Chemo- and biocatalytic strategies to obtain phenylisoserine, a lateral chain of Taxol by asymmetric reduction

Article abstract of DOI:10.1016/j.tetasy.2011.11.017

Enriched ethyl 3-benzamido-2-hydroxy-3-phenylpropanoate (protected phenylisoserine), the chiral side chain of Taxol, was obtained via asymmetric reduction with transition metal-diphoshine complexes or with whole cells of non-conventional yeasts. Asymmetri

Full text of DOI:10.1016/j.tetasy.2011.11.017

Tetrahedron: Asymmetry 22 (2011) 2110–2116
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemo- and biocatalytic strategies to obtain phenylisoserine, a lateral chain
of Taxol by asymmetric reduction
Isabella Rimoldi a, , Michela Pellizzoni c, Giorgio Facchetti a, Francesco Molinari b, Daniele Zerla a,
⇑
Raffaella Gandolfi c
a Università degli Studi di Milano, Facoltà di Farmacia, Dipartimento di Chimica Inorganica, Metallorganica e Analitica ‘L. Malatesta’ e Istituto CNR-ISTM, Via Venezian 21,
20133 Milano, Italy
b Università degli Studi di Milano, Facoltà di Agraria, DISTAM-Sez. Microbiologia Industriale, Via Celoria 2, 20133 Milano, Italy
c Università degli Studi di Milano, Facoltà di Farmacia, Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Sez. Chimica Organica ‘A. Marchesini’, Via Venezian 21,
20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Enriched ethyl 3-benzamido-2-hydroxy-3-phenylpropanoate (protected phenylisoserine), the chiral side
chain of Taxol, was obtained via asymmetric reduction with transition metal–diphoshine complexes or
with whole cells of non-conventional yeasts. Asymmetric hydrogenation was carried out using different
approaches: hydrogenation of the tetra-substituted double bond of (E)-1-benzamido-3-ethoxy-3-oxo-1-
phenylprop-1-en-2-yl ethyl oxalate 1 with Ir(I)–diphosphine complexes in the presence of TEA, hydroge-
Received 26 October 2011
Accepted 30 November 2011
Available online 4 January 2012
nation of the carbonyl group of racemic ethyl 3-benzamido-2-oxo-3-phenylpropanoate
2 with
Ru(II)–diphoshine complexes in the presence of a Lewis acid and finally a two-step enzymatic transforma-
tion of (E)-1-benzamido-3-ethoxy-3-oxo-1-phenylprop-1-en-2-yl ethyl oxalate 1 catalyzed by whole cells
of yeasts bearing cell-bound esterases and dehydrogenases.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
O
HO
Drugs that come from plants have always had an important role
in the treatment of an enormous array of pathologies, including
Cancer. Today the most used antineoplastic drug in therapy is
Paclitaxel (Fig. 1), particularly in breast and ovary Cancer and ad-
vanced forms of Kaposi’s sarcoma.1,2
The major differences between the Taxol skeleton and the other
200 members of its family are the presence of a side chain at the C-
13 position, esterified by an N-benzoyl-phenyl-isoserine group and
an oxetanic ring attached to C 4–5 of the cyclohexane ring. Both
groups are necessary for the biological activity.
OH
O
NH
O
O
H
O
OAc
HO
O
O
OH
Taxol can be isolated from the bark of Taxus brevifolia, a tree
that grows slowly, making Taxol production insufficient for world
demand. Thus there is a need to develop new strategies for the
large-scale synthesis of this drug. Different approaches have been
studied: total synthesis,3–5 production by vegetable crops,6 prepa-
ration from mushrooms,7 extraction from the leaves of the Taxus
species,8 the semi-synthesis from 10-deacetyl baccatine III,9 and
the stereoselective synthesis of the Taxol side chain.10–14 From
these methods, only the last three approaches have become indus-
trially applied, one of which has been patented by Bristol-Myers
Squibb.15,16
Figure 1. Paclitaxel.
This synthesis provides the esterification of modified 10-deacetyl
baccatine III, a tetra substituted diterpene extract from T. brevifolia
leaves, with the lateral chain (2R,3S)-N-benzoyl-O-(1-ethoxyeth-
yl)-3-phenylisoserine. Different strategies have been developed for
the synthesis of the lateral chain in an enantiomerically pure form.
