from the European Social Fund
POSDRU number 107/1.5/S/78702 is also acknowledged. This work
is also related to the scientific program of ‘Talent care and cultivation
in the scientific workshops of BME’ project (TÁMOP-4.2.2.B-10/1–
in the optically active alcohol (R)-2 (119 mg, 49%) and acetate
R)-3 (142 mg, 48%) as clear oils. The 1H and C NMR spectra of
13
(
the optically active products (R)-2 and (S)-3 were indistinguishable
from those of their racemates.
2010–0009), supported by the New Hungary Development Plan.
2
D
5
), ee = 96% by HPLC {lit.33
(
R)-2: ½
a
ꢂ
¼ þ51:0 (c 1.5, CHCl
3
:
:
25
References
½
½
a
ꢂ
ꢂ
¼ þ44:1 (c 1, CHCl
3
), Ee >99%}; HPLC, see Section 4.5.
D
2
D
5
), ee = 96% by GC {lit.34
(
S)-3:
½
a
ꢂ
¼ ꢀ1:1 (c 1.5, CHCl
3
1
.
1
5
7
a
¼ ꢀ1:2 (neat)}; GC [on permethylated b-cyclodextrin, b-PM
46
column (30 ꢁ 0.32 mm ꢁ 0.25
lM); carrier gas: nitrogen; oven