Asymmetric synthesis of (R)-(-)-chlozolinate through a chemoenzymatic procedure

Article abstract of DOI:10.1016/S0957-4166(01)00040-4

A new asymmetric synthesis of (R)-chlozolinate 1, an important antifungal agent, based on the enzymatic asymmetrization of diethyl 2-benzyloxy-2-methylmalonate, is reported.

Full text of DOI:10.1016/S0957-4166(01)00040-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 271–277
Asymmetric synthesis of (R)-(−)-chlozolinate through a
chemoenzymatic procedure
Giuseppe Guanti,a,b,* Luca Banfi,a,b Katharine Powles,a Marcello Rasparini,a Carlo Scolasticoc and
Novella Fossatid
aDipartimento di Chimica e Chimica Industriale, via Dodecaneso 31, I-16146 Genoa, Italy
bC.N.R., Centro di Studio per la Chimica dei Composti Cicloalifatici ed Aromatici, via Dodecaneso 31, I-16146 Genoa, Italy†
cDipartimento di Chimica Organica e Industriale, via Venezian 21, 20133 Milan, Italy
dIsagro Ricerca S.r.l., Dipartimento di Biologia e Agronomia, Via Fauser 4, 28100 Novara, Italy
Received 4 December 2000; accepted 22 January 2001
Abstract—A new asymmetric synthesis of (R)-chlozolinate 1, an important antifungal agent, based on the enzymatic asymmetriza-
tion of diethyl 2-benzyloxy-2-methylmalonate, is reported. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
forms. Separation or resolution at the final product
stage was foreseen to be difficult, due to the absence of
suitable functionalities and the easy degradation of
chlozolinate in solution.
Botrytis cinerea is one of the worst and most wide-
spread fungal diseases in viticulture. The most useful
pesticides currently used in order to prevent the growth
of this microorganism belong to the dicarboximide
family. These products were found to control Botrytis
fairly well and additionally do not affect the alcoholic
fermentation process.1–5 Chlozolinate 16 is particularly
useful since it is rapidly degraded in soil and in wine.
This ensures the absence of possible toxic residues in
the final product, even when the statutory procedures
are not followed (Scheme 1).2
Thus, we decided to develop an asymmetric synthesis
based on the enzyme catalyzed asymmetrization of a
substituted prochiral malonate of the type 2–4.
There is only one literature report on asymmetrization
of unprotected malonate 2.7 The results, however, were
far from satisfactory in terms of enantiomeric excess
(46%) of the resulting monoester. More recently a
moderately good e.e. (up to 80.6% for the (R)-enan-
tiomer) has been achieved in the asymmetrization of a
series of 2-methyl-2-(2-nitrophenoxy)malonates.8 How-
ever, the aryl groups bonded to oxygen in that study
cannot be easily removed and thus are not suitable for
chlozolinate synthesis.
Until now, chlozolinate has been produced and sold
only in racemic form. In order to evaluate any differ-
ences in the biological behavior of its two enantiomers,
we needed to develop a practical way to prepare multi-
gram quantities of at least one of the two antipodal
Scheme 1.
* Corresponding author. E-mail: guanti@chimica.unige.it
†
Associated to the National Institute of the C.N.R. for the Chemistry of Biological Systems.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00040-4

272
G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
In the present study, we chose to employ malonates
protected at the tertiary hydroxyl with an easily remov-
able group. This strategy was adopted for two reasons:
firstly, we expected that the protecting group may
better differentiate the substituents at the prostereo-
genic center, thus allowing higher e.e.s to be achieved;
and secondly, it was felt that the presence of the
protecting group might facilitate the transformation of
monoesters 5–6 into chlozolinate.
more efficient than the previously known syntheses,
especially in the case of 10.
Table 1 reports the selected results of enzymatic asym-
metrization of diesters 3 and 4. The reactions were
quenched (except for entry 5) when the starting mal-
onate was no longer visible by TLC analysis. The
reactions usually tended to stop after mono-hydrolysis.