The first asymmetric synthesis of the lateral chain was realised
by Greene9 in 1986, utilising a Sharpless epoxidation. Other strat-
egies have been developed by Ojima et al. using b-lactam,11 and by
Sharpless using methyl cinnamate.12 Another example includes the
chemo-enzymatic Kayser approach,13 which combined a chemical
synthesis with biocatalysis reactions using yeasts.
⇑
Corresponding author. Tel.: +39 02 503 14609; fax: +39 02 503 14615.
E-mail address: isabella.rimoldi@unimi.it (I. Rimoldi).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.11.017

I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
2111
An enzymatic resolution with Ps. cepacia was adopted by Bris-
tol-Myers Squibb from a racemic mixture of azetin-2-one acetate.
Bristol-Myers Squibb’s catalytic approach involved the reduction
and resolution of 2-keto-3-(N-benzoylamino)-3-phenylpropionic
acid ethyl ester, as reported in the U.S. patient.16 Herein, we have
evaluated different chemo and enzymatic strategies for improving
the yields and the selectivity of the conversion of 1 into 3.
completely inactive in the reduction of 1 (Table 2, entry 1), but
very active in the reduction of 2 (Table 2, entry 2).
Since all of the results previously reported showed that iridium
complexes were inactive on tetra-substituted double bonds with-
out the addition of TEA, and given that substrate 1 was hydrolyzed
into substrate 2, the reduction of an a-keto ester was investigated
with ruthenium diphosphine complexes as described in Table 3.
Ruthenium phosphines complexes, prepared according to stan-
dard procedures31 from [Ru(DMF)nCl2]2 gave the
a-hydroxy-N-
2. Results and discussion
benzoyl phenyl isoserine 3 in quantitative yield and with good
de but with modest to no enantioselectivity (Table 3, entries 1–
3). In order to improve the enantiodifferentiation, several Lewis
acids (AlCl3, MnCl2, CrCl3, and FeCl3) and HBF4 were evaluated32,33
(Table 3, entries 4–15). The investigation of additive effects was
studied on complexes derived from atropoisomeric ligands and ex-
tended to some chiral phosphorous ligands with sp3 stereogenic
carbon atoms (Fig. 3).
Asymmetric hydrogenation is one of the most applied methods
to produce enantiomerically pure amino acids.17–20 In order to re-
duce the number of synthetic steps for the production of enantio-
merically pure N-benzoyl-phenyl-isoserine 3, the first substrate
investigated in the asymmetric reduction was 3-benzoylamino-3-
phenyl-(ethyl, 2-oxalyl) propenoic acid ethyl ester 1, already used
as a synthon in Bristol-Myers Squibb’s synthesis (Fig. 2).
When atropoisomeric diphosphines were used, the addition of
the Lewis acids to the catalyst generally decreased the de, which
changed from a maximum of 53% (entry 6) to less than 10% (entry
7). On the other hand addition of Lewis acid increased the ee of
both diastereoisomers (Table 3, entries 4–11 vs 1–3).
Generally, tetra-substituted double bonds are difficult to re-
duce, particularly in the presence of substituents such as –OCO-
COOEt and –NHCOPh. Our first attempts were made with
[Rh(Me-Duphos)(COD)+ClO4ꢀ], one of the catalysts of choice in
the reduction of a tetra-substituted double bond,21–23 but this ap-
proach was unsuccessful. Subsequently, iridium complexes with
diphosphines, characterised by an atropoisomeric chirality, were
used: the commercially available BINAP24 and TolBinap25 and the
particular electron rich TetraMe-Bitiop26 (Fig. 3).
Iridium(I) complexes, prepared from [Ir(COD)Cl2]2 and the afore-
mentioned atropoisomeric diphosphines, are very active especially
in the reductions of imines.27–29 However, in this case, they proved
ineffective (Table 1, entries 1 and 2); the catalysts were only able to
reduce substrate 1 after the addition of a base such as TEA. The
reduction in the presence of TEA with an iridium(I) catalyst pro-
ceeded with good diastereoselectivity for the anti-isomers but in
an almost racemic form (Table 1, entries 3–5). Table 1 illustrates
the effect of the addition of the base: the reactivity increased when
the amount of base was set up at the optimal ratio of 5:1 (base/sub-
strate), but decreased when the ratio was raised to 10:1. TEA base
helped with the efficiency of the catalyst by hydrolysis of substrate
1 to give keto-ester 2.
