Practical synthesis of optically active α,α-disubstituted malonamic acids through asymmetric hydrolysis of malonamide derivatives with Rhodococcus sp. CGMCC 0497

Article abstract of DOI:10.1021/jo026691u

A variety of α,α-disubstituted malonamides undergo enantioselective hydrolysis with Rhodococcus sp. CGMCC 0497 to give challenging enantiopure α,α-disubstituted malonamic acids with up to >99% enantiomeric excesses and 98% chemical yields. The enantiosele

Full text of DOI:10.1021/jo026691u

In our previous studies, the strain Rhodococcus sp.
CGMCC 0497 has been screened out in our laboratory
and proved to be a powerful nitrile hydratase/amidase-
containing microorganism. It has also been demonstrated
that the strain was an efficient enantioselective biocata-
lytic system and was able to transform a variety of
R-substituted arylacetonitriles into optically pure R-sub-
stituted arylacetamides and R-substituted arylacetic acid
such as the famous nonsteroidal antiinflammatory drug
(S)-naproxen with excellent enantiomeric excesses.8 We
recently have focused our attention on the asymmetric
hydrolysis of prochiral R,R-disubstituted malononitriles,
owing to the interest in the synthesis of enantiopure R,R-
dialkylated R-amino acids through enzymatic method.
P r a ctica l Syn th esis of Op tica lly Active
r,r-Disu bstitu ted Ma lon a m ic Acid s th r ou gh
Asym m etr ic Hyd r olysis of Ma lon a m id e
Der iva tives w ith Rh od ococcu s sp .
CGMCC 0497
Zhong-Liu Wu and Zu-Yi Li*
State Key Laboratory of Bioorganic & Natural Products
Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai
200032, China
lizy@pub.sioc.ac.cn
Received November 12, 2002
This class of nonproteinogenic R-amino acids has
attracted increasing attention in recent years due to their
potential to induce particular conformations when incor-
porated into a polypeptidic chain. They can therefore be
used as enzyme inhibitors and offer opportunities for
drug discovery.9 However, compared to the synthesis of
optically active R-monosubstituted amino acids, highly
efficient methods to the asymmetric synthesis of R,R-
dialkylated R-amino acids are relatively rare and their
extensive use is limited by the availability of enantiopure
compounds in large scale.10 Considered to be an attractive
approach, enzyme-catalyzed resolution has been success-
fully applied to the production of R-monosubstituted
amino acid, but the attempt to extending the methodology
to R,R-dialkylated R-amino acids was not satisfying,
because the process is generally slow and less stereo-
selective and the undesired enantiomer cannot be race-
mized.10 In this paper, we report a practical enzymatic
method for the synthesis of optically pure R,R-disubsti-
tuted malonamic acids, precursors of R,R-dialkylated
R-amino acids, by asymmetric hydrolysis of diamides with
Rhodococcus sp. CGMCC 0497.
Abstr a ct: A variety of R,R-disubstituted malonamides
undergo enantioselective hydrolysis with Rhodococcus sp.
CGMCC 0497 to give challenging enantiopure R,R-disubsti-
tuted malonamic acids with up to >99% enantiomeric
excesses and 98% chemical yields. The enantioselectivity
originated from the effects of a highly enantioselective
amidase. The products could be converted to valuable (R)-
or (S)-R,R-dialkylated amino acids after routine conversions.
Nitrile-converting enzymes have been known for sev-
eral decades1 and demonstrated great potential in organic
synthesis and chemical industry,2 but the substrates
studied are still very limited and the ability of the
enzymes to catalyze stereoselective conversion remains
largely unexploited.3,4 So far, most studies focus on the
enantioselective conversion of racemic nitriles, such as
R-alkyl nitriles,5a,5b R-hydroxy nitriles,5c,5d R-acyloxy
nitriles,5e R-amino nitriles,5f,5g and â-acetoxy nitriles,5h
while only a few focus on prochiral nitriles especially
malononitrile derivatives.6,7
Yokoyama et al. have reported the hydrolysis of
prochiral disubstituted malononitriles by Rhodococcus
rhodochrous ATCC 21197, but the substrate was limited
to 2-butyl-2-methylmalononitrile.7 In our study, first,
dinitriles were used as substrate. In accordance with the
literature, 2-butyl-2-methylmalononitrile can be con-
verted to (R)-2-butyl-2-methylmalonamic acid neatly by
Rhodococcus sp. CGMCC 0497. However, when 2-benzyl-
2-methyl-malononitrile 1 was used as substrate (0.125%
w/v), most probably due to the steric hindrance, the
products isolated were a complex mixture of hydrolysis
intermediates (Scheme 1). After 66 h, the reaction give
(1) (a) Thimann, K. V.; Mahadervan, S. Arch. Biochem. Biophys.
