Biocatalyzed enantioselective reduction of activated C=C bonds: Synthesis of enantiomerically enriched α-halo-β-arylpropionic acids

Article abstract of DOI:10.1002/ejoc.201100537

The enantioselective biocatalyzed reduction of the C=C bond of some (Z)-methyl α-halo-β-arylacrylates was investigated. The reaction was performed by baker's yeast fermentation and Old Yellow Enzymes 1-3 mediated biotransformations. The final products wer

Full text of DOI:10.1002/ejoc.201100537

FULL PAPER
DOI: 10.1002/ejoc.201100537
Biocatalyzed Enantioselective Reduction of Activated C=C Bonds: Synthesis of
Enantiomerically Enriched α-Halo-β-arylpropionic Acids
Elisabetta Brenna,*[a] Francesco G. Gatti,[a] Alessia Manfredi,[a] Daniela Monti,[b] and
Fabio Parmeggiani[a]
Keywords: Enzyme catalysis / Biocatalysis / Reduction / Asymmetric synthesis / Biotransformations
The enantioselective biocatalyzed reduction of the C=C bond
of some (Z)-methyl α-halo-β-arylacrylates was investigated.
The reaction was performed by baker’s yeast fermentation
and Old Yellow Enzymes 1–3 mediated biotransformations.
The final products were, respectively, enantiomerically en-
riched (S)-α-halo-β-arylpropionic acids and their methyl es-
ters, and ester hydrolysis was promoted in the whole cell sys-
tem. High conversions and enantioselectivity values were
observed when the aromatic ring was substituted by an elec-
tron-withdrawing group. Further manipulation of two of
these enantiomerically enriched (S)-haloacids afforded p-
substituted D-phenylalanines.
age, may occur in the whole cell system because of the pres-
ence of enzymes other than enoate reductases. If these reac-
tions are undesired, cloned and overexpressed enoate re-
ductases can be employed together with the suitable meta-
bolic set of redox enzymes for the recycling of the nicotin-
amide cofactor.
The data collected up to now, mainly on BY fermenta-
tions, establish that an electron-withdrawing substituent has
to be present on the double bond to activate it towards
biocatalyzed reductions. Unsaturated aldehydes and
ketones have been identified as the optimal substrates for
enoate reductases.[1] Such substrates are reduced very ef-
ficiently with high enantioselectivities and conversions. Re-
cent work on the activity of cloned and overexpressed eno-
ate reductases[5] has shown that a greater diversity of
compounds containing C=C bonds can be bioreduced than
was previously recognized. These investigations have been
performed quite exclusively on model substrates, with only
a very few examples of preparative biotransformations be-
ing presented.[6]
To add such bioreductions to the toolbox available for
enantioselective synthesis and to implement biocatalysis in
industrial manufacturing processes, it is necessary to ex-
plore the synthetic capabilities of enoate reductases on new
activated substrates. It is important to investigate the effects
upon enantioselectivity and conversion rates that might be
exerted by structural modifications to substituents while
still preserving the nature of those necessary to allow biore-
duction. All these investigations should be aimed at ob-
taining useful enantiopure synthons for the preparation of
relevant chiral molecules. This is especially the case, as ex-
pectations are that the proportion of products manufac-
tured using biotechnology may reach ~20% of global chem-
icals by 2020 as a consequence of demands for more sus-
Introduction
The creation of a stereogenic center in a preferential con-
figuration by reduction of a suitably substituted C=C bond
by combining the control of E/Z configuration is a very
attractive preparative procedure. Biocatalysis can offer a
highly enantioselective variant of this process,[1] which
could be complementary or alternative to chemical pro-
cedures based on chiral metal complexes.[2] Enzyme-cata-
lyzed reductions of activated C=C bonds can be performed
at room temperature under atmospheric pressure in aque-
ous medium, generally allowing energy consumption and
waste streams to be minimized.
These kinds of reactions, promoted by enoate reductases,
have been known for a long time,[3] but their full synthetic
potential remains underappreciated and underexploited.
Many of these enoate reductases, belonging to the family
of Old Yellow Enzymes (OYEs), have been isolated and
characterized; OYE1 from Saccharomyces pastorianus (for-
merly carlsbergensis) and OYE2–3 from S. cerevisiae are ex-
cellent examples.[4] However, most such transformations,
when done on a preparative scale, have been performed by
using microbial cell systems, especially Saccharomyces cere-
visiae (baker’s yeast) to avoid the requirement for external
cofactor recycling and protein purification.
Baker’s yeast (BY) fermentations can be carried out very
simply, but sometimes side reactions, such as carbonyl re-
duction, ester hydrolysis, acyloin reaction, and acetal cleav-
[a] Politecnico di Milano, Dipartimento di Chimica,
Materiali, Ingegneria Chimica,
Via Mancinelli 7, 20131 Milano, Italy
Fax: +39-02-23993180
E-mail: elisabetta.brenna@polimi.it
[b] Istituto di Chimica del Riconoscimento Molecolare – CNR,
Via Mario Bianco, 9 20131 Milano, Italy
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E. Brenna, F. G. Gatti, A. Manfredi, D. Monti, F. Parmeggiani
FULL PAPER
tainable manufacturing processes.[7] In particular, the effec-
tive exploitation of enoate reductases is on the wish list of
companies involved in the production of small-molecule
pharmaceuticals, pharma intermediates, and fine chemicals
for the life sciences area.[7]
Thus, we envisaged the possibility of exploiting the bio-
catalyzed reduction of the C=C bond of (Z)-methyl α-halo-
β-arylacrylates of type I with Z = aryl (Scheme 1) to pre-
pare enantiopure (S)-α-halo-β-arylpropionic acids II. We
showed the synthetic significance of this biotransformation
by converting two of these enantiomerically enriched
haloacids II into non-natural d-amino esters of type III,
according to an efficient three-step procedure. We report
herein on the results of this research.