Whatever Lewis acid was added, BINAP furnished a syn-diaste-
reodifferentiation, opposite to the other two atropoisomeric
diphosphines; the closely related Tolbinap and the more basic Tet-
raMe-BITIOP gave the anti-diastereoisomers with good ee (Table 3,
entries 4–8 vs 1–3 and 9–11).
Moving from pure atropisomeric ligands to BDPP,34 character-
ised by an sp3 stereogenic carbon atom and to (R,Rax)-Isaphos
diphosphine bearing both the chiralities,35 the anti-diastereoiso-
mers were predominant following the behavior of Tolbinap and
TetraMe-BITIOP (Table 3, entries 12–15).
The results of the asymmetric homogeneous catalysis with
transition metal complexes suggested that both hydrolytic and
reductive activities were required for the one-pot reduction of sub-
strate 1 into 3 to by-pass a step of the synthesis reported by Bris-
tol-Myers Squibb’s patent.
This conversion can be realized in two different pathways:
reduction of the alkene-group of 1 to give 4 which after hydrolysis
furnishes 3, or hydrolysis of 1 to form ketone 2 which is then re-
duced to 1 (Scheme 1).
We attempted to perform the two-step transformation in one
pot. Different yeasts known for the simultaneous occurrence of
cell-associated esterase and carbonyl reductase activities36–38 were
evaluated for the biotransformation of 1 (Table 4).
The syn- and anti-stereoisomers were obtained depending on
the yeast used; high diastereoselectivity was sometimes observed.
In particular, Pachysolen tannophilus CBS 4044 (entry 7) and Toru-
lopsis molischiana CBS 837 (entry 9) gave only anti-3 with modest
enantioselectivities, while Lindnera fabiani (entry 13) gave only
syn-3 with notable enantioselectivity. Other yeasts (entries 10–
12) gave poor diastereoselectivity in the formation of syn-3, but
high enantioselectivity. In most cases, the pharmacologically rele-
vant steroisomer (2R,3S)-syn-3 was produced with an ee ranging
In Table 2 it can be seen that TEA was necessary for high activity
and diastereoselectivity (compare entry 3 to entry 2) but no
enantioselectivity. For further insight, a partial characterisation of
the Ir/diphosphine/TEA complex was performed. The [Ir(COD)(P–
P)Cl] complex, prepared from [Ir(COD)Cl2]2 and (S)-BINAP and
characterised by two doublets at ꢀ2.2 ppm (J = 18.3 Hz) and
ꢀ11.6 ppm in the 31P NMR spectrum,30 was able to reduce sub-
strate 2 with modest efficiency (Table 2, entry 3).
The addition of 1 equiv of TEA had no effect on the 31P NMR
spectrum and the catalyst was completely inactive. When the
amount of TEA was raised to 10 equiv, the 31P NMR was changed
and showed two doublets at 1.0 ppm (J = 21.4 Hz) and at
13.0 ppm. Adding a further excess of TEA (12.5 equiv) and heating
at 60 °C to mimic the reaction conditions, caused two doublets at
3.6 ppm (J = 17.4 Hz) and ꢀ1.2 ppm to appear; this complex was
COOEt
O
COOEt
O
O
COOEt
OH
PhOCHN
PhOCHN
PhOCHN
COOEt
1
2
3
Figure 2. Substrates and products used and obtained in asymmetric hydrogenation.

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I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
CH3
S
PPh2
PPh2
H3C
H3C
PAr2
PAr2
S
CH3
Ar = Ph
L1 = (S)-BINAP
Ar = 4-Me-Ph L2 = (S)-Tol-BINAP
L3 = (R)-(-)-TetraMe-Bitiop
OR'
O
O
PPh2 PPh2
P
R =
N
R
L
4 = (S,S)-BDPP
R' = PPh2
5 = (RRax)-Isaphos C1
Figure 3. Chiral chelating diphosphines used in the asymmetric hydrogenation of 2.