1964, 105, 133. (b) Asano, Y.; Fujishiro, K.; Tani, Y.; Yamada, H. Agri.
Biol. Chem. 1982, 46, 1165.
(2) (a) Kobayashi, M.; Nagasawa, T.; Yamada, H. TIBTECH 1992,
10, 402. (b) Kobayashi, M.; Shimizu, S. Curr. Opin. Chem. Biol. 2000,
4, 95.(c) Peter, M.; Kiener, A. Green Chem. 1999, 4, 99. (d) Kiener, A.;
Roduit, J .-P.; Tinschert, A.; Heinzmann, K. Synlett. 1994, 10, 814. (e)
Glo¨ckler, R.; Roduit, J .-P. Chimia 1996, 50, 413. (f) Hann, E. C.;
Eisenberg, A.; Fager, S. K.; Perkins, N. E.; Gallagher, F. G.; Cooper,
S. M.; Gavagan, J . E.; Stieglitz, B.; Hennesey, S. M.; DiCosimo, R.
Bioorg. Med. Chem. 1999, 7, 2239.
(3) (a) Wieser, M.; Nagasawa T. In Stereoselective Biocatalysis; Patel,
R. N., ED.; Dekker: New York, NY, 2000; pp 461-486. (b) Sugai, T.;
Yamazaki, T.; Yokoyama, M.; Ohta, H. Biosci., Biotechnol., Biochem.
1997, 61, 1419.
(4) (a) Faber, K. Biotransformations in organic chemistry: A text-
book, 4th ed.; Spring-Verlag: Berlin, 2000; Chapter 2. (b) Crosby, J .;
Moilliet, J .; Parratt, J . S.; Turner, N. J . J . Chem. Soc., Perkin Trans.
1 1994, 1679.
(6) (a) Beard, T.; Cohen, M. A.; Parratt, J . S.; Turner, N. J .; Crosby,
J .; Moilliet, J . Tetrahedron: Asymmetry 1993, 4, 1085. (b) Kakeya; H.;
Sakai, N.; Sano, A.; Yokoyama, M.; Sugai, T.; Ohta, H. Chem. Lett.
1991, 1823.
(7) Yokoyama, M.; Sugai, T.; Ohta, H. Tetrahedron: Asymmetry
1993, 4, 1081.
(8) (a) Wu, Z.-L.; Li, Z.-Y. Tetrahedron: Asymmetry 2001, 12, 3305.
(b) Wu, Z.-L.; Li, Z.-Y. Biotechnol. Appl. Biochem. 2002, 35, 61.
(9) (a) Trost, B. M.; Ariza, X. Angew. Chem., Int. Ed. Engl. 1997,
36, 2635 and references therein. (b) Ma, D.; Ding, K. Org. Lett. 2000,
2, 2515. (c) Clark, J . S.; Middleton, M. D. Org. Lett. 2002, 4, 765. (d)
Ooi, T.; Takeuchi, M.; Kameda, M.; Maruoka, K. J . Am. Chem. Soc.
2000, 122, 5228.
(5) (a) Wang, M.-X.; Lu, G.; J i, G.-J .; Huang, Z.-T.; Meth-Cohn O.;
Colby, J . Tetrahedron: Asymmetry 2000, 11, 1123. (b) Payne, S.; Wu,
S.; Fallon, R. D.; Tudor, G.; Stieglitz, B.; Turner J . M.; Nelson, M. J .
Biochemistry 1997, 36, 5447. (c) Yamamoto, K.; Oishi, K.; Fujimatsu
I.; Komatus, K. I. Appl. Environ. Microbiol. 1991, 57, 3028. (d)
DeSantis, G.; Zhu, Z.; Greenberg, W. A.; Wong, K.; Chaplin, J .; Hanson,
S. R.; Farwell, B.; Nicholson, L. W.; Rand, C. L.; Weiner, D. P.;
Robertson, D. E.; Burk, M. J . J . Am. Chem. Soc. 2002, 124, 9024. (e)
Layh, N.; Stolz, A.; Forster, S.; Effenberger F.; Knackmuss, H.-J . Arch.