Scheme 2. Reagents and conditions: (i) NCS or NBS, THF, –20 °C,
under N2; (ii) K2CO3, ArCHO, 12 h, r.t.
The results of the bioreductions of compounds 1–8 are
reported in Table 1. (S)-Halopropionic acids 9–14
(Scheme 3) could be recovered from the BY fermentation
medium and isolated by column chromatography; ester hy-
drolysis occurred inevitably in the reaction medium. The
reduction products of OYE-mediated biotransformations
were saturated (S)-methyl ester derivatives, which were iden-
tified by GC–MS in the crude reaction mixtures.
Scheme 1.
The absolute configurations of compounds 9, 10, and 12
were determined by comparing chiral GC analyses of the
corresponding methyl esters, obtained by diazomethane
treatment, with those of authentic samples of (S)-methyl
propionates, prepared by van Slyke reaction of the corre-
sponding l-amino acid (see the Experimental Section). The
Results and Discussion
Bioreduction of Methyl α-Halo-β-arylacrylates
Compounds of type II are synthetically useful chiral syn- absolute configurations of compounds 11 and 14 were es-
thons that can be converted into a broad range of deriva- tablished by chemical correlation to known phenylalanine
tives by stereospecific nucleophilic substitution reactions derivatives.
with several nitrogen,[8] oxygen,[9] and sulfur nucleo-
No bioreduction was observed for arylacrylates 5 and 8,
philes.[10] The known methods for obtaining enantiopure whereas compounds 6 and 7, bearing an electron-with-
compounds II are limited to the conversion of natural drawing substituent on the aromatic ring, were almost
amino acids through the agency of diazonium salts with quantitatively converted with high enantiomeric excess val-
retention of configuration, and to dynamic resolution pro- ues by both BY and OYE3. In the case of compound 4, the
cedures within nucleophilic substitution reactions.[11] It is presence of an electron-donating group on the aromatic
known that the C=C bond of α,β-unsaturated esters and ring did not prevent bioreduction, but it did lower the
acids cannot be reduced by baker’s yeast, but that the pres- enantioselectivity of reduction. The comparison of the bio-
ence of a chlorine atom on the α carbon atom makes the transformation data for compounds 1 and 2 shows that the
biotransformation occur enantioselectively. To date only a presence of a bromine atom is not detrimental to conver-
few examples have been reported in the literature and these sion and enantioselectivity. Importantly, bromine can be
are limited to olefin scaffolds in which Z = alkyl or chlo- more useful than chlorine as a leaving group in synthetic
roalkyl moieties and X = Cl.[12]
procedures. In particular, the BY reduction of substrate 2
We prepared a series of substrates with Z = aryl and was characterized by a much higher conversion and a
X = Cl or Br and subjected them to bioreduction by BY slightly lower enantioselectivity than that of compound 1.
fermentation and by means of cloned and overexpressed
OYE 1–3 to investigate the robustness of this reaction to have no great influence on the enantiomeric excess value
towards substituent modifications. of the resulting reduced product, and this could be a great
The diastereomeric purity of the starting acrylate seems
Compounds 1–8 (Scheme 2) were prepared by reaction advantage for practical applications. However, this result is
of the corresponding aromatic aldehyde with chloro- or quite surprising because the BY reduction of (E)- and (Z)-
bromophosphoylides, according to a known one-pot pro- chloride derivatives of type I is described to afford (R) and
cedure[13] based on the in situ generation of the haloylide (S)-α-chloroalkanoic acids.[12] A tentative explanation is
and subsequent condensation with the carbonyl compound. that the C=C bond with the (Z) configuration is preferen-
The (Z)-stereoisomer was always obtained as the main com- tially converted by the enzyme to give the (S)-haloacid and
ponent (60–100%de determined by GC analysis after col- concomitant isomerization of the (E)-olefin into the more
umn chromatography) in high isolated yields (63–75%).
favored (Z)-isomer occurs in the reaction medium.
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biocatalyzed Enantioselective Reduction of Activated Olefins
Table 1. Results of BY fermentations and OYE1–3 biotransformations of substrates 1–8.
Entry
Substrate
dei[a] [%]
def BY [%]
def OYE [%]
BY
OYE 1
c[c] [%]
ee[d] [%]
OYE 2
c[c] [%]
ee[d] [%]
OYE 3
c[c] [%]
ee[d] [%]
c[b] [%]
ee[d] [%]
1
2
3
4
Ar = C6H5, X = Cl (1)
Ar = C6H5, X = Br (2)
62
28
96 (S)
4
5
38
99 (S)
64
68, 70, 64
84
82 (S)
80 (S)
71
94 (S)
10
94 (S)
1
37
94 (S)
5
82
89 (S)
6
7
8
9
10
11
12
13
14
15
16
17
18
98, 98, 98
80
Ar = 4-FC6H4, X = Cl (3)
Ar = 4-CH3OC6H4, X = Cl (4)
20
93(S)
0
3
58
97(S)
86
94(S)
80, 82, 87
80
15
80 (S)
14
60 (S)
6
20
88 (S)
86
62 (S)
66, 58, 50
100
Ar = 4-HOC6H4, X = Br (5)
Ar = 2-NO2C6H4, X = Cl (6)
4[e]
100
0
38
93
28
97 (S)
1
0
15
92
0
91
99
73
96 (S)
9
100
98[e]
100
Ar = 4-CNC6H4, X = Cl (7)
100
5
94 (S)
90 (S)
2
Ar = naphthyl, X = Cl (8)
100
1[e]
[a] Diastereomeric excess values (GC) of the starting acrylate (dei), of the acrylate left unreacted after BY fermentation (def BY) and
after OYEs biotransformations (def OYE). [b] Conversion calculated by GC analysis of the crude mixture treated with CH2N2 solution
after 72 h reaction time. [c] Conversion calculated by GC analysis of the crude mixture after 24 h reaction time. [d] Calculated by GC
analysis on a chiral column. [e] Absolute configuration not determined.