However, the lower yields obtained in some cases may
be due to the formation of significant quantities of
diacid (which was difficult to extract). As can be seen
from entry 1, in the case of compound 3 PLE gave,
under the usual conditions, poor results in terms of
enantioselection, as reported by Tamm for the unpro-
tected malonate 2.7 It should, however, be stressed that
in Tamm’s work the main enantiomer was probably
(S)-configured although the absolute configuration was
not demonstrated. By contrast, in this work, the (R)-
enantiomer was always preferentially formed.
2. Results and discussion
The required malonates 3 and 4 were prepared as
depicted in Scheme 2. The benzyl protected diethyl ester
49 was easily obtained from known 2-hydroxy-2-
methylmalonate 7,10,11 which is an intermediate for the
industrial synthesis of racemic chlozolinate.
For dimethyl malonate 3, which had never been pre-
pared before, we developed two new synthetic routes
involving only two or three steps from low cost, com-
mercially available, substrates (Scheme 2). The first step
involved, for both routes, the one-pot conversion of a
2-bromoacid into the 2-benzyloxy esters 8 and 10 by an
efficient procedure recently discovered in our laborato-
ries. This involved treating an alkoxide with sodium
bromoacetate, followed by reaction with an alkyl chlo-
roformate in the presence of a catalytic amount of
alkoxide. A mechanism for this conversion is proposed
in Scheme 3. Chloroformate forms a mixed anhydride,
which is then attacked by the catalytic alkoxide (in this
case sodium methoxide). During this reaction, methox-
ide is reformed, and thus it is truly catalytic. Although
compounds 812,13 and 1014 are known in the literature,
this one-pot procedure is convenient and comparable or
Since it is well known15 that the presence of co-solvents
can dramatically influence the enantioselectivity of PLE
catalyzed reactions, we tried to optimize the e.e. by
carrying out the reactions in mixed solvent systems
(entries 2–4). No significant improvements were seen.
We then explored other enzymes. However, of the
many tested (lipases from Candida anctarctica, Pseu-
domonas cepacia, Pig liver acylase Amano 30.000,
esterase from Mucor miehei), only lipase from Candida
cylindracea did react, although quite slowly, with low
e.e. A breakthrough was eventually made when we
tested horse liver acetone powder (HLAP) (entry 6),
which led to an encouraging e.e. of 89%.
Since for chlozolinate synthesis a monoethyl ester was
expected to be more useful, we also attempted mono-
Scheme 2.
Scheme 3.

G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
273
Table 1. Results of enzymatic asymmetrization of malonates 3–4 to give (R)-monoesters 5–6a
Entry
Subst.
Enzymeb
Bufferc
Co-solvent (%)
Time (h)
% Conversiond
% Yielde
% E.e.f
1
2
3
4
5
6
7
8
3
3
3
3
3
3
4
4
4
4
4
4
4
PLE
PLE
PLE
PLE
A
A
A
A
B
A
B
B
B
B
B
B
B
None
6
6
4.5
4.5
24
3
15
3
7
45
18
25
19
\95
\95
\95
\95
15
\95
\95
\95
\95
\95
\95
\95
\95
85
82
79
86
10
75
84
88
92
62
54
78
80
53
59
31
44
46
89
86
67
83
96
96
93
57
DMSO (10)
t-BuOH (10)
DMF (10)
None
None
None
t-BuOH (10)
DMF (10)
i-Pr2O (10)
EtOH (10)
n-Heptane (10)
None
CCL
HLAP
HLAP
HLAP
HLAP
HLAP
HLAP
HLAPg
BLAP
9
10
11
12
13
a All reactions were carried out at 20°C.
b PLE=pig liver esterase (600 U/mmol of substrate); CCL=Candida cylindracea lipase (300 mg/mmol of substrate); HLAP=horse liver acetone
powder (500 mg/mmol of substrate); BLAP=bovine liver acetone powder (500 mg/mmol of substrate).
c A=phosphate buffer, pH 7; B=Tris buffer, pH 7.5.
d Means the percentage of initial diester which has reacted.
e Isolated yields after chromatography.
f Determined by 1H NMR in the presence of (−)-ephedrine. The (R)-enantiomer was in all cases predominant.
g For preparative purposes 250 mg/mmol of substrate were used.