Microbiol. 1992, 158, 405. (f) Macadam, A. M.; Knowles, C. J .
Biotechnol. Lett. 1985, 7, 865. (g) Wang, M.-X.; Lin, S.-J . J . Org. Chem.
2002, 67, 6542. (h) Idogaki, H.; Kasai, N.; Takeuchi, M.; Hatada, M.;
Suzuki, T. Tetrahedron: Asymmetry 2001, 12, 369.
(10) Cativiela, C.; Diaz-de-Villegas, M. D. Tetrahedron: Asymmetry
1998, 9, 3517 and references therein.
10.1021/jo026691u CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/14/2003
J . Org. Chem. 2003, 68, 2479-2482
2479

TABLE 1. En a n tioselective Hyd r olysis of Va r iou s
r-Su bstitu ted -r-m eth ylm a lon a m id es by Rh od ococcu s sp .
CGMCC 0497
SCHEME 1. Hyd r olysis of r-Ben zyl-r-m eth yl
Ma lon on itr ile 1
entrya substrate
R
T (°C) Yield (%) ee (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
4a
4a
4b
4b
4c
4c
4d
4d
4e
4e
4f
C6H5
C6H5
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30
94
94
94
92
94
91
92
95
97
98
95
93
95
95
97
94
94
92
94
97
97
o-ClC6H4
o-ClC6H4
m-ClC6H4
m-ClC6H4
p-ClC6H4
p-ClC6H4
p-CH3C6H4
p-CH3C6H4
p-MeOC6H4
p-MeOC6H4
p-FC6H4
97c
97c
95
96
>99
>99
>99
>99
>99c
>99c
>99
>99
>99
>99
>99
>99
91c
a mixture of (S)-2-cyano-2-methyl-3-phenylpropanamide
2 (56%, 93% ee), (R)-2-cyano-2-methyl-3-phenylpropanoic
acid 3 (33%, 52% ee), and 2-benzyl-2-methyl-malonamide
4a (8%). Similar results were obtained for the hydrolysis
of 2-(2′-chlorobenzyl)-2-methylmalononitrile, 2-(3′-chlo-
robenzyl)-2-methylmalononitrile, 2-(4′-chlorobenzyl)-2-
methylmalononitrile, and 2-(4′-methoxybenzyl)-2-meth-
ylmalononitrile. By prolonging the reaction time of 1 to
112 h, the mixture converted to (R)-2-benzyl-2-methyl-
malonamic acid 5a (52%, 95% ee) and 3 (44%, 1.2% ee).
The high enantiomeric excess of 5a encouraged us to
explore a new approach to improve its chemical yield.
It is well-known that enzyme-catalyzed hydrolysis of
nitriles proceeds by two routes: by nitrilase or by a
combination of nitrile hydratase (NHase) and amidase
through an intermediate amide.4 Rodococcus sp. CGMCC
0497 acts mainly by the latter route. It is clear that
2-benzyl-2-methylmalonamic acid 5a may be derived in
two ways: one involved 2-cyano-2-methyl-3-phenylpro-
pionic acid 3 by NHase and the other involved 2-benzyl-
2-methylmalonamide 4a by amidase. Further experi-
ments were carried out using racemic 3a and 4a as
substrates, respectively (0.125% w/v). The transformation
of racemic 3a did not occur at all after 1 day and 3a was
recovered quantitively, while the diamide 4a was con-
verted to 5a in >99% yield and 94% ee, which demon-
strated that 5a was derived mostly from diamide 4a with
high efficiency and enantioselectivity.
It is especially noticeable that, using 4a as substrate,
the reaction concentration could reach to 1% (w/v) and
5a was achieved in 95% yield and 97% ee after 1 day at
30 °C with Rodococcus sp. CGMCC 0497. Compared to
the above-mentioned hydrolysis of 1, the hydrolysis of
4a was much faster and more efficient. The quantitive
conversion of 4a to 5a made recrystallization feasible for
the purification process and flash chromatograph could
be avoided, which made large-scale production possible.