Scheme 3. BY and OYE1–3 mediated reductions of substrates 1–8.
The data highlights that OYE3 is the most efficient cata-
d-Amino acids are incorporated into active pharmaceuti-
lyst of this set of enoate reductases; the reducing activity of cal ingredients as key structural fragments, or in peptides,
BY seems to be due primarily to this enzyme rather than to confer higher resistance to hydrolysis by peptidases and
to OYE2.
proteases.[23] The skeleton of p-fluoro-d-phenylalanine, for
example, has been recently introduced[24] into the molecular
structure of compound 15 (Figure 1), a potent, selective,
and orally bioavailable melanocortin subtype-4 receptor
partial agonist developed by Merck researchers to treat
Conversion of (S)-α-Chloro-β-arylpropionic Acids into p-
Substituted D-Phenylalanines
We investigated the synthetic potential of these enantio- obesity without potentiating erectile activity. Many low mo-
enriched halopropionic acids by converting some of them lecular weight thrombin inhibitors,[25] such as prototype
into p-substituted d-phenylalanines. The synthesis of this 16[26] (Figure 1), incorporate a d-phenylalanine fragment
class of amino acids is still a challenge for organic chem- bearing an amidino unit in the para position, which is cre-
ists.[14] Many enzymatic approaches have been exploited for ated by suitable reactions of a nitrile group. Thus, we con-
their preparation including (i) deracemization[15] or kinetic verted 4-fluoro- and 4-cyanophenyl propionic acids (S)-11
resolution of chemically modified forms of racemic amino and (S)-14 into the corresponding phenylalanine deriva-
acids[16] using amidases,[17] hydantoinases,[18] and acyl- tives.
ases;[19] and (ii) stereoselective synthesis of enantiopure
Chloropropionic acids (S)-11 and (S)-14, recovered from
amino acids from oxo acids[20] using dehydrogenases[21] and BY fermentation, were treated with SOCl2 in methanol to
transaminases.[22]
give the corresponding methyl esters (S)-17 and (S)-18
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E. Brenna, F. G. Gatti, A. Manfredi, D. Monti, F. Parmeggiani
FULL PAPER
and with good yields, working in aqueous solution under
very mild reaction conditions. The fermentation medium
promotes ester hydrolysis, whereas employment of isolated
enzymes preserves the ester functionality. Hence, it is pos-
sible to access both optically active α-halo acids or esters,
according to the particular requirements of the synthetic
procedure of which the biotransformation is a part. The
reaction is particularly favored by the presence of electron-
withdrawing substituents on the aromatic ring and does not
require diastereomerically pure substrates.
Figure 1. Compounds 15 and 16.
Experimental Section
(Scheme 4). Nucleophilic substitution with NaN3 in DMF
afforded azides (R)-19 and (R)-20, which were then submit-
ted to hydrogenation. When 10% Pd/C in AcOEt solution
was employed at room temperature, (R)-19 was transformed
into d-21, whereas the CN moiety of substrate (R)-20 was
converted into a methyl group concomitantly with azido
reduction, affording d-22. With the aim of preserving the
nitrile functionality, the reduction was performed under
mild conditions, specifically, 5% wet Pd/C in AcOEt/EtOH
(1:1) solution at 0 °C.[27] Thus, by exploiting the higher re-
activity of the azido group towards hydrogenation, 4-cyano-
substituted derivative d-23 could be recovered. Treatment
of the crude hydrogenation mixtures with HCl gas in diethyl
ether afforded the hydrochloride salts of p-substituted phen-
ylalanines d-21, d-22, and d-23 in 12, 45, and 42% yield,
respectively, from BY fermentation.
General Methods: Baker’s yeast from Lesaffre Italia (code number
30509) was employed. TLC analyses were performed on Merck
Kieselgel 60 F254 plates. All chromatographic separations were
carried out on silica gel columns. The classical van Slike pro-
cedure[28] was employed to prepare reference samples of methyl α-
halopropionates for GC analysis on a chiral column. Commercial
l- and d,l-phenylalanine, l- and d,l-tyrosine, and p-fluoro-d,l-
phenylalanine were employed to prepare methyl esters of com-
pounds 9, 10, 12, and 11. Racemic 2-nitro- and 4-cyanophenylal-
anines were synthesized according to the literature[29] to prepare
racemic methyl esters of compounds 13 and 14. H and 13C NMR
1
spectra were recorded with a 400 MHz spectrometer. The chemical
shift scale was based on internal tetramethylsilane. GC–MS analy-
ses were performed with a HP-5MS column (30 m ϫ0.25 mm
ϫ0.25 μm). The following temperature program was employed: 60°
(1 min)/6°/min/150° (1 min)/12°/min/280° (5 min). The enantio-
meric excess values were determined by GC analysis, performed
with a DAcTBSil.BetaCDX 0.25 μm ϫ0.25 mm ϫ25 m column
(Mega, Italy), installed on a HP 6890 gas chromatograph, accord-
ing to the following conditions: (i) 60 °C (3 min)/5 °C min–1/180 °C
(2 min)/30 °C min–1/220 °C; methyl ester of compound 9: tR
=
17.47 (R), 17.59 (S) min; methyl ester of compound 11: tR = 18.83
(R), 19.06 (S) min. (ii) 80 °C (3 min)/2 °C min–1/140 °C (2 min)/
30 °C min–1/220 °C, methyl ester of compound 10: tR = 24.24 (R),
24.42 (S) min; methyl ester of compound 12: tR = 34.21 (R), 34.29
(S) min; methyl ester of compound 13: tR = 35.50 (R), 35.54
(S) min. (iii) 130 °C (2 min)/1.5 °C min–1/180 °C/30 °C min–1/
220 °C, methyl ester of compound 14: tR = 29.82 (R), 29.99 (S) min.