Scheme 4.
finally because it is highly likely that HLAP shows the
same enantiopreference for both ethyl and methyl
esters.
hydrolysis of 4 with HLAP. Interestingly (entry 7) the
expected decrease in the reaction rate on changing from
a methyl to an ethyl diester was not dramatic, and the
e.e. was still good. We further optimized this reaction
for an ethyl diester substrate. In this case, the addition
of co-solvents had a remarkable effect on the rate of
reaction, the product e.e. and the substrate selectivity
(entries 8–12). For example, the presence of tert-BuOH
increased the rate but decreased the enantioselectivity.
The opposite effect was seen when iso-Pr2O was added.
Ethanol gave a very good e.e., but substrate selectivity
(and thence the yield) dropped significantly because
substantial amounts of diacid formed. The best co-sol-
vent was found to be n-heptane, which afforded a high
e.e. (93%) together with good yield and workable reac-
tion times.
We then studied the conversion of mono-acid 6 into
(R)-chlozolinate (Scheme 4). Our plan involved trans-
formation of the free carboxylic acid into an active
ester. The choice of active ester was not obvious
because it was required to be active enough to be
completely differentiated from the ethyl ester, but also
be stable under the conditions required for benzyl ether
deprotection by hydrogenolysis. After various experi-
ments involving substituted phenyl esters, we found
that the simple phenyl ester itself fulfilled all of our
requirements, and hydrogenolysis of 11 took place
without side-reactions to give 12. Treatment of the
latter with 3,5-dichlorophenyl isocyanate1 in the pres-
ence of excess Et3N afforded (R)-chlozolinate 1 in good
yield. No analogous phenyl ester (from competitive
cyclization onto the ethyl ester) was observed. 1H NMR
analysis of (R)-1 in the presence of Eu(hfc)3 showed (by
comparison with a racemic sample) that no racemiza-
tion had occurred during the sequence from 6 to 1.
Moreover, crystallization from absolute ethanol was
found to increase the e.e. to ]96%.
The absolute configuration of 6 was unambiguously
established as (R) by [h]D correlation with (R)- and
(S)-6, that have been previously obtained by resolution
and correlated with citramalic acid.9,16 In the case of 5
the absolute configuration is also thought to be (R) on
the basis of similar NMR behavior in the presence of
(−)-ephedrine, on the same sign of specific rotation, and

274
G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
(R)-Chlozolinate 1 was then tested on two strains of
Botrytis cinerea (IR004 and IR058). The former is
sensitive to dicarboximide pesticides while the second
one is resistant. As a control the same tests were carried
out on racemic chlozolinate. The results, shown in
Table 2, indicate no significant difference in activity
between the two compounds. These data seem to sug-
gest that the two enantiomers interact with the fungus
equally well.
(10.01 g, 52.6 mmol) in dry DMF (10 mL) was added
dropwise over 30 minutes.
After stirring the mixture for 15 minutes, benzyl bro-
mide (6.40 mL, 52.6 mmol) was added and the mixture
was stirred at room temperature for 5 hours, then
quenched with H2O/saturated aqueous NH4Cl 1:1 (200
mL) and extracted with PE/Et2O 1:1 (3×60 mL). The
organic layer was then washed with water (100 mL).
After drying (Na2SO4) and evaporation, the crude
product was finally purified by silica chromatography
with PE/Et2O 75:25. The main product band to elute
was evaporated to afford 4 as an oil (12.84 g, 87%).
In conclusion an efficient asymmetric synthesis of (R)-
(−)-chlozolinate was developed, based on an enzymatic
asymmetrization step catalyzed by horse liver acetone
powder.
