Enzyme-catalyzed preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate: A key intermediate for (R)-metalaxyl

Article abstract of DOI:10.1016/j.tetasy.2005.01.033

A biocatalytic approach for the production of (R)-metalaxyl, mefenoxam, has been developed. A practical synthesis of methyl (R)-N-(2,6-dimethylphenyl) alaninate, a key intermediate for (R)-metalaxyl, has been developed by the use of lipase-catalyzed hydrolytic kinetic resolution and chemical racemization of the remaining ester. At high concentrations in aqueous media (300 g/L) lipases were stable and gave moderate to good conversions and excellent enantioselectivities (>98% ee). A simple extraction procedure was used to separate the acid product from the remaining ester and the acid was esterified with methanol to give methyl (R)-N-(2,6-dimethylphenyl)alaninate without any reduction in enantiomeric excess (>98% ee). Subsequent chemical coupling with methoxyacetyl chloride provided enantiomerically pure (R)-metalaxyl (>98% ee) without racemization.

Full text of DOI:10.1016/j.tetasy.2005.01.033

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 1221–1225
Enzyme-catalyzed preparation of methyl (R)-N-(2,6-
dimethylphenyl)alaninate: a key intermediate for (R)-metalaxyl
Oh-Jin Park,a,* Sang-Hyun Lee,a Tae-Yoon Park,a Sang-Who Leeb and Koon-Ho Chob
aLG Chem, Ltd/Research Park, 104-1 Moonji-dong, Yusung-gu, Daejon 305-380, Republic of Korea
bLG Life Sciences Ltd/Research Park, 104-1, Moonji-dong, Yusung-gu, Daejon 305-380, Republic of Korea
Received 3 January 2005; accepted 25 January 2005
Abstract—A biocatalytic approach for the production of (R)-metalaxyl, mefenoxam, has been developed. A practical synthesis of
methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-metalaxyl, has been developed by the use of lipase-catalyzed
hydrolytic kinetic resolution and chemical racemization of the remaining ester. At high concentrations in aqueous media (300 g/L)
lipases were stable and gave moderate to good conversions and excellent enantioselectivities (>98% ee). A simple extraction proce-
dure was used to separate the acid product from the remaining ester and the acid was esterified with methanol to give methyl (R)-N-
(2,6-dimethylphenyl)alaninate without any reduction in enantiomeric excess (>98% ee). Subsequent chemical coupling with
methoxyacetyl chloride provided enantiomerically pure (R)-metalaxyl (>98% ee) without racemization.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1.Introduction
transformations. Enantioselective hydrolysis of esters
and enantioselective esterification of carboxylic acids
using hydrolases are among the most commonly used
methods for the preparation of enantiomerically pure
alcohols, acids, and esters. Among various commercial
enzymes, lipases and esterases are attractive in terms
of ready availability, no need of cofactors, high stability,
and activity in organic media. Lipase-catalyzed hydro-
lytic kinetic resolutions are still popular for manufactur-
ing chiral chemicals.6
A large number of chiral chemicals in the pharmaceuti-
cal sector, and increasingly also in the agrochemical and
nonlife science sectors, contain at least one stereogenic
center.1 Chiral chemicals are gaining increasing atten-
tion in the context of biological activity.2,5 This has
led to the discovery of a wide range of chiral techno-
logies, such as large-scale chiral chromatography and
catalytic asymmetric reactions and resolutions, both
chemical and enzymatic. Catalytic asymmetric synthesis
has distinct advantages over stoichiometric reactions for
economic and environmental reasons. Resolution ap-
proaches have played a central role in the production
of optically active compounds despite the inherent
disadvantage of a maximum yield of 50% based on a
racemic starting material. Classical resolution uses
stoichiometric amounts of a chiral resolving agent. Ki-
netic resolutions employing chiral catalysts or reagents
to produce enantiomerically enriched forms must always
be evaluated against any asymmetric synthesis.1 En-
zymes have been used to catalyze reactions with high
chemo-, regio-, and stereoselectivity. Furthermore, en-
zyme-catalyzed reactions are less hazardous, polluting,
and energy-intensive than conventional chemistry-based
Metalaxyl is a fungicide, which is effective in the control
of phytopathogenic fungi and one of the few pesticides
marketed in a single enantiomer formulation (as well
as a racemic formulation). The biological activity resides
with (R)-metalaxyl, mefenoxam, (R)-1 (Fig. 1).2
Currently racemic metalaxyl is being replaced in many
OMe
N
O
O
OMe
(R)-1 [(R)-Metalaxyl]
Figure 1. Structure of (R)-metalaxyl.