L
Table 1
Table 3
Asymmetric reduction of 1 with [Ir(COD)Cl2]2, (S)-BINAP and different amounts of
Chiral chelating diphosphines used in the asymmetric hydrogenation of 2
TEA
COOEt
COOEt
OH
COOEt
O
O
COOEt
OH
PhOCHN
PhOCHN
PhOCHN
PhOCHN
O
COOEt
[Ir(COD)Cl2]2, (S)-BINAP
TEA; H2
[Ru(DMF)Cl2]2 ; PP*
H2; with or without Lewis acid
3
1
3
2
Entry
TEA/substrate
Conversion (%)
syn:anti (%)
ee anti (%)
Entry
Lewis acid
Ligand
ed (%)
ee syn(%)
ee anti (%)
1
2
3
4
5
—
0.5:1
1:1
5:1
10:1
0
0
30
100
73
—
—
0:100
14:86
12:88
—
—
0
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
—
—
—
L1
L2
L3
L1
L1
L1
L1
L1
L2
L3
L3
L4
L4
L5
L5
62 anti
76 anti
58 anti
30 syn
20 syn
53 syn
9 syn
25 syn
24 anti
21 anti
47 anti
12 anti
24 anti
8 anti
0
0
9 (2S,3S)
32 (2R,3R)
19 (2S,3S)
60 (2S,3S)
53 (2S,3S)
55 (2S,3S)
19 (2S,3S)
63 (2S,3S)
56 (2S,3S)
39 (2R,3R)
7 (2S,3R)
1 (2S,3S)
0
30 (2R,3S)
15 (2S,3R)
38 (2S,3R)
51 (2S,3R)
57 (2S,3R)
52 (2S,3R)
31 (2S,3R)
76 (2S,3R)
52 (2R,3S)
40 (2R,3S)
26 (2S,3R)
9 (2S,3R)
25 (2S,3R)
50 (2S,3R)
FeCl3
HBF4
CrCl3
MnCl2
AlCl3
FeCl3
FeCl3
CrCl3
FeCl3
CrCl3
FeCl3
CrCl3
Reaction conditions: Solvent: toluene; reaction time 24 h except for entry 4 (5 h);
substrate/[Ir] = 50:1; substrate concentration = 0.012 mmol/mL; T = room temper-
ature; P = 30 atm H2. The ee and ed values were determined by chiral HPLC with a
Daicel Chiralpak AD column (80:20 = hexane–isopropanol, flow = 1.0 mL/min).
Table 2
Asymmetric reduction of 1 and 2 with [Ir(COD)Cl2]2, (S)-BINAP and TEA complex
45 (2S,3S)
34 (2S,3S)
Entry
Substrate
Conversion (%)
syn:anti
ee anti (%)
1
1
2
2
0
100
20
—
15:85
10:90
—
0
0
Reaction conditions: Solvent: EtOH–CH2Cl2 = 50:50; reaction time 48 h; conversion
>99%; substrate/[RuCl2(DMF)n(PP⁄)] = 50:1; catalyst/additive = 1:10, substrate con-
centration = 0.012 M; T = 60 °C; P = 30 atm H2. The ee and de values were deter-
mined by chiral HPLC with a Daicel Chiralpak AD (80:20 = hexane–isopropanol,
flow = 1.0 mL/min).
2
3a
Reaction conditions: Solvent: toluene; reaction time 5 h; substrate/[Ir] = 50:1;
substrate concentration = 0.012 mmol/mL; T = room temperature for substrate 1
and T = 60 °C for substrate 2; P = 30 atm H2. The ee and ed values were determined
by chiral HPLC with a Daicel Chiralpak AD column (80:20 = hexane–isopropanol,
flow = 1.0 mL/min).
a
the double bond (substrate 1) by hydrogenation with Pd/C in the
heterogeneous phase was performed. Preliminary studies with
Pd/C as the catalyst showed a low molar conversion. The addition
of NH3 via ammonium formate allowed the total conversion of the
substrate (Fig. 4).39,40
The strong dependence of the solvent used has been high-
lighted: in the presence of aprotic solvents (i.e. AcOEt) the desired
oxalyl ester 4 was obtained, while when protic solvents (i.e. MeOH
Reaction was performed with [Ir(COD)Cl2]2, (S)-BINAP without TEA in 24 h.
from 59% up to 99%, in accordance with Prelog’s rule. The best
performing yeast in terms of diastereoselectivity was L. fabiani,
previously used for the stereoselective reduction of 2 into 3.