Moreover, R,R-disubstituted malonamides can be easily
synthesized from the corresponding malonamic acid
diesters by routine reactions.11 Since R,R-disubstituted
malonamides have advantages over the corresponding
dinitriles as substrates in the production R,R-disubsti-
tuted malonamic acids, the asymmetric hydrolysis of R,R-
disubstituted malonamides instead of malononitriles was
investigated with whole cells of Rodococcus sp. CGMCC
0497. To the best of our knowledge, the study on the
asymmetric desymmetrization of R,R-disubstituted ma-
lonamides to afford optically pure malonamic acid de-
rivatives is very rare and the catalytic method directly
to the enantiopure R,R-disubstituted malonamic acids has
4f
4g
4g
4h
4h
4i
p-FC6H4
p-BrC6H4
p-BrC6H4
C6H5CH2
C6H5CH2
C3H7
4i
4j
a
Carried out for 1 day with a substrate concentration of 2.5
b
g/L. Determined by HPLC on a Chiralpak AS column with
hexane/2-propanol mixtures unless stated otherwise. c Determined
by HPLC on a Chiralcel OJ column.
SCHEME 2. Asym m etr ic Hyd r olysis of
r,r-Disu bstitu ted Ma lon a m id es 4
never been reported. The characteristics of the two
functional groups of the amide acid enable the conversion
to both enantiomers of amino acids in 100% theoretic
yield.
The results of the enantioselective hydrolysis of various
R,R-disubstituted malonamides by Rhodococcus sp.
CGMCC 0497 at 20 or 30 °C are summarized in Table 1.
As shown, in all cases, the enzymes maintain high
activity at 20 and 30 °C and the reactions proceeded
smoothly and completely during 1 day (Scheme 2).
Excellent enantioselectivities and isolated yields were
obtained with a variety of substrates. Rhodococcus sp.
CGMCC 0497 tolerates aromatic ring substituents in the
ortho-, meta-, and para-positions. All para-substituted
substrates gave products with excellent enantiomeric
excesses of >99%, slightly higher than ortho and meta
ones. With a monosubstituted benzene ring, 4i gave
product 5i with >99% ee, while 4a gave product 5a with
97% ee. Dialkyl-substituted malonamide 4j gave product
5j with a slightly lower ee of 91%.
The hydrolysis of all the above substrates was neat and
concordant. However, the effects between the substrates
and enzymes in Rhodococcus sp. CGMCC 0497 are very
complex. When 2-benzyl-2-ethylmalonamide 4k and 2-bu-
tyl-2-ethylmalonamide 4l were subjected to the reaction,
there were only trace products observed after 2 days,
while the hydrolysis of 2-allyl-2-benzylmalonamide 4m
gave the deamidized product 2-allylphenylpropionate 6
in a yield of 78% after 4 days. It seemed that the results
were not only induced by steric hindrance but by an
electronic effect of the substrates as well. Other enzymes
in the whole cell may also come into effect under certain
(11) Sandler, S. R.; Karo, W. Organic functional group preparations,
2nd ed. Academic Press: New York. 1983.
2480 J . Org. Chem., Vol. 68, No. 6, 2003

tuted malonamic acid 5 (0.1 mmol, obtained from flash chro-
matography) in DMF (0.1 mL) was added bromoethane or
iodomethane (2 mmol) and anhydrous K2CO3 (2 mmol). The
reaction was carried out at room temperature for 1 day to
achieve the esters, and the esters were subjected to chiral HPLC.
(R)-2-Ben zyl-2-m eth yl-m a lon a m ic a cid 5a : white solid, mp
SCHEME 3. Syn th esis of eith er (R)- or
(S)-r,r-Dia lk yla ted Am in o Acid a
117.9-118.9 °C, lit.12 mp 120-121 °C; [R]15 -15.01 (c 1.07,
D
MeOH), 94% ee {lit.12 [R]D -4.4 (c 0.5, MeOH), R}; 1H NMR (300
MHz, [D6]acetone) δ 7.29-7.19 (m, 5H), 3.61 (br s, 3H), 3.25 (d,
1H, J ) 13.8 Hz), 3.20 (d, 1H, J ) 13.5 Hz), 1.37 (s, 3H); IR
(KBr): ν 3423, 3204 (NH), 3034 (br OH), 1745, 1659 (CdO); MS
m/z (%) 207 (M+, 2), 91 (100).