Rf=0.75 (Et2O/PE 1:1+2% CH3COOH). Anal. found:
C, 64.2; H, 7.2%. C15H20O5 requires: C, 64.27; H,
7.19%. GC–MS: Rt=6.76 min; m/z: 207 (M+−73,
0.6%), 174 (35.5), 128 (31.6), 100 (19.7); 92 (8.9), 91
3. Experimental
1
(100), 65 (8.5), 43 (9.4). H NMR: l 7.46–7.22 [5H, m,
1H and 13C NMR spectra were taken in CDCl3, at 200
and 50 MHz, respectively. Chemical shifts are reported
in ppm (l scale), using TMS as an internal standard. In
ABX systems, the proton A is considered downfield
and B upfield. GC–MS was carried out on an HP-
5971A instrument, using an HP-1 column (12 m long,
0.2 mm wide), electron impact at 70 eV, a mass temper-
ature of about 167°C and starting the mass range from
m/z=33. Analyses were performed with a constant He
flow of 0.9 mL/min, starting at 100°C for 2 min and
then raising the temperature by 20°C/min; Rt are mea-
sured in minutes from injection. IR spectra were mea-
sured with a Perkin–Elmer 881 instrument. TLC
analyses were carried out on silica gel plates, which
were developed by UV or by dipping into a solution of
(NH4)4MoO4 tetrahydrate (21 g) and Ce(SO4)2 tetra-
hydrate (1 g) in H2SO4 (31 mL) and H2O (469 mL) and
warming. Rf values were measured after an elution of
7–9 cm. Chromatography was carried out on 220–400
mesh silica gel using the ‘flash’ methodology. Petroleum
ether (40–60°C) is abbreviated as PE. All reactions
employing dry solvents were carried out under a nitro-
gen atmosphere. Candida cylindracea lipase, Pig liver
esterase, Horse liver acetone powder and Bovine liver
acetone powder were purchased from Sigma and used
as received.
ArH]; 4.67 [2H, s, CH2Ph]; 4.25 [4H, q, CH2CH3,
J=7.3 Hz]; 1.72 [3H, s, CCH3]; 1.29 [6H, t, CH2CH3,
J=7.3 Hz].
3.2. Preparation of methyl 2-(benzyloxy)acetate 8
Sodium hydride (60% suspension in mineral oil, 8.47 g,
212 mmol) was placed in a two-necked flask equipped
with a dropping funnel. It was washed twice with dry
n-hexane (20 mL) under nitrogen, suspended in dry
DMF (90 mL) and cooled to 0°C. A solution of benzyl
alcohol (10 ml, 96.6 mmol) in dry DMF (20 mL) was
then added over 20 min through a dropping funnel.
After stirring for a further 15 min, a solution of bro-
moacetic acid (13.51 g, 97.2 mmol) in dry DMF (20
mL) was added over 20 min through the dropping
funnel. The suspension was stirred for 30 min at 0°C,
then for 3 h at rt, and a further 2 h at 60°C. After
cooling again to 0°C, methyl chloroformate (7.5 mL, 97
mmol) was added over 15 min and the mixture stirred
for 10 min. Sodium methoxide (500 mg, 9.3 mmol) was
added. The suspension was allowed to warm to room
temperature and stirred overnight. It was then cau-
tiously poured into a mixture of saturated aqueous
NH4Cl (100 mL)+H2O (100 mL). Three extractions
with Et2O, followed by washing of the ethereal extract
with brine gave, after drying (Na2SO4) and evaporation
to dryness, a crude oil which was distilled to afford
pure 8 as a colorless liquid (12.19 g, 70%). Bp 85–90°C
(at 0.6 mbar); Rf=0.67 (Et2O/PE 4:6). Anal. found: C,
66.45; H, 6.8%. C10H12O3 requires: C, 66.65; H, 6.71%.
GC–MS: Rt=4.52 min; m/z: 180 (M+, 0.55); 121 (3.2);
107 (77.7); 105 (3.0); 92 (9.2); 91 (100); 89 (4.8); 79
3.1. Preparation of diethyl 2-(benzyloxy)-2-methyl-
malonate 4
To a suspension of NaH (60% in mineral oil) (2.11 g,
52.6 mmol) in dry DMF (80 mL) cooled in an ice bath,
a solution of diethyl 2-hydroxy-2-methylmalonate 7
Table 2. Activity of (R) and racemic 1 toward Botrytis cinerea strains
IR004 (sensible strain)
IR058 (resistant strain)
Inhibition % (R)-1 Inhibition % (R,S)-1
Dose (ppm)
Inhibition % (R)-1
Inibition % (R,S)-1
Dose (ppm)
1
0.5
0.25
0.125
84
50
12
5
86.5
65.5
13.5
5.5
50
25
12.5
6.25
96.5
90.5
71.5
9.5
95.5
93.5
77.5
19

G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
275
(11.3); 77 (5.9); 74 (41.6); 65 (16.0); 51 (5.2); 43 (22.3);
39 (6.4). 1H NMR: l 7.40–7.25 [5H, m, ArH]; 4.64 [2H,
s, CH2Ph]; 4.11 [2H, s, CH2CO2Me]; 3.76 [3H, s,
OCH3].
times with Et2O/PE 1:1 and once with AcOEt. After
drying (Na2SO4) and evaporation to dryness, the crude
product was chromatographed (PE/Et2O 6:4) to give
pure 3 as an oil (1.605 g, 69%).