Optical rotations were measured with a Dr. Kernchen Propol digi-
tal automatic polarimeter at 589 nm.
Scheme 4. Reagents and conditions: (i) NaN3, DMF, 60 °C; (ii) H2,
Pd/C 10%, AcOEt; (iii) HCl(g) in Et2O; (iv) H2, 5% Pd/C, AcOEt/
EtOH, 0 °C.
General Procedure for the Synthesis of Methyl (Z)-2-Halo-2-acryl-
ates 1–8: NBS or NCS (0.11 mol) was added portionwise under a
nitrogen atmosphere to a solution of methyl(triphenylphosphor-
anylidene)acetate (33.4 g, 0.10 mol) in CH2Cl2 (300 mL) at –20 °C
The mixture was stirred at –20 °C for 1 h and then warmed to room
temperature. The aromatic aldehyde (0.10 mol) and K2CO3 (34.5 g,
0.25 mol) were added to the mixture, which was stirred for 16 h.
The reaction mixture was poured into water (300 mL). The organic
phase was dried with Na2SO4 and the solvent was removed under
reduced pressure to give a solid, which was purified by silica gel
column chromatography (hexane/AcOEt).
Conclusions
In order to promote a real increase in the practicality of
biocatalyzed C=C reductions on an industrial scale, and to
effectively exploit all the advantages of sustainability related
to enzyme processes, it is essential to enlarge the structural
class of compounds that can be accepted as substrates by
enoate reductases. Also necessary are investigations into the
effects of structural modifications on enantioselectivity and
conversion in such biocatalytic processes.
(Z)-Methyl 2-Chloro-3-phenylacrylate [(Z)-1]: Yield: 13.9 g, 71%;
1
62%de. H NMR[30] (400 MHz, CDCl3): δ = 7.91 [s, 1 H, H-C(3)],
7.85–7.81 (m, 2 H, aromatic), 7.45–7.39 (m, 3 H, aromatic), 3.90
1
(s, 3 H, COOCH3) ppm; representative H NMR signals of minor
This work highlights the possibility of employing either
BY or isolated enoate reductases to prepare (S)-α-halo-β-
(E)-diastereoisomer: δ = 7.36–7.26 (m, 5 H, aromatic), 7.19 [s, 1
H, H-C(3)], 3.74 (s, 3 H, COOCH3) ppm. 13C NMR (100.6 MHz,
arylpropionic acid derivatives in high enantiomeric purity CDCl3): δ = 163.8, 137.1, 132.8, 130.7, 130.1, 128.4, 121.7,
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Biocatalyzed Enantioselective Reduction of Activated Olefins
53.2 ppm. GC–MS:, tR = 18.96 min; m/z (%) = 196 (97) [M]+, 161
(100), 102 (87).
washed and dried organic phase was chromatographed with in-
creasing amounts of AcOEt in hexane to afford 2-halopropionic
acids 9–14.
(Z)-Methyl 2-Bromo-3-phenylacrylate [(Z)-2]: Yield: 18.0 g, 75%;
1
84%de. H NMR[31] (400 MHz, CDCl3): δ = 8.20 [s, 1 H, H-C(3)],
(S)-2-Chloro-3-phenylpropanoic Acid [(S)-9]: From (Z)-1 (8.0 g,
0.041 mol). Yield: 1.58 g, 21%; 96%ee (GC on a chiral column of
the corresponding methyl ester). [α]2D0 = +7.5 (c = 1.1, MeOH)
{ref.[34] for (R)-9 with 88%ee: [α]2D0 = –6.8 (c = 1, MeOH)}. 1H
NMR[28] (400 MHz, CDCl3): δ = 7.35–7.20 (m, 5 H, aromatic),
4.49 [dd, J = 7.8, 6.8 Hz, 1 H, H-C(2)], 3.39 [dd, J = 14.1, 6.8 Hz, 1
H, H-C(3)], 3.19 [dd, J = 14.1, 7.8 Hz, 1 H, H-C(3)] ppm. 13CNMR
(100 Hz, CDCl3): δ = 174.9, 135.5, 129.3, 128.6, 127.4, 57.2,
40.7 ppm. GC–MS as methyl ester: tR = 15.97 min; m/z (%) = 198
(1) [M]+, 162 (71), 131 (100).
7.83 (m, 2 H, aromatic), 7.41 (m, 3 H, aromatic), 3.90 (s, 3 H,
COOCH3) ppm. 13C NMR[31] (100.6 MHz, CDCl3): δ = 163.8,
141.1, 133.6, 130.3, 128.9, 128.4, 112.5, 53.5 ppm. GC–MS: tR
=
20.20 min; m/z (%) = 240 (33) [M]+, 209 (13), 161 (100), 102 (50).
(Z)-Methyl 2-Chloro-3-(4-fluorophenyl)acrylate [(Z)-3]:[32] Yield:
1
14.8 g, 69%; 80%de. H NMR (400 MHz, CDCl3): δ = 7.87 [s, 1
H, H-C(3)], 7.85 (m, 2 H, aromatic), 7.12 (m, 2 H, aromatic), 3.90
(s, 3 H, COOCH3) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 163.8,
163.4 (d), 135.9, 132.8 (d), 129.1 (d), 121.5 (d), 115.7 (d), 53.3 ppm.