(S)-2-Eth oxyca r bon yla m in o-2-m eth yl-3-p h en ylp r op a n o-
ic Acid Eth yl Ester 7. To a solution of (R)-2-benzyl-2-methyl-
malonamic acid 5a (33 mg, 0.16 mmol) in dry DMF (0.3 mL)
were added ethyl bromide (0.32 mmol) and solid K2CO3 (0.32
mmol) at room temperature. After 24 h, the resulting mixture
was poured into water, extracted with ether, washed, and dried
on Na2SO4. The solvent was removed under reduced pressure
and the resulting product was purified by flash chromatograph
(98% yield). The product was dissolved in dry DMF (1.5 mL).
Hg(OAc)2 (61 mg, 0.19 mmol), dry ethanol (220 mg, 5 mmol),
and NBS (37 mg, 0.21 mmol) were added at room temperature.
After 16 h, the resulting mixture was poured into water,
extracted with ether, washed, and dried on Na2SO4. The solvent
was removed under reduced pressure and the resulting product
was purified by flash chromatograph to afford (S)-7 (96%
yield): oil; [R]18D +31.02 (c 2.15, CHCl3); the enantiomeric excess
was determined by HPLC on a Chiralcel OD column with
a
Conditions: (a) DMF, EtBr, K2CO3, rt; (b) DMF, Hg(OAc)2,
NBS, EtOH, rt; (c) 20% HCl, reflux; (d) P2O5, toluene; (e) 3 N
NaOH, THF, rt; (f) SOCl2, NaN3, MeOH.
conditions. Substrates 4a -4j, with methyl group as one
of the two substituents at the R-position, are more
favorable in the hydrolysis reaction, and 4m , with
unsaturated allyl group, may result in side reactions. Our
further work will be focused on the modification of the
strain at the molecular level in order to enhance its
adaptability toward diamide substrates.
The products of the enantioselective hydrolysis of R,R-
disubstituted malononitriles, (R)-R,R-disubstituted ma-
lonamic acids 5, could afford either (R)- or (S)-R,R-
dialkylated amino acids after routine conversion. For
example, (R)-2-benzyl-2-methylmalonamic acid 5a was
transferred to (R)-10 or (S)-10. (Scheme 3). R-Methylphen-
ylalanine 10 is an efficient â-turn and helix former, much
better than its nonmethylated parent compound phen-
ylalanine.9
1
hexane/2-propanol mixtures 8:2; H NMR (300 MHz, CDCl3) δ
7.30-7.23(m, 3H), 7.08-7.05(m, 2H), 5.37 (brs, 1H), 4.27-4.10
(m, 4H), 3.43 (d, 1H, J ) 13.8 Hz), 3.18 (d, 1H, J ) 13.2 Hz),
1.64 (s, 3H), 1.33-1.23 (m, 6H); IR (film) ν 3358 (NH), 1721 (Cd
O), 704; MS m/z (%) 279 (M+, 0.8), 234 (2.5), 206 (M+ - COOEt,
43.3), 190 (28.0), 188 (100), 142 (53.5), 116 (53.6), 91 (58.8), 88
(35.7), 42 (43.0); HRMS calcd for (C15H21NO4)+ 279.14706, found
279.15017.
In conclusion, we have demonstrated a successful
asymmetric hydrolysis of R,R-disubstituted malonamides
to afford enantiopure (R)-R,R-disubstituted malonamic
acids using the strain Rodococcus sp. CGMCC 0497 at
20 or 30 °C. The products can be converted to valuable
products such as either (R)- or (S)-R,R-dialkylated amino
acids or other biologically active compounds.12 The mild
and practical reaction condition, high substrate concen-
tration, and excellent enantioselectivity and chemical
yield as well as the convenient purification procedure
provide a good prerequisite for large-scale production.
Currently, the applications of the technologies of cell
immobilization and bisphasic systems in enzymatic hy-
drolysis of diamides are being actively investigated in this
laboratory, and we believe that the method could be
developed into a valuable alternative for both enanti-
omers of R,R-dialkylated amino acids.