3.3. Preparation of dimethyl 2-(benzyloxy)malonate 9
Method B: A solution of lithium di-iso-propylamide in
THF/n-hexane was prepared under Ar at −15°C from
n-BuLi (1.6N in n-hexane, 4.0 mL, 6.4 mmol),
(i-Pr)2NH (1.00 mL, 7.1 mmol) and dry THF (11 mL).
To this solution, cooled to −78°C, was added a solution
of ester 10 (1.04 g, 5.4 mmol) in THF (20 mL) dropwise
over 5 min. After 15 min, methyl chloroformate (0.50
mL, 6.5 mmol) was added. After 30 min, saturated
NH4Cl (15 mL) was added. After warming to rt, the
mixture was extracted with AcOEt. Purification as
above gave pure 3 as an oil (737 mg, 54%).
A solution of lithium di-iso-propylamide in THF/n-
hexane was prepared under Ar, at −15°C, from n-BuLi
(1.6 M in n-hexane, 14.5 mL, 23.2 mmol), (i-Pr)2NH
(3.70 mL, 26 mmol) and dry THF (28 mL). To this
solution, cooled to −78°C, a solution of ester 8 (3.22 g,
17.9 mmol) in THF (5 mL) was added dropwise over 5
min. In the meantime, methyl chloroformate (1.80 ml,
23.3 mmol) was dissolved under Ar in dry THF (20
mL) and cooled to −78°C in another flask, equipped
with a dropping funnel. The dropping funnel was
cooled by an acetone/dry ice mantle at −78°C. After
complete addition of 8 to LDA (in 15 min), the result-
ing solution was transferred into the cooled dropping
funnel and added over 5 min to the chloroformate
solution. After 30 min, saturated NH4Cl (100 mL) was
added. After warming to rt, the mixture was extracted
with Et2O. After washing with brine, drying (Na2SO4)
and evaporation to dryness, the crude product was
chromatographed (PE/Et2O 6:4“1:1) to give pure 9 as
an oil (2.50 g, 59%). Rf=0.42 (Et2O/ETP 4:6). Anal.
found: C, 60.25; H, 6.1%. C12H14O5 requires: C, 60.50;
H, 5.92%. GC–MS: Rt=6.28 min; m/z: 179 (M+−59,
0.21%); 132 (77.3); 107 (14.5); 101 (8.6); 100 (46.3); 91
(100); 79 (6.6); 77 (4.5); 74 (5.6); 69 (14.0); 65 (15.3); 59
Rf=0.37 (Et2O/ETP 4:6). Anal. found: C, 61.65; H,
6.50%. C13H16O5 requires: C, 61.90; H, 6.39%. GC–MS:
Rt=6.27 min; m/z: 193 (M+−59, 0.44%); 146 (46.1); 114
(44.0); 91 (100); 86 (13.3); 65 (12.0); 43 (10.1). 1H
NMR: l 7.46–7.25 [5H, m, ArH]; 4.65 [2H, s, CH2Ph];
3.78 [6H, s, OCH3]; 1.73 [3H, s, CH3]. 13C NMR: l
169.82 [CꢀO]; 137.51 [quat. aromatic]; 128.28, 127.94,
127.74 [aromatic CH]; 81.94 [C-OBn]; 68.52 [CH2Ph];
52.76 [OCH3]; 21.24 [CH3-C-O].