GC–MS: tR = 18.73 min; m/z (%) = 214 (45) [M]+, 179 (47), 147
(47), 120 (100).
(S)-2-Bromo-3-phenylpropanoic Acid [(S)-10]: From (Z)-2 (8.5 g,
0.035 mol). Yield: 4.5 g, 57%; 94%ee (GC on a chiral column of
the corresponding methyl ester). [α]2D0 = –10.4 (c = 1.2, MeOH)
{ref.[35] [α]2D0 = –11.4 (c = 2, MeOH)}. 1H NMR[35] (400 MHz,
CDCl3): 1HNMR (400 Hz, CDCl3): δ = 7.30–7.15 (m, 5 H, aro-
matic), 4.40 [t, J = 7.6 Hz, 1 H, H-C(2)], 3.43 [dd, J = 14.3, 8.0 Hz,
1 H, H-C (3)], 3.21 [dd, J = 14.3, 7.3 Hz, 1 H, H-C (3)] ppm. 13C
NMR (100.6 MHz, CDCl3): δ = 174.8, 136.4, 129.1, 128.7, 127.4,
44.8, 40.7 ppm. GC–MS as methyl ester: tR = 17.65 min; m/z (%)
= 242 (0.5) [M]+, 211 (3), 163 (94), 131 (100).
(Z)-Methyl 2-Chloro-3-(4-methoxyphenyl)acrylate [(Z)-4]:[33] Yield:
1
14.7 g, 65%; 80%de. H NMR[30] (400 MHz, CDCl3): δ = 7.91 [s,
1 H, H-C(3)], 7.85–7.80 (m, 2 H, aromatic), 7.46–7.40 (m, 2 H,
aromatic), 3.89 and 3.86 (2 s, 6 H, COOCH3 + OCH3) ppm. 13C
NMR (100.6 MHz, CDCl3): δ = 164.2, 161.2, 136.8, 132.7, 130.5,
125.6, 114.1, 55.3, 53.2 ppm. GC–MS: tR = 22.85 min; m/z (%) =
226 (100) [M]+, 191 (37), 159 (50), 132 (100).
(Z)-Methyl 2-Bromo-3-(4-hydroxyphenyl)acrylate [(Z)-5]:[33] Yield:
16.1 g, 63%; 100%de. H NMR (400 MHz, CDCl3): δ = 8.16 [s, 1
(S)-2-Chloro-3-(4-fluorophenyl)propanoic Acid [(S)-11]: From (Z)-3
(8.0 g, 0.037 mol). Yield: 1.19 g, 16%; 93%ee (GC on a chiral col-
umn of the corresponding methyl ester). [α]2D0 = +6.6 (c = 1.1,
MeOH). 1H NMR (400 MHz, CDCl3): δ = 7.23–7.19 (m, 2 H, aro-
matic), 7.01 (m, 2 H, aromatic), 4.46 [dd, J = 7.6, 6.8 Hz, 1 H, H-
C(2)], 3.38 [dd, J = 14.3, 6.7 Hz, 1 H, H-C(3)], 3.18 [dd, J = 14.3,
7.6 Hz, 1 H, H-C(3)] ppm. 13C NMR (100.6 MHz, CDCl3, 100 Hz,
CDCl3): δ = 173.9, 162.2 (d), 131.2, 130.9 (d), 115.5 (d), 57.0,
40.0 ppm. GC–MS as methyl ester: tR = 16.03 min; m/z (%) = 216
(1) [M]+, 180 (67), 149 (73), 109 (100).
1
H, H-C(3)], 7.85 (d, J = 8.7, 2 H, aromatic), 6.89 (d, J = 8.7, 2 H,
aromatic), 3.88 (s, 3 H, COOCH3) ppm. 13C NMR (100.6 MHz,
CDCl3):
δ = 164.3, 157.6, 140.6,132.7, 126.3, 115.5, 109.7,
53.5 ppm. GC–MS: tR = 24.34 min; m/z (%) = 256 (36) [M]+, 225
(25), 177 (100), 145 (54).
(Z)-Methyl 2-Chloro-3-(2-nitrophenyl)acrylate [(Z)-6]:[33] Yield:
1
16.4 g, 68%; 100%de. H NMR (400 MHz, CDCl3): δ = 8.23 [s, 1
H, H-C(3)], 8.18 (m, 1 H, aromatic), 7.70 (m, 2 H, aromatic), 7.58
(m, 1 H, aromatic), 3.93 (s, 3 H, COOCH3) ppm. 13C NMR
(100.6 MHz, CDCl3): δ = 162.7, 147.4, 133.9, 133.4, 131.2, 129.9,
129.1, 125.4, 124.8, 55.5 ppm. GC–MS: tR = 22.97 min; m/z (%) =
241 (1) [M]+, 206 (17), 182 (67), 89 (100).