Although the reduction of tetra-substituted double bonds by
yeasts is not common, to discard this possibility, the reduction of

I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
2113
path a
COOEt
O
O
PhOCHN
COOEt
COOEt
O
O
4
COOEt
OH
PhOCHN
PhOCHN
COOEt
COOEt
O
PhOCHN
1
3
2
path b
Scheme 1. Different pathways to obtain 3 from 1.
Table 4
Preparation of 3 by the bioreduction of 1 with different yeasts
Entry
Yeast
de (%)
ee syn (%)
ee anti (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
Kluyveromyces marxianus CBS 1553
Kluyveromyces marxianus var. lactis CL69
Pichia etchellsii MIM
Pichia glucozyma CBS 5766
Pichia pastoris CBS 2612
22 anti
20 anti
42 anti
30 anti
40 anti
66 anti
>99 anti
74 anti
>99 anti
16 syn
>99 (2R,3S)
80 (2R,3S)
59 (2R,3S)
9 (2S,3R)
20 (2S,3R)
>99 (2R,3S)
0
69 (2R,3S)
0
>99 (2R,3S)
>99 (2R,3S)
>99 (2R,3S)
82 (2R,3S)
41 (2S,3S)
10 (2S,3S)
47 (2R,3R)
42 (2S,3S)
43 (2S,3S)
52 (2R,3R)
48 (2S,3S)
38 (2R,3R)
10 (2S,3S)
43 (2S,3S)
68 (2S,3S)
59 (2S,3S)
__
Pichia henricii CBS 5765
Pachysolen tannophilus CBS 4044
Torulopsis magnoliae MIM 42
Torulopsis molischiana CBS 837
Saccharomyces cerevisiae Zeus
Torulopsis castelli MIM 1705
Sporobolomyces salmonicolor MIM
Lindnera fabiani CBS 5640
22 syn
26 syn
>99 syn
The ee and de were determined after complete conversion of 1 in 3 obtained in 72 h.
O
COOEt
O
PhOCHN
COOEt
1
H2; Pd/C
H2; Pd/C
HCOONH4
HCOONH4
MeOH/EtOH
EtOAc
COOEt
OH
COOEt
O
PhOCHN
NHCOPh
PhOCHN
O
COOEt
+
COOEt
3
6
4
Figure 4. Reduction of 2 with Pd/C and HCOONH2.
or EtOH) were used, a mixture of 3 and 3-benzamido-3-phenylpro-
panoate 6 was obtained.
The formation of 3 and 6 can be explained by the proposed
mechanisms in Scheme 2. Compound 4 is formed by hydrogena-
tion of 1 and can undergo subsequent alcoholysis to produce 3
(path a); alternatively alcoholysis can occur on the ethyl ester pres-
ent on the oxalyl residue, followed by decarboxylation and the
formation of 3 and the ethyl carbonate (path b).

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I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
H
R
O
COOEt
COOEt
O
O
O
PhOCHN
PhOCHN
OEt
COOEt
O
1
a
O
b
a
COOEt
OH
COOEt
O
PhOCHN
PhOCHN
OEt
O
O
+ ROCOCOOEt
3
O
H
R
O
COOEt
O
COOEt
PhOCHN
PhOCHN
OEt
O
b
O
CO2 + ROCOOEt
+
R
H
5
6
Scheme 2. Possible mechanism for the production of 3 and 6.
The biotransformations are carried out in a buffer so that the
production of 3 could be justified but the total absence of 6 shows
that the hypothesis mentioned above is the only one acceptable.
The biotransformation with Sporobolomyces salmonicolor was
optimized for improving the diastereoselectivity. Co-substrate type
and concentration, co-solvent type and concentration, substrate
concentration, and pH were chosen as control variables. Yields
and diastereoselectivities after 48 h were used as response targets.
Optimization was carried out by employing the MultisimplexÒ 2.0
software.41 S. salmonicolor completely converted 1 into (2R,3S)-
syn-3 within 48 h with a de >70% and an ee of 98% using DMSO
(2%) as the co-solvent, an imidazole buffer 0.1 M pH 7.8, xylose
as the co-substrate (50 g/L) and 4.2 g/L of substrate.