*
Corresponding author. Fax: +8242861 3647; e-mail:
lgchem.com
parko@
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.01.033

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O.-J. Park et al. / Tetrahedron: Asymmetry 16 (2005) 1221–1225
Table 1. Enzyme screening for enantioselective hydrolysis of racemic
ester 3
countries by metalaxyl-M, the product enriched with the
(R)-enantiomer, although the use of generic racemic
metalaxyl may continue in some countries. Metalaxyl-
M typically consists of 97.5% of the (R)-isomer and
2.5% of (S)-isomer. Asymmetric catalytic hydrogenation
of prochiral imine and enamide was reported to give
(R)-1 of 95.6% ee, which is not satisfactory in terms of
enantiomeric excess.3–5 Herein we report a practical
lipase-catalyzed synthesis of optically active methyl
(R)-N-(2,6-dimethylphenyl)alaninate (R)-3, a key inter-
mediate for (R)-metalaxyl, mefenoxam, (R)-1 (>98% ee).
Enzyme
Source
Conversion (%)
1 day
2days
Alcalase
Lipase PS
Bacillus licheniformis
Burkholderia cepacia
Candida antarctica
Candida rugosa
Alcaligenes sp.
60
50
83
56
—
57
76
65
—
—
Novozym 435
Lipase OF
100
47
Lipase QLM
Acylase Amano
Protease PS
PLE-AL Amano
60
51
Aspergillus melleus
Bacillus sp.
Porcine liver
100
100
The conversion of the ester was analyzed by HPLC on a C18 reverse
phase column.
2.Results and discussion
Readily accessible racemic methyl ester 3 was used in the
preliminary enzyme screening with enzymes chosen from
the collection of more than 20 hydrolytic enzymes (lip-
ases, esterases, and proteases), which are known to
hydrolyze ester bonds. The hydrolytic enzyme reactions
were performed by incubating a racemic substrate
(50 mg) with enzymes (25 mg) in a 0.1 M phosphate buf-
fer (pH 7.0) at 30 ꢁC. Reactions were stopped at inter-
vals to check the conversion and enantiomeric excess
of the remaining ester and the product acid 2. Although
under the enzyme screening the substrate was suspended
in the aqueous mixture (the substrate esters are liquid),
the reactions proceeded because of lipophilicity of the
lipases. There was no reaction with organic solvents
added. Determination of the enantiomeric excess of
the remaining ester and the acid product was carried
out with HPLC on a Chiralcel OD column. The reduced
reaction rate after 50% conversion indicated the selecti-
vity of the enzyme used (Table 1). Separate tests of
enzyme screening with methyl (R)-N-(2,6-dimethyl-
phenyl)alaninate (R)-3 and methyl (R)-N-(2,6-dimethyl-
phenyl)alaninate (S)-3 showed that Lipase PS had
preference for (R)-3.
remaining ester 3 [enriched as (S)-3] were extracted with
ethyl acetate. These two compounds can be easily sepa-
rated by partitioning in ethyl acetate or toluene/H2O.
This simple extraction/partition procedure is especially
convenient and suitable for a large-scale operation.
The remaining ester can be recycled by chemical racemi-
zation. Racemization was achieved through treatment
with acid–butyraldehyde mixture in toluene (Scheme 1).