(S)-2-Chloro-3-(4-methoxyphenyl)propanoic Acid [(S)-12]: From
(Z)-4 (8.5 g, 0.037 mol). Yield: 0.90 g, 11%; 80%ee (GC on a chiral
column of the corresponding methyl ester). 1H NMR (400 MHz,
CDCl3): δ = 7.15 (m, 2 H, aromatic), 6.85 (m, 2 H, aromatic), 4.45
[t, J = 7.3 Hz, 1 H, H-C(2)], 3.79 (s, 3 H, OCH3), 3.33 [dd, J =
14.1, 7.3 Hz, 1 H, H-C(3)], 3.14 [dd, J = 14.1, 7.3 Hz, 1 H, H-
C(3)] ppm. 13C NMR (100.6 MHz, CDCl3): δ = 173.6, 158.8, 130.4,
(Z)-Methyl 2-Chloro-3-(4-cyanophenyl)acrylate [(Z)-7]:[33] Yield:
16.6 g, 75%; 100%de. 1H NMR (500 MHz, CDCl3): δ = 7.90 (d, J
= 8.5, 2 H, aromatic), 7.89 [s, 1 H, H-C(3)], 7.71 (d, J = 8.5, 2 H,
aromatic), 3.93 (s, 3 H, COOCH3) ppm. 13C NMR (100.6 MHz,
CDCl3): δ = 163.0, 137.1, 134.9, 132.1, 130.7, 125.1, 118.1, 113.2,
53.5 ppm. GC–MS: tR = 22.86 min; m/z (%) = 221 (67) [M]+, 186
(100), 162 (34), 154 (44).
127.7, 114.0, 60.7, 55.2, 40.0 ppm. GC–MS as methyl ester: tR
=
20.69 min; m/z (%) = 228 (4) [M]+, 192 (41), 161 (22), 121 (100).
2-Chloro-3-(2-nitrophenyl)propanoic Acid (13): From (Z)-6 (8.0 g,
0.039 mol). Yield: 5.7 g, 75%; 98%ee (GC on a chiral column of
the corresponding methyl ester). [α]2D0 = +31 (c = 1, MeOH). 1H
NMR (400 MHz, CDCl3): δ = 8.05 (m, 1 H, aromatic), 7.60 (m, 1
H, aromatic), 7.48 (m, 2 H, aromatic), 4.76 [dd, J = 8.5, 5.8 Hz, 1
H, H-C(2)], 3.80 [dd, J = 14.1, 5.8 Hz, 1 H, H-C(3)], 3.42 [dd, J =
14.1, 8.5 Hz, 1 H, H-C(3)] ppm. 13C NMR (100.6 MHz, CDCl3): δ
= 173.2, 149.2, 133.9, 133.3, 130.9, 128.8, 125.3, 55.9, 38.2 ppm.
GC–MS as methyl ester: tR = 21.84 min; m/z (%) = 208 (11) [M –
35]+, 197 (100), 176 (78), 130 (39).
(Z)-Methyl 2-Chloro-3-(naphthalen-2-yl)acrylate [(Z)-8]: Yield:
15.5 g, 63%; 100%de. 1H NMR[32] (400 MHz, CDCl3): δ = 8.33 [s,
1 H, H-C(3) or aromatic], 8.08 [s, 1 H, aromatic or H-C(3)], 8.00–
7.80 (m, 2 H, aromatic), 7.60–7.45 (m, 2 H, aromatic), 3.92 (s, 3 H,
COOCH3) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 163.9, 137.3,
133.9, 132.9, 131.5, 129.8, 128.7, 128.1, 127.6, 127.5, 126.9, 126.6,
123.5, 53.3 ppm. GC–MS: tR = 25.45 min; m/z (%) = 246 (86)
[M]+, 210 (21), 152 (100).
(S)-2-Chloro-3-(4-cyanophenyl)propanoic Acid [(S)-14]: From (Z)-7
(8.5 g, 0.038 mol). Yield: 5.6 g, 70%; 94%ee (GC on a chiral col-
umn of the corresponding methyl ester). [α]2D0 = –11 (c = 0.62,
MeOH). 1H NMR (400 MHz, CDCl3): δ = 7.62 (m, 2 H, aromatic),
7.39 (m, 2 H, aromatic), 4.52 [dd, J = 7.9, 6.2 Hz, 1 H, H-C(2)],
3.47 [dd, J = 14.3, 6.2 Hz, 1 H, H-C(3)], 3.26 [dd, J = 14.3, 8.1 Hz,
1 H, H-C(3)] ppm. 13C NMR (100.6 MHz, CDCl3): δ = 172.1,
141.3, 132.4, 130.3, 118.5, 111.3, 60.7, 40.6 ppm. GC–MS as methyl
ester: tR = 21.76 min; m/z (%) = 188 (93) [M – 35]+, 156 (100), 128
(43), 116 (57).
General Procedure for Baker’s Yeast Fermentation: To a stirred mix-
ture of baker’s yeast (100 g) and d-glucose (40 g) in tap water (1 L)
at 30 °C was dropwise added a solution of the substrate (0.025 mol)
in ethanol (10 mL). After 72 h under these conditions, acetone
(500 mL) was added, followed by AcOEt/hexane (4:1, 500 mL). The
mixture was filtered in a large Buchner funnel through a thick Ce-
lite pad previously washed with acetone. The two phases were then
separated, and the aqueous phase was extracted with AcOEt/hex-
ane (2ϫ). The oily residue obtained upon evaporation of the
Eur. J. Org. Chem. 2011, 4015–4022
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4019

E. Brenna, F. G. Gatti, A. Manfredi, D. Monti, F. Parmeggiani
FULL PAPER
Synthesis of 4-Fluoro-D-phenylalanine Methyl Ester Hydrochloride = 6.8 Hz, 1 H, H-C(2)], 3.68 (s, 3 H, COOCH3), 3.14 [m, 2 H, 2H-
(D
-21) and 4-Methyl-
D
-phenylalanine Methyl Ester Hydrochloride C(3)] ppm. 13C NMR (100.6 MHz, [D6]DMSO): δ = 169.1, 161.4
(D-22)
(d), 131.3 (d), 115.2 (d), 53.1, 52.5, 34.8 ppm.