purification. 1H NMR spectra were recorded on a Bruker DRX Avance
300 MHz equipped with a non-reverse probe and on a Bruker DRX
Avance 400 MHz. HPLC analysis: Merck-Hitachi L-7100 equipped
with Detector UV-6000LP and Diacel Chiralpak AD. MS analyses
were performed by using a Thermo Finnigan (MA, USA) LCQ Advan-
tage system MS spectrometer with an electrospray ionisation source
and an ‘Ion Trap’ mass analyzer. The MS spectra were obtained by
the direct infusion of a sample solution in a mixture MeOH/H2O/
AcOH 10:89:1 under ionization, ESI positive. Literature data were
used for the assignment of the absolute configuration of the isomers
of N-benzoyl phenyl isoserine.42,16
4.1. Analytical methods
3. Conclusion
Analysis was performed on HPLC Merck-Hitachi L-7100 with
UV-6000LP Thermo Finnigan and CHIRALPACK AD (4.6 mm ꢁ
250 mm). The analysis of 1 and its products was performed with
a solvent system consisting of hexane and iPrOH in a ratio of
80:20. The flow-rate was 1.0 mL minꢀ1; injection volume was
Different approaches for the preparation of phenylisoserine, the
lateral chain of Taxol, were investigated by homogeneous asym-
metric catalysis using Ir(I)- and Ru(II)–phosphine complexes and
by an enzymatic approach with non-conventional yeasts. While
iridium complexes gave good diastereoselectivities, the enantiose-
lectivities were poor. Lewis acids added to ruthenium-diphos-
phines complexes in a 1:10 ratio gave good enantioselectivities.
The best results (up to 76% ee) were obtained using FeCl3 as the Le-
wis acid.
20
equipped with GC Trace (SE 52 column: length 25 m/a int.
0.32 mm, film 0.4–0.45 m) and MS analyses were performed by
lL. GC–MS spectra were recorded on Thermo Finnigan MD 800
l
using a Thermo Finnigan (MA, USA). The column temperature was
kept at 150 °C and increased by 8 °C minꢀ1 up to 270 °C.
Yeasts, with cell-associated esterase and carbonyl reductase
activities, allowed for the two-step, one-pot preparation of the syn-
thon (2R,3S)-N-benzoyl-3-phenyl isoserine ethyl ester with high
yields, furnishing higher yields and a shorter route than the previ-
ously reported chemoenzymatic synthesis of 3.
4.2. Synthesis of 1-benzamido-3-ethoxy-3-oxo-1-
phenylpropan-2-yl ethyl oxalate 1
This compound was prepared in accordance with a literature
procedure.16 1H NMR(CDCl3): d = 1.30 (t, CH3, J = 7.3 Hz); 1.32 (t,
CH3, J = 7.0 Hz); 4.29 (q,CH2, J = 7.3 Hz); 4.33 (q, CH2, J = 7.3 Hz);
7.44–7.68 (m, aromatic); 7.95 (d, aromatic, J = 6.4 Hz); 11.52 (s,
NH); 13C NMR (CDCl3): d = 14.3 (CH3), 14.4 (CH3), 62.1 (CH2), 64.1
(CH2), 128.4–134.0 (CH, aromatic), 133.0 (C@C), 133.44 (C@C),
156.7 (C@O, ester), 156.8 (C@O, ester), 162.5 (C@O, ester), 165.8
(C@O, amide); IR (film) 3274, 2995, 1972, 1906, 1781, 1740, 1699,
4. Experimental
Catalytic reactions were performed in a 100 mL glass autoclave
equipped with a magnetic stirrer. Unless otherwise stated, materials
were obtained from commercial source and used without further

I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
2115
1685, 1622 cmꢀ1; HRMS of C22H21NO7 (m/z): calcd 411.23, found
500 mL Erlenmeyer flasks containing 80 mL of the medium and
incubated for 48 h at 28 °C on a reciprocal shaker (100 spm). The
yeasts were grown on a malt extract with 5 g Lꢀ1 Difco yeast ex-
tract. Fresh cells from submerged cultures were centrifuged
(5000 rpm per 100) and washed with 0.1 M phosphate buffer pH
7 prior to use.
434.1 (M++Na).
4.3. Synthesis of ethyl 3-benzamido-2-oxo-3-phenylpropanoate
2
This compound was prepared in accordance with a literature
procedure.16 1H NMR (CDCl3): d = 1.35 (t, CH3, J = 7.0 Hz); 4.23 (q,
CH2, J = 5.5 Hz); 6.44 (d, CH, J = 6.6 Hz); 7.24 (br, NH), 7.26–7.54
(m, aromatic); 7.79 (d, aromatic, J = 6.6 Hz). 13C NMR (CDCl3):
d = 13.6 (CH3), 14.0 (CH3), 45.6 (CH2), 60.6 (CH), 127.4–134.6 (CH
aromatic), 159.9 (C@O, ester), 166.8 (C@O, carbonyl), 189.3 (C@O,
amide). HRMS of C18H17NO4 (m/z): calcd 311.12, found 334.2
(M++Na).