3.6. Preparation of (R)-2-benzyloxy-2-methylmalonic
acid monomethyl ester 5
Diester 3 (305 mg, 1.21 mmol) was suspended in pH 7.5
Tris buffer (60 mL) (prepared dissolving 3.64 g of
Tris–base in 50 mL of H2O, adjusting to pH 7.5 with
1N HCl, and finally diluting to 100 mL). Horse liver
acetone powder (300 mg) was then added and the
suspension was stirred at rt for 6 h. It was then acidified
with 0.5 M citric acid to pH 3, saturated with NaCl,
and filtered through a Celite pad washing with Et2O.
The phases were separated and the aqueous extracted
three times with Et2O always keeping pH<3. In order to
prevent the build-up of emulsions a few drops of EtOH
were added. After drying (Na2SO4) and evaporation to
dryness, chromatography (PE/Et2O 1:1“PE/Et2O/
AcOH 29:69:2) gave pure monoacid 5 as an oil (217
mg, 75%). Rf=0.23 (Et2O/PE/AcOH 39:59:2). [h]D:
+5.27 (c 2.1, CHCl3). Anal. found: C, 60.7; H, 6.05%.
C12H14O5 requires: C, 60.50; H, 5.92%. 1H NMR: l
7.42–7.30 [5H, m, ArH]; 4.62 [2H, s, CH2Ph]; 3.81 [3H,
s, OCH3]; 1.79 [3H, s, CH3-C]. 13C NMR: l 172.58,
169.22 [CꢀO]; 136.89 [quat. aromatic], 128.43, 128.05,
127.96 [aromatic CH]; 81.76 [C-OBn]; 68.60 [CH2Ph];
53.10 [CH3O]; 20.66 [CH3-C-O].
1
(5.6); 39 (5.2). H NMR: l 7.40–7.25 [5H, m, ArH];
4.70 [2H, s, CH2Ph]; 4.57 [1H, s, CH-CO2Me]; 3.79
[6H, s, OCH3].
3.4. Preparation of methyl 2-(benzyloxy)propanoate 10
Prepared using the same methodology employed for
ester 8, this time using 2-bromopropanoic acid instead
of bromoacetic acid. The crude product was purified by
chromatography (PE/Et2O 6:4). Yield: 64% (oil). Rf=
0.44 (Et2O/ETP 4:6). Anal. found: C, 68.1; H, 7.4%.
C11H14O3 requires: C, 68.02; H, 7.27%. GC–MS: Rt=
4.53 min; m/z: 194 (M+, 0.07%); 135 (3.0); 107 (9.9); 91
(100); 88 (55.7); 65 (8.8); 57 (5.6). 1H NMR: l 7.40–7.25
[5H, m, ArH]; 4.70 [2H, AB syst., CH2Ph, JAB 11.7 Hz];
4.07 [1H, q, CH-CH3, J=6.9 Hz]; 3.75 [3H, s, OCH3];
1.44 [3H, d, CH3-CH, J=6.9 Hz].
3.5. Preparation of dimethyl 2-(benzyloxy)-2-methyl-
malonate 3
Method A: Dry t-BuOH (50 ml) was placed under N2
in a two-necked flask equipped with a dropping funnel.
n-BuLi (1.6 M in hexanes, 6.75 mL, 10.8 mmol) was
added. After 5 min, a solution of ester 9 (2.20 g, 9.25
mmol) in t-BuOH (30 mL) was added. During the
additions, the flask was externally water-cooled when
needed. After 15 min, methyl iodide (675 mL, 1.08
mmol) was added. After stirring for 6 h at rt, the
reaction was quenched with saturated aqueous NH4Cl
(60 mL). Most of the t-BuOH was evaporated under
reduced pressure. Then the mixture was extracted three
1
E.e. was measured by H NMR in the presence of 1.5
equiv. of (−)-ephedrine. Under these conditions the
singlets given by OCH3 were split with a Dl of 0.037
ppm. Integration gave an e.e. of 89%.
3.7. Preparation of (R)-2-benzyloxy-2-methylmalonic
acid monoethyl ester 6
A solution of diester 4 (1.04 g, 3.71 mmol) in n-heptane
(10 mL) was treated with pH 7.5 Tris buffer (100 mL)

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G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
(prepared as described above), and with horse liver
acetone powder (1.02 g). The two-phase suspension was
stirred at rt for 25 h. It was then acidified with 0.5 M
citric acid to pH 3, saturated with NaCl, and filtered
through a Celite pad washing with Et2O. The phases
were separated and the aqueous extracted three times
with Et2O, keeping pH<3. In order to prevent the
build-up of emulsions a few drops of EtOH were
added. After drying (Na2SO4) and evaporation to dry-
ness chromatography (PE/Et2O 6:4“PE/Et2O/AcOH
39:59:2) gave pure monoacid 6 as an oil (730 mg, 78%).