(S)-Methyl 2-Chloro-3-(4-fluorophenyl)propanoate [(S)-17]: Thionyl (R)-Methyl 2-Amino-3-(4-tolyl)propanoate Hydrochloride [(R)-22]:
chloride (0.53 mL, 7.3 mmol) was added dropwise to a solution of
(S)-11 (1.0 g, 5 mmol) in methanol (15 mL), and the mixture was
stirred at room temperature for 16 h. The reaction mixture was
concentrated, poured into water, and extracted with CH2Cl2
According to the same procedure described for the conversion of
(R)-19 into (R)-21·HCl, (R)-20 (2.50 g, 0.011 mol) gave (R)-22·HCl
(2.1 g, 85%). [α]2D0 = –28.6 (c = 0.55, EtOH) {ref.[37] for l-22·HCl,
98.4%ee: [α]2D0 = +29.9 (c = 1, EtOH) }. 1H NMR (400 MHz,
(2ϫ200 mL). The organic phase was washed with water and dried D2O): δ = 7.28 (m, 2 H, aromatic), 7.18 (m, 2 H, aromatic), 4.40 [m,
with Na2SO4. The solvent was removed under reduced pressure to
1 H, H-C(2)], 3.82 (s, 3 H, COOCH3), 3.29 [dd, J = 14.4, 5.2 Hz, 1
H, H-C(3)], 3.21 [dd, J = 14.2, 6.9 Hz, 1 H, H-C(3)], 2.32 (s, 3 H,
give (S)-17 (0.85 g, 84%). [α]2D0 = +4.3 (c = 1.2, CHCl3). H NMR
1
(400 MHz, CDCl3): δ = 7.18 (m, 2 H, aromatic), 6.99 (m, 2 H, CH3) ppm. 13C NMR (100.6 MHz, D2O): δ = 170.7, 138.9, 131.2,
aromatic), 4.41 [t, J = 7.1 Hz, 1 H, H-C(2)], 3.74 (s, 3 H, CO-
130.4, 129.9, 54.8, 54.2, 35.8, 20.8 ppm. GC–MS (as a free base):
OCH3), 3.33 [dd, J = 14.2, 7.1 Hz, 1 H, H-C(3)], 3.15 [dd, J = 14.2, tR = 18.62 min; m/z (%) = 193 (2) [M]+ 134 (97), 88 (100).
7.1 Hz, 1 H, H-C(3)] ppm. 13C NMR (100.6 MHz, CDCl3): δ =
(R)-Methyl 2-Amino-3-(4-cyanophenyl)propanoate Hydrochloride
[(R)-23]: A solution of (R)-20 (0.250 g, 1.1 mmol) in AcOEt/EtOH
(1:1, 5 mL) and wet 5% Pd/C catalyst (5 mg) was stirred under a
hydrogen atmosphere at 0 °C until complete reduction. The solu-
169.5, 162.1 (d), 131.6, 130.9 (d), 115.5 (d), 57.2, 52.9, 40.2 ppm.
GC–MS: tR = 16.03 min; m/z (%) = 216 (1) [M]+, 180 (67), 149
(73), 109 (100).
(S)-Methyl 2-Chloro-3-(4-cyanophenyl)propanoate [(S)-18]: Accord-
ing to the same procedure described for the conversion of (S)-11
tion was filtered and concentrated under reduced pressure to give
a residue, which was treated with a saturated solution of HCl(g) in
into (S)-17, (S)-14 (4.0 g, 0.019 mol) gave (S)-18 (3.4 g, 79%). Et2O (2 mL). (R)-23·HCl was recovered by filtration (0.21 g, 79%).
[α]2D0 = –11.4 (c = 0.77, CHCl3). H NMR (400 MHz, CDCl3): δ =
7.62 (m, 2 H, aromatic), 7.35 (m, 2 H, aromatic), 4.47 [dd, J = 7.9,
6.5 Hz, 1 H, H-C(2)], 3.77 (s, 3 H, COOCH3), 3.43 [dd, J = 14.2,
6.5 Hz, 1 H, H-C(3)], 3.24 [dd, J = 14.2, 7.9 Hz, 1 H, H-C(3)] ppm.
[α]2D0 = –44.9 (c = 1.0, EtOH) {ref.[38,39] for (R)-23 HCl [α]2D0 = –48.3
1
1
(c = 0.84, EtOH)}. H NMR[38,39] (400 MHz, D2O): δ = 7.72 (m,
2 H, aromatic), 7.41 (m, 2 H, aromatic), 4.46 [m, 1 H, H-C(2)],
3.38 (s, 3 H, COOCH3), 3.38 [dd, J = 14.5, 6.3 Hz, 1 H, H-C(3)],
13C NMR (100.6 MHz, CDCl3): δ = 169.1, 141.2, 132.3, 130.1, 3.28 [dd, J = 14.5, 7.3 Hz, 1 H, H-C(3)] ppm. 13C NMR
118.4, 111.5, 56.4, 53.1, 40.8 ppm. GC–MS: tR = 21.76 min; m/z
(100.6 MHz, D2O): δ = 172.4, 142.5, 135.8, 132.9, 122.1, 113.4,
56.4, 56.3, 38.4 ppm. GC–MS (as the free base): tR = 22.08 min;
m/z (%) = 204 (0.5) [M]+, 145 (57), 88 (100).
(%) = 188 (93) [M – 35]+, 156 (100), 128 (43), 116 (57).