For a large-scale preparation, the activity of Lipase PS
with methyl ester 3 was still too low. In some cases,
the reaction rate and substrate specificity depend on
the substituents by partially modifying the structure of
the substrate.7,8 We prepared various racemic esters 4–
7. Their syntheses were achieved by esterification of
racemic acid chloride with alcohols. Under the substrate
screening with Lipase PS, the reaction rates highly de-
pend on the kind of racemic esters employed (Table
2). These interesting results indicate that an electronega-
tive moiety can increase the reaction rates. However,
ester substituents do not influence the enantiomeric
excess significantly in this case. After 50% conversion,
the enantiomeric excess of the acid product decreases
as observed in other enzyme-catalyzed kinetic resolu-
tions. 2-Chloroethyl ester 4, allyl ester 5, methoxyethyl
ester 6, and ethoxyethyl ester 7 all gave higher reaction
rate than methyl ester 3.
Lipase PS was chosen from the enzyme screening for
further study. Lipase PS from Burkholderia cepacia,
one of the most frequently employed lipases in kinetic
resolutions, was selected based on enantiopreference
and its readily availability. Lipase PS preparation con-
tains insoluble inorganic Celite and active proteins. Sep-
arate preparation of (R)-3 and (S)-3, and reaction of
them with Lipase PS showed that (R)-3 was hydrolyzed
much faster than (S)-3 (data not shown). After enzy-
matic hydrolysis, both the acid product (R)-2 and the
There are other reports in which haloesters and vinyl
esters were employed as activated esters for enzymatic
acylation of alcohols.8 However, the use of activated
esters for hydrolytic kinetic resolutions has been less
applied. Recently various esters of acetyl-N-tert-leucine
Racemization (n-butyraldehyde, benzoic acid, toluene)
OR
O
OH
O
OR
O
Lipases and esterases
NH
NH
NH
(R)-2 (MAP-acid)
(S)-3-7 esters
Racemic esters 3-7
Scheme 1. Lipase-catalyzed hydrolysis of racemic esters and chemical racemization.

O.-J. Park et al. / Tetrahedron: Asymmetry 16 (2005) 1221–1225
1223
Table 2. Results of Lipase PS-catalyzed hydrolysis of racemic esters
3–7
Lipase PS and chemical racemization of the remaining
ester. The kind of esters strongly influenced the reaction
rate and the methoxyethyl ester was found to be the best
substrate in terms of reaction rate and enantiomeric ex-
cess. This example along with recent examples of hydro-
lytic kinetic resolutions of drug intermediates supports
the usefulness of lipases and esterases in organic synthe-
sis.6,9 The method is easy to perform with standard
equipment and can be widely applied. We will report the
immobilization of enzymes, recycling of the enzymes,
and scale-up results of the process with racemic 6 in
due course.
Esters (R)
Conversiona (%)
Enantiomeric
excess (eep)b (%)
3 h
17.1
2 4 h
50.0
3 h
2 4 h
3 CH3
4 ClC2H4
96.6
97.0
99.6
99.0
97.2
90.2
98.0
92.0
48.3
32.5
39.6
48.1
52.6
50.5
5 H2C@CHCH2
6 CH3OC2H4
7 C2H5OC2H4
52.1
51.298.8
95.4
a c = ees/(ees + eep), where ees and eep represent the enantiomeric excess
of starting ester and acid product, respectively.
b The enantiomeric excess of the remaining ester and acid was deter-
mined by HPLC on a Chiralcel OD column.
4.Experimental
4.1. General remarks
were compared with alcalase-catalyzed hydrolytic reso-
lutions.7 Faster reaction rates of acylation of racemic
amines were also reported by changing the acylating
agents as well.7 We suppose the enabling effect of the
methoxy-group is caused by the enhanced activity of
the carbonyl-group induced by the electronegative sub-
stituent. With methoxyethyl N-(2,6-dimethylphenyl)-
alaninate 6, the enzymatic reaction was optimized.
Even at high concentrations of 6 (300 g/L), the Lipase
PS-catalyzed reaction gave enantiomerically pure (R)-2
(>98% ee), and the recovery of acid product with extrac-
tion and esterification with methanol gave (R)-3 (>98%
ee) without racemization.