4.6. Bioreduction conditions
General procedure for the screening: reductions were carried
out in 10 mL screw-capped test tubes with a reaction volume of
3 mL with cells (20 g Lꢀ1, dry weight) suspended in 0.1 M phos-
phate buffer pH 7 containing 5% of glucose and 4 g Lꢀ1 of 3-ben-
zoylamino-3-phenyl-(ethyl, 2-oxalyl) propenoic acid ethyl ester
1. The reactions were carried out at 30 °C under magnetic stirring.
Samples (200 lL) were taken at intervals, extracted with an equal
4.4. General procedure for the asymmetric hydrogenation
volume of ethyl acetate and the organic layer was dried under
Na2SO4, filtered, and then evaporated. The extracts were analyzed
by thin-layer chromatography (CH2Cl2–diisopropylether = 2:8) and
by chiral HPLC and GC–MS.
In a Schlenk tube under argon, the substrate was added to the
catalyst, iridium or ruthenium complex, followed by 20 ml of sol-
vent, with or without an appropriate amount of Lewis base (TEA)
or acid. The solution was stirred for 15 min at room temperature
and then transferred with a cannula in an autoclave. The stainless
steel autoclave (200 mL), equipped with temperature control and
magnetic stirrer, was purged five times with hydrogen. After the
transfer of the reaction mixture, the autoclave was pressurized.
At the end the autoclave was vented and the mixture was analyzed
by NMR spectra and HPLC.
4.7. Optimisation of the biotransformation carried out with
Sporobolomyces salmonicolor MIM
The conditions of the sequential experimental trials were se-
lected employing the MultisimplexÒ 2.0 software (F. H. Walters,
L. R. Parker, S. L. Morgan, S. N. Deming, (1991) In Sequential Simplex
Optimization, Boca Raton: CRC Press). The control variables were
pH (5.5 < pH > 8), co-solvent (DMSO, CH3CN 1–2% v/v), substrate
1 concentration (0.5–5 g Lꢀ1) and co-substrate (glucose, xylose
30–70 g Lꢀ1). The three response variables were chosen for the
optimization: the molar conversion after 48 h, diastereomeric,
and enantiomeric excess. The biotransformations were carried
out in 25 mL flasks under magnetic stirring; optimization was per-
formed using 5 mL of total volume.
4.4.1. Preparation of Ir(COD)[(S)-BINAP]Cl
A solution of [Ir(COD)Cl2]2 (12 mg, 0.0236 mmol) and (S)-BINAP
(29.6 mg, 0.0475 mmol) in 8 mL of toluene was stirred at room
temperature for 4 h. The solution was concentrated under reduced
pressure and the yellow-orange solid obtained was washed with
hexane. The complex was dissolved in 0.7 mL of toluene/CDCl3
7:3 solution in a NMR tube, increasing the amount of TEA added
to the complex solution and analyzed by 31P NMR.
31P NMR of Ir(COD)[(S)-BINAP]Cl (CDCl3): d = ꢀ2.2 (d,
J = 18.3 Hz), ꢀ11.6 (d).
4.8. Reduction by Pd/C with ammonium formate
In a stainless steel autoclave (20 ml), equipped with temperature
control and a magnetic stirrer, was purged five times with hydro-
gen, a solution of 1 in ethyl acetate with 5% of Pd/C after which
1 equiv of ammonium formate was transferred. The autoclave was
pressurized at 10 atm and kept at room temperature. The reaction
was monitorated by GC–MS analysis. The mixture was filtered on
Celite and the solvent was evaporated in vacuo. A mixture of 4
and 6 was obtained. The products were isolated by preparative
TLC on silica gel using an eluent CH2Cl2–diisopropylether = 2:8.
31P NMR of Ir(COD)[(S)-BINAP]Cl with 10 equiv of TEA (CDCl3):
d = 1.0 (d, J = 21.4 Hz), 13.0 (d).