(R)-Methyl 2-Azido-3-(4-fluorophenyl)propanoate [(R)-19]: Sodium
azide (0.36 g, 5.6 mmol) was added portionwise to a solution of
(S)-17 (0.80 g, 3.7 mmol) in DMF (25 mL), and the mixture was
stirred at 60 °C for 3 h. The reaction mixture was poured into water
(300 mL) and extracted with AcOEt (2ϫ200 mL). The organic
phase was dried with Na2SO4, and the solvent was removed under
reduced pressure to give (R)-19 (0.78 g, 95%). [α]2D0 = +39 (c = 1.1,
Strains: All the enzymes employed were overexpressed in E. coli
BL21 (DE3) strains harboring a specific plasmid prepared accord-
ing to standard molecular biology techniques: pET30a-OYE1 from
the original plasmid kindly provided by Neil C. Bruce,[40] pET30a-
OYE2 and pET30a-OYE3 from S. cerevisiae BY4741 and pKTS-
GDH from B. megaterium DSM509 (detailed steps submitted for
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.19 (m, 2 H, aromatic), publication[41]).
7.00 (m, 2 H, aromatic), 4.05 [dd, J = 8.4, 5.5 Hz, 1 H, H-C(2)],
3.77 (s, 3 H, COOCH3), 3.13 [dd, J = 14.1, 5.4 Hz, 1 H, H-C(3)],
2.98 [dd, J = 14.1, 8.5 Hz, 1 H, H-C(3)] ppm. 13C NMR
(100.6 MHz, CDCl3): δ = 170.1, 161.9 (d), 131.6, 130.7 (d), 115.4
(d), 63.1, 52.5, 36.7 ppm. GC–MS: tR = 18.15 min; m/z (%) = 223
(1) [M]+, 195 (54), 135 (50), 109 (100).
Overexpression of the Enzymes in E. coli BL21 (DE3): A 5 mL cul-
ture in LB medium containing the appropriate antibiotic (50 μg/
mL kanamycin for pET-30a, 100 μg/mL ampicillin for pKTS) was
inoculated with a single colony from a fresh plate and grown over-
night at 37 °C. This starter culture was used to inoculate a 200 mL
culture, which was in turn grown overnight at 37 °C and used to
inoculate a 1.5 L culture. The latter was vigorously aerated at 37 °C
until OD600 reached 0.4–0.5 and then induced adding 0.1 mm IPTG
(50 ng/mL anhydrotetracycline was also added for the pKTS-GDH
plasmid). After 5–6 h the cells were harvested, resuspended in an
equal volume of lysis buffer (20 mm phosphate buffer pH 7.0,
300 mm NaCl, 20 mm imidazole) and homogenized (Haskel high-
pressure homogenizer). The CFE, after centrifugation (20000 g,
(R)-Methyl 2-Azido-3-(4-cyanophenyl)propanoate [(R)-20]: Accord-
ing to the same procedure described for the conversion of (S)-17
into (R)-19, (S)-18 (3.0 g, 0.013 mol) gave (R)-20 (2.78 g, 90%).
1
[α]2D0 = +25.2 (c = 2.0, CHCl3). H NMR (400 MHz, CDCl3): δ =
7.62 (m, 2 H, aromatic), 7.35 (m, 2 H, aromatic), 4.14 [dd, J = 8.5,
5.2 Hz, 1 H, H-C(2)], 3.80 (s, 3 H, COOCH3), 3.21 [dd, J = 14.1,
5.2 Hz, 1 H, H-C(3)], 3.05 [dd, J = 14.1, 8.5 Hz, 1 H, H-C(3)] ppm.
13C NMR (100.6 MHz, CDCl3): δ = 169.7, 141.5, 132.3, 130.1, 1 h, 4 °C), was chromatographed on IMAC stationary phase (Ni-
118.5, 111.4, 62.6, 52.8, 37.4 ppm. GC–MS: tR = 22.79 min; m/z
Sepharose Fast Flow, GE Healthcare) with a mobile phase com-
posed of 20 mm phosphate buffer, pH 7.0, 300 mm NaCl and a 20–
500 mm imidazole gradient. The fractions were collected according
to the chromatogram and dialyzed twice against 1 L of 20 mm
phosphate buffer pH 7.0 (12 h each, 4 °C) to remove imidazole and
salts.
(%) = 202 (64) [M –28]+, 142 (72), 116 (100).
(R)-Methyl 2-Amino-3-(4-fluorophenyl)propanoate Hydrochloride
[(R)-21]: A solution of (R)-19 (0.70 g, 3.1 mmol) in AcOEt (25 mL)
and 10% Pd/C catalyst (5 mg) was stirred under a hydrogen atmo-
sphere until complete reduction. The solution was filtered and con-
centrated under reduced pressure to give a residue, which was
treated with a saturated solution of HCl(g) in Et2O (5 mL). (R)-
21·HCl was recovered by filtration (0.65 g, 89%). [α]2D0 = –31.5 (c
= 1.01, EtOH) {ref.[36] for (R)-21·HCl: [α]2D0 = –34.3 (c = 1.025,
EtOH)}. 1H NMR (400 MHz, [D6]DMSO): δ = 8.62 (br. s, 3 H,
General Procedure for the OYE Mediated Bioreduction: Unsatu-
rated methyl ester 1–8 (5 μmol) dissolved in DMSO (10 μL) was
added to a solution of glucose (1 m, 20 μL, overall 20 mm), NADP+
(10 mm, 10 μL, overall 0.1 mm), GDH (10 μL sol., overall ab. 20 μg/
mL protein conc.) and the required OYE (80 μL sol., overall ab.
NH3+), 7.29 (m, 2 H, aromatic), 7.16 (m, 2 H, aromatic), 4.25 [t, J 40 μg/mL protein conc.) in phosphate buffer (870 μL, 50 mm,
4020 Eur. J. Org. Chem. 2011, 4015–4022
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biocatalyzed Enantioselective Reduction of Activated Olefins
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Received: April 17, 2011
Published Online: June 17, 2011
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Eur. J. Org. Chem. 2011, 4015–4022