Rf=0.30 (Et2O/PE/AcOH 49:49:2). [h]D: +9.54 (c 2.2,
EtOH); +7.13 (c 2.2, CHCl3) (lit.: +9.65, c=2, EtOH).16
Anal. found: C, 62.1; H, 6.4%. C13H16O5 requires: C,
3.9. Preparation of (S)-2-hydroxy-2-methylmalonic acid
ethyl ester phenyl ester 12
A solution of phenyl ester 11 (1.005 g, 3.06 mmol) in
96% ethanol (50 mL) containing AcOH (0.5 mL) was
hydrogenated using 10% Pd/C (200 mg) for 7.25 h.
After removal of the catalyst and evaporation, the
crude product was chromatographed (PE/AcOEt
85:15“6:4) to give pure 12 as an oil (569 mg, 78%).
Rf=0.55 (PE/Et2O 1:1). [h]D: −0.45 (c 2.1, CHCl3).
Anal. found: C, 60.45; H, 5.85%. C12H14O5 requires: C,
60.50; H, 5.92%. GC–MS: Rt=8.94 min; m/z: 210
(M+−28, 2.8%); 165 (1.21); 95 (14.7); 94 (100); 77 (5.4);
1
66 (4.4); 65 (5.1); 51 (3.3); 43 (51.8); 39 (5.4). H NMR:
1
61.90; H, 6.39%. H NMR: l 7.40–7.25 [5H, m, ArH];
l 7.40 [2H, t, H meta to O, J=7.8 Hz]; 7.26 [1H, t, H
para to O, J=7.3 Hz]; 7.09 [2H, d, H ortho to O, J=8.0
Hz]; 4.35 [2H, q, CH2-CH3, J=7.1 Hz]; 3.92 [1H, s,
OH]; 1.78 [3H, s, CH3-C]; 1.35 [3H, t, CH3-CH2]. 13C
NMR: l 170.79, 169.39 [CꢀO]; 150.32, 129.57, 126.40,
120.93 [aromatics]; 76.27 [C-OH]; 62.85 [CH3-CH2];
21.62 [CH3-CH2]; 14.10 [CH3-C-OH]. IR (CHCl3): wmax
3527, 2986, 2940, 1743, 1590, 1478, 1445, 1379, 1261,
1159, 1106, 1015, 956 cm−1.
4.61, 4.59 [2H, AB syst., CH2Ph, JAB 12.7 Hz]; 4.29
[2H, q, CH2-CH3, J=7.2 Hz]; 1.79 [3H, s, CH3-C]; 1.32
[3H, t, CH2CH3, J=7.3 Hz]. 13C NMR: l 173.59,
168.72 [CꢀO]; 136.97 [quat. aromatic]; 128.20, 127.76
[aromatic CH]; 81.68 [C-OBn]; 68.40 [CH2Ph]; 62.20
[CH3CH2O]; 20.68 [CH3-C-O]; 13.79 [CH3CH2O].
1
E.e. was measured by H NMR in the presence of 1.5
equiv. of (−)-ephedrine. Under these conditions the
signals given by CH2CH3 were splitted with a Dl of
0.034 ppm. The signal of the (R)-enantiomer was
downfield. Relative integration was done irradiating the
CH2CH3 signal at 1.27 ppm. In this way the CH2CH3
signals become sharp singlets. Also, the signals due to
CH3-CH2 are split (Dl=0.038 ppm; (R)-enantiomer
downfield). They can be integrated as well upon irradi-
ation at 4.23 ppm. However, traces of BHT coming
from the solvents used for chromatography can alter
the measurement. In this case e.e. was 93%.