31P NMR of Ir(COD)[(S)-BINAP]Cl with 12.5 equiv of and heating
at 60 °C (CDCl3): d = 3.6 (d, J = 17.4 Hz), ꢀ1.2 (d).
4.4.2. Ethyl 3-benzamido-2-hydroxy-3-phenylpropanoate 3
syn-3: 1H NMR (CDCl3): d = 1.28 (t, CH3, J = 7.0 Hz); 4.28 (q, CH2,
J = 5.0 Hz); 4.63 (d, CH, J = 2.2 Hz); 5.75 (dd, CH, J = 1.8, J = 6.9 Hz);
7.0 (d, NH, J = 8.0); 7.23–7.59 (m, aromatic); 7.80 (d, aromatic,
J = 2.2 Hz). 13C NMR (CDCl3): d = 14.1 (CH3), 53.3 (CH), 61.8 (CH2),
71.4 (CH), 125.4–133.7 (CH aromatic), 169 (C@O, ester), 173.1
(C@O, amide).
4.8.1. Ethyl 3-benzamido-2-((ethoxycarbonyl)oxy)-3-
phenylpropanoate 4
1H NMR (CDCl3) d = 1.26 (t, CH3, J = 7.1 Hz), 1.39 (t, CH3,
J = 7.0 Hz), 4.28 (q, CH2, J = 5.0 Hz), 4.35 (q, CH2, J = 5.2 Hz), 5.47
(d, CH, J = 2.6 Hz), 5.98 (dd, CH, J1 = 2.6, J2 = 9.5 Hz), 7.14 (d, NH,
J = 9.1 Hz), 7.27–7.58 (m, aromatic), 7.77 (d, aromatic, J = 1.5 Hz).
13C NMR (CDCl3): d = 13.6 (CH3), 14.1 (CH3), 53.3 (CH), 61.8 (CH),
63.2 (CH2); 63.7 (CH2); 127.4–134.7 (CH, aromatic); 160.3 (C@O,
ester), 162.8 (C@O, ester) 168.4 (C@O, carbonyl); 171.2 (C@O,
amide). HRMS of C22H23NO7 (m/z): calcd 413.15, found 436.1
(M++Na).
anti-3: 1H NMR (CDCl3): d = 1.25 (t, CH3, J = 7.0); 4.14 (q, CH2,
J = 5.0 Hz); 4.68 (d, CH, J = 2.2 Hz); 5.60 (dd, CH, J = 1.8,
J = 6.9 Hz); 7.16 (d, NH, J = 8.0); 7.23–7.59 (m, aromatic); 7.83 (d,
aromatic, J = 2.2 Hz). 13C NMR (CDCl3): d = 13.9 (CH3), 53.3 (CH),
61.2 (CH2), 71.8 (CH), 125.4–133.6 (CH, aromatic), 168.6 (C@O, es-
ter), 173.1 (C@O, amide).
IR (film) 3348, 3063, 3033, 2980, 2962, 2904, 1960, 1985, 1780,
1718, 1644, 1109 cmꢀ1 C18H19NO4 Exact Mass: 313.13, found 336.2
(M++Na).
4.8.2. Ethyl 3-benzamido-3-phenylpropanoate 6
1H NMR (CDCl3) d = 1.17 (t, CH3, J = 7.0 Hz), 2.98 (t, CH2,
J = 3.0 Hz), 4.10 (q, CH2, J = 4.6 Hz), 5.63 (m, CH), 7.29–7.52 (m, aro-
matic), 7.83 (d, aromatic, J = 1.5 Hz). 13C NMR (CDCl3): d = 14.3
(CH3), 40.1 (CH2), 50.0 (CH), 61.1 (CH2), 126.5–139.8 (CH, aro-
matic); 160.0 (C@O, ester), 171.8 (C@O, amide). HRMS of
4.5. Microorganisms: culture conditions
Strains from official collections or from our collection (Microbi-
ologia Industriale Milano) were routinely maintained on a malt ex-
tract (8 g Lꢀ1, agar 15 g Lꢀ1, pH 5.5). In order to obtain cells for the
biocatalytic activity tests, the microorganisms were cultured in
C
18H19NO3 (m/z): calcd 297.14, found 192.2 (M+ꢀC6H5CO).

2116
I. Rimoldi et al. / Tetrahedron: Asymmetry 22 (2011) 2110–2116
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