1H NMR spectra were recorded on a NMR spectrome-
ter (400 MHz) using tetramethylsilane as the internal
standard. Column chromatography was conducted
using silica gel 60 (230–400) mesh. All reactions were
periodically monitored by TLC and worked up after
the complete consumption of starting materials unless
specified otherwise. GC analyses were performed on a
chromatograph equipped with an FID detector using
the capillary column AT-5 (Altech). Mass spectra were
recorded using an electrospray (4000 V, positive mode)
as ionization source. Optical rotation was measured
with JASCO Digital Polarimeter at 589 nm and 24 ꢁC.
Mass spectra were recorded using an electrospray
(4000 V, positive mode) as ionization source. Enzymatic
reactions were performed in a 0.1 M phosphate buffer
(1.0 mL, pH 7.0) with 50 mg substrate at 30 ꢁC with an
adequate amount of enzymes. HPLC was carried out
on a C18 Capcell Pak column for the substrate conver-
sion (250 · 4.6 mm, acetonitrile/water/trifluoroacetic
acid = 70/30/0.1) and a Chiralcel OD column for enan-
tiomeric excess [250 · 4.6 mm, n-hexane/i-PrOH/trifluo-
roacetic acid = 95/5/0.1 for acid, n-hexane/i-PrOH =
100/1 for esters, and n-hexane/i-PrOH = 30/70 for
(R)-1 and (S)-1] at UV 254 nm. The conversion can be
also calculated from the equation c = ees/(ees + eep),
where ees and eep represent the enantiomeric excess of
the starting ester and the acid product, respectively.10
The retention times of the two pairs of enantiomers 1,
2, 3, and 6 are shown in parentheses (min): (R)-1
(10.6), (S)-1 (4.7), (R)-2 (7.7), (S)-2 (9.5), (R)-3 (7.9),
(S)-3 (8.9), (R)-6 (11.2), and (S)-6 (12.4).
Chemical coupling of (R)-3 with methoxyacetyl chlo-
ride was carried out in toluene and gave (R)-1 with
quantitative yield without racemization. Racemic
(RS)-N-(2,6-dimethylphenyl)alaninate and their ester
compounds are useful as precursors for the synthesis
of benalaxyl, furalaxyl, etc. having antifungal activity
with different amide moiety other than methoxyacetyl
as well as metalaxyl. Hence methyl (R)-N-(2,6-dimeth-
ylphenyl)alaninate (R)-3 can be a common intermediate
of these enantiomerically enriched fungicides (Scheme
2).
3.Conclusion
In conclusion, we have successfully developed a conve-
nient and scalable chemoenzymatic procedure of the
synthesis of enantiomeric version of metalaxyl (R)-1,
taking advantage of the excellent enantioselectivity of
OH
OMe
O
OMe
O
i
ii
NH
O
NH
N
O
OMe
(R)-2 (MAP-acid)
(R)-3
(R)-1 (Mefenoxam)
Scheme 2. Synthesis of mefenoxam from enzymatically-prepared acid. Reagents and conditions: (i) methanol, thionyl chloride, reflux, 3 h; (ii)
NaHCO3, toluene, methoxyacetyl chloride, rt, 1 h.

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O.-J. Park et al. / Tetrahedron: Asymmetry 16 (2005) 1221–1225
4.2. Procedure for the preparation of the compounds 4–7
4.2.6. Methyl (R)-N-(2,6-dimethylphenyl)alaninate (R)-
3. N-(2,6-Dimethylphenyl)alaninate (R)-2 (19.32g,
0.1 mol, R/S = 99/1) prepared by enzymatic hydrolysis,
was dissolved in methanol (58 g). Thionyl chloride
(13.5 g, 0.1 mol) was added dropwise at 0 ꢁC for
10 min and the reaction mixture refluxed for 3 h. After
the reaction the mixture was concentrated under re-
duced pressure. The residue was dissolved in ethyl ace-
tate (60 mL) and washed with 5% Na2CO3 (30 mL · 2
times) and H2O (20 mL). The layers were separated
and the organic layer dried over anhydrous MgSO4.