3.10. Preparation of (R)-chlozolinate 1
A solution of alcohol 12 (1.088 g, 4.56 mmol) in dry
n-hexane (60 mL) was treated, under N2, with triethyl-
amine (0.636 ml, 4.56 mmol) and 3,5-dichloro-
phenylisocyanate 131 (1.20 g, 6.38 mmol). After stirring
at rt for 30 min, the mixture was refluxed overnight.
After cooling, the suspension was filtered and the
filtrate evaporated to dryness. Trituration with absolute
1
ethanol gave 1 as a solid (987 mg, 65%), pure by H
NMR and TLC, mp 127.2–130.0°C, [h]D: −14.9 (c 2.0,
CHCl3). Recrystallization from EtOH gave a purer
sample (691 mg) with mp=135.5–136.1°C and [h]D=
−16.79 (c 2.0, CHCl3). The mother liquors had [h]D=
−11.1. The enantiomeric excess of the sample obtained
just after trituration was determined by 1H NMR in the
presence of Eu(hfc)3 by observation of the split CH3-C
signals, and showed an e.e. of 90%. In the case of the
recrystallized sample, the signal of the minor enan-
tiomer was no longer visible. Thus, we estimated an e.e.
of ]96%. This compound was found to be identical to
3.8. Preparation of (S)-2-benzyloxy-2-methylmalonic
acid ethyl ester phenyl ester 11
A solution of monoester 6 (982 mg, 3.89 mmol) in
CH2Cl2 (8 mL), phenol (440 mg, 4.68 mmol) and
4-dimethylaminopyridine (48 mg, 0.39 mmol) was
cooled in an ice bath and a solution of dicyclohexylcar-
bodiimide (964 mg, 4.67 mmol) in CH2Cl2 (23 mL) was
added. The solution was stirred at 0°C for 30 min then
at room temperature for 5 h, then filtered and concen-
trated under reduced pressure. The residue was purified
by chromatography (PE/Et2O 95:5“7:3) to give pure
11 (1.03 g, 81%) as an oil. Rf=0.42 (PE/Et2O 85:15).
[h]D: +9.46 (c 1.65, CHCl3). Anal. found: C, 69.35; H,
6.1%. C19H20O5 requires: C, 69.50; H, 6.14%. GC–MS:
Rt=8.94 min; m/z: 255 (M+−73, 0.04%); 222 (8.5); 184
(6.8); 128 (5.2); 94 (28.2); 92 (10.1); 91 (100); 65 (10.7);
43 (8.9). 1H NMR: l 7.50–7.20 [8H, m, ArH]; 7.09 [2H,
d, H ortho to O, J=8.2 Hz]; 4.84, 4.75 [2H, AB syst.,
CH2OPh, JAB=11.1 Hz]; 4.32 [2H, q, CH2-CH3, J=7.1
Hz]; 1.87 [3H, s, C-CH3]; 1.34 [3H, t, CH2-CH3, J=7.2
Hz]. 13C NMR: l 168.94, 168.24 [CꢀO]; 150.36, 137.59
[quat. aromatics]; 129.54, 128.37, 127.85, 127.82,
126.26, 121.16 [aromatic CH]; 82.09 [C-OBn]; 68.78
[CH2Ph]; 62.15 [CH3CH2O]; 21.46 [CH3-C-O]; 14.15
[CH3CH2O]. IR (CHCl3): wmax 3023, 1745, 1591, 1185,
1138, 1100, 1019 cm−1.
1
an authentic sample of the racemate by TLC, MS, H
NMR and IR. GC–MS: Rt=8.16 min; m/z: 333
(11.6%); 331 (M+, 17.7); 261 (18.4); 259 (28.2); 216
(5.23); 190 (13.4); 189 (15.5); 188 (32.8); 187 (22.2); 186
(24.4); 124 (11.0); 43 (100). 1H NMR: l 7.45, 7.26
[2×1H, 2s, aromatics]; 4.34 [2H, q, CH2-CH3, J=7.2
Hz]; 1.91 [3H, s, CH3-C]; 1.34 [3H, t, CH3–CH2, J=7.2
Hz].
Acknowledgements
We wish to thank Isagro Ricerca, University of
Genoa, and M.U.R.S.T. (COFIN 98) for financial
assistance.

G. Guanti et al. / Tetrahedron: Asymmetry 12 (2001) 271–277
277
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