The organic layer was filtered and concentrated in vacuo
to give a pale yellow oil, methyl (R)-N-(2,6-dimethylphen-
yl)alaninate (R)-3 (19.3 g, 93% yield, R/S = 99/1). 1H
NMR (400 MHz, CDCl3) d 1.39 (d, 3H), 2.30 (s, 6H),
Racemic methyl N-(2,6-dimethylphenyl)alaninate 3 was
hydrolyzed with aqueous HCl and the acid, N-(2,6-dime-
thylphenyl)alaninate was extracted with ethyl acetate.
After evaporation, thionyl chloride was added dropwise
in the presence of each alcohol. N-(2,6-Dimethylphen-
yl)alaninate was esterified with each alcohol in the pres-
ence of thionyl chloride. Various esters were obtained in
quantitative yield. The purity of compounds was verified
with GC and MS. After work-up, the silica gel column
chromatography gave each racemic ester (confirmed
with GC).
4.2.1. Chloroethyl N-(2,6-dimethylphenyl)alaninate 4. 1H
NMR (400 MHz, CDCl3) d 1.43 (d, 3H), 2.30 (s, 6H),
3.61 (m, 2H), 3.72 (br, 1H), 4.08 (q, 1H), 4.36 (m,
2H), 6.82 (m, 1H), 6.97 (m, 2H), ESIMS m/z: 2 56.2
[M+H]+.
3.67 (s, 3H), 4.01 (q, 1H), 6.82(m, 1H), 6.97 (m, 2H),
24
D
½aŠ ¼ þ32:1 (c 1.08, CH3OH), ESIMS m/z: 208.2
[M+H]+.
4.2.7. Racemization of methyl N-(2,6-dimethylphen-
yl)alaninate (S)-3. A solution of methyl N-(2,6-dime-
thylphenyl)alaninate (S)-3 (15 g, 72.4 mmol, R/S = 2/98)
in toluene (20 mL) was added to a mixture of n-butyral-
dehyde (5.22 g, 72.4 mmol) and benzoic acid (3.54 g,
28.9 mmol, 0.4 equiv). The mixture was refluxed for
2h under N 2 gas. After the reaction, the mixture was
cooled to 20 ꢁC and washed with 5% Na2CO3
(20 mL · 3 times) and H2O (10 mL). The layers were
separated and the organic layer dried over anhydrous
MgSO4. The organic layer was filtered and concentrated
under reduced pressure. The residue (a reddish brown
oil) was distilled (1 Torr) at 110 ꢁC to give a pale yellow
oil (13.8 g, 92% yield, R/S = 50/50).
4.2.2. Allyl N-(2,6-dimethylphenyl)alaninate 5. 1H
NMR (400 MHz, CDCl3) d 1.41 (d, 3H), 2.30 (s, 6H),
3.77 (br, 1H), 4.02 (q, 1H), 4.58 (m, 2H), 5.26 (m,
2H), 5.87 (m, 1H), 6.82 (m, 1H), 6.97 (m, 2H), ESIMS
m/z: 234.2 [M+H]+.
4.2.3. Methoxyethyl N-(2,6-dimethylphenyl)alaninate 6.
1H NMR (400 MHz, CDCl3) d 1.41 (d, 3H), 2.30 (s,
6H), 3.33 (s, 3H), 3.53 (m, 2H), 3.79 (br, 1H), 4.04 (q,
1H), 4.26 (m, 2H), 6.81 (m, 1H), 6.97 (m, 2H), ESIMS
m/z: 252.2 [M+H]+.
4.2.4. Ethoxyethyl N-(2,6-dimethylphenyl)alaninate 7.
1H NMR (400 MHz, CDCl3) d 1.20 (t, 3H), 1.40 (d,
3H), 2.30 (s, 6H), 3.50 (m, 2H), 3.57 (m, 2H), 3.78 (br,
1H), 4.06 (q, 1H), 4.24 (m, 2H), 6.81 (m, 1H), 6.97 (m,
2H), ESIMS m/z: 266.2 [M+H]+.
4.2.8. Methyl (R)-N-(2,6-dimethylphenyl)-N-(methoxy-
acetyl)alaninate (R)-1. NaHCO3 (2.43 g, 28.9 mmol,
1.2equiv) was added to a solution of methyl ( R)-N-
(2,6-dimethylphenyl)alaninate (R)-3 (5 g, 24.1 mmol,
R/S = 99/1, in toluene 10 g) and the temperature re-
duced to 0 ꢁC. Methoxyacetyl chloride (2.88 g,
26.5 mmol, 1.1 equiv) was slowly added at 0 ꢁC and
the mixture stirred for 1 h at room temperature. The
mixture was washed with 5% Na2CO3 (20 mL) and
H2O (20 mL). The layers were separated and the
organic layer dried over anhydrous MgSO4. The organic
layer was filtered and concentrated in vacuo to give a
pale brown oil, methyl (R)-N-(2,6-dimethylphenyl)-N-
(methoxyacetyl)alaninate (R)-1 (6.7 g, 99% yield, R/S =
99/1). 1H NMR (400 MHz, CDCl3) d 1.02(d, 3H),
2.16 (s, 3H), 2.47 (s, 3H), 3.34 (s, 3H), 3.63 (dd, 2H),
4.2.5. (R)-N-(2,6-Dimethylphenyl)alaninate (R)-2. Race-
mic methyl N-(2,6-dimethylphenyl)alaninate 3 (20 g,
96.5 mmol) and Lipase PS (750 mg) were added to
distilled water (30 mL). Triton X-100 (0.1 mL) was
added for effective mixing. The reaction was performed
at 40 ꢁC with magnetic stirring. The pH of the reac-
tion mixture was adjusted to pH 7.0 by adding 1 M
NaOH solution with Mettler Toledo DL 70 pH stat.
The progress of the reaction was monitored by HPLC.
After 40% conversion, the reaction mixture was filtered
and both the acid product 2 and the remaining ester 3
were extracted with ethyl acetate. The pH of the aqueous
layer was adjusted to pH 9.0 by the careful addition of
1 M NaOH. Ethyl acetate was removed under reduced
pressure to give the crude 3. The pH of aqueous
layer was adjusted to pH 2–3 with concentrated HCl
and extracted with ethyl acetate. The layers were
separated and the organic layer was dried with
anhydrous MgSO4. Ethyl acetate was removed in vacuo
to give a pale brown solid (R)-N-(2,6-dimethylphen-
yl)alaninate (R)-2. (6.1 g, 32.7% yield, R/S = 98.2/1.4).
1H NMR (400 MHz, CDCl3) d 1.39 (d, 3H), 2.30 (s,
3.80 (s, 3H), 4.54 (q, 1H), 7.10–7.24 (m, 3H),
24
D
½aŠ ¼ ꢀ55:4 (c 1.88, CH3COCH3), ESIMS m/z: 280.4
(10%) [M+H]+, 302.3 (15%) [M+Na]+, 581.1 (100%)
[2M+H]+.
Acknowledgments
We wish to thank Amano Enzymes Inc. and Meito San-
gyo for kindly supplying enzyme samples for the re-
search. We especially thank Dr. J. H. Chung, W. K.
Chung, B. W. Seo, and J. H. Bang, for scientific assis-
tance and for fruitful discussions.
6H), 4.01 (q, 1H), 6.82(m, 1H), 6.97 (m, H2),
24
D
½aŠ ¼ þ11:7 (c 1.325, C2H5OH), ESIMS m/z: 194.1
[M+H]+.

O.-J. Park et al. / Tetrahedron: Asymmetry 16 (2005) 1221–1225
1225
F.; Ogawa, K.; Ohno, T.; Otsuka, K.; Sakota, A. K.;
Shirasaka, N.; Uzura, A.; Uzura, K. Org. Process Res.
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