(R)-2-Cya n o-2-m et h yl-3-p h en ylp r op ion ic Acid E t h yl
Ester 8.13 To a solution of (R)-2-benzyl-2-methylmalonamic acid
5a (33 mg, 0.16 mmol) in dry DMF (0.3 mL) were added ethyl
bromide (0.32 mmol), and solid K2CO3 (0.32 mmol) at room
temperature. After 24 h, the resulting mixture was poured into
water, extracted with ether, washed, and dried on Na2SO4. The
solvent was removed under reduced pressure and the resulting
product was purified by flash chromatograph (98% yield). The
product was dissolved in dry toluene (2 mL). Phosphorus
pentoxide (0.3 mmol) was added. The resulting mixture was
refluxed for 4 h, cooled to room temperature, poured into water,
and extracted with ethyl acetate. The solvent was removed under
reduced pressure and the resulting product was purified by flash
chromatograph to afford (R)-8 (93% yield): oil; [R]25 -23.8 (c
D
0.51, CHCl3); the enantiomeric excess was determined by HPLC
on a Chiralcel OJ column with hexane/2-propanol mixtures 9:1;
1H NMR (300 MHz, CDCl3) δ 7.39-7.23 (m, 5H), 4.19 (q, 2H, J
) 7.2 Hz), 3.23 (d, 1H, J ) 13.5 Hz), 3.08 (d, 1H, J ) 13.5 Hz),
1.62 (s, 3H), 1.24 (t, 3H, J ) 7.1 Hz); IR (film) ν 2245 (CN), 1743
(CdO); MS m/z (%) 217 (M+, 3), 144 (M+ - COOEt, 5), 91 (100).
(R)-2-Meth oxycar bon ylam in o-2-m eth yl-3-ph en ylpr opan o-
n itr ile 9. To a solution of (R)-2-cyano-2-methyl-3-phenylpropi-
onic acid ethyl ester 7 (0.27 mmol) in THF (1.5 mL) was added
3 N NaOH (1.5 mL). The mixture was stirred at room temper-
ature for 30 min and then acidified, extracted with ethyl acetate,
and dried on MgSO4. After evaporation, the residue was purified
by flash chromatograph. To the product was added SOCl2 (0.3
mL) and the reaction mixture was stirred at 40 °C for 3 h. The
excess SOCl2 was removed at reduced pressure. The residue was
dissolved in cyclohexane and evaporated again. The acid chloride
was then cooled to room temperature and dissolved in dry
acetone (1 mL). Then a solution of sodium azide (26 mg, 0.4
mmol) in water (0.2 mL) was added and the stirring was
continued for 1 h. The mixture was poured into water, extracted
with ether, and dried on MgSO4. After filtration dry methanol
Exp er im en ta l Section
Gen er a l P r oced u r e for r,r-Disu bstitu ted Ma lon a m ic
Acid s (5) a n d Deter m in a tion of En a n tiom er ic Excess. A
suspension of 5 g of washed wet cells of Rodococcus sp. CGMCC
0497 (available from the China General Microbiological Culture
Colletion Center) and 40 mL of 0.1 mM potassium phosphate
buffer (pH ) 7.0) was incubated at 30 or 20 °C for 30 min with
continuous magnetic stirring before the addition of 4 (100-400
mg in 100-200 µL DMSO). The reaction was quenched with 3
N HCl after 1 day and centrifuged. The resulting supernatant
was extracted with ethyl acetate and dried over Na2SO4. After
concentration, the residue was purified by flash chromatography
on silica gel (elutent, petroleum ether/EtOAc/AcOH 100:100:1)
or recrystallization (CH2Cl2/PE). To a solution of R,R-disubsti-
(12) Lee, M.; J in, Y.; Kim, D. H. Bioorg. Med. Chem. 1999, 7, 1755.
(13) Meier, M.; Ruechardt, C. Chem. Ber. 1987, 120, 1.
J . Org. Chem, Vol. 68, No. 6, 2003 2481

(1 mL) was added. The solution was stirred at 80 °C for 2 h and
vacuum. To the residue was added distilled water (3 mL) and
the solution was evaporated again. The residue was purified by
Dowex 50 × 2-400 ion-exchange resin to yield 95% of a white
powder: [R]17D -22.0 (c 0.61, H2O), S; [R]17D +21.8 (c 0.73, H2O),
R {lit.15 [R]D -22 (c 1, H2O), S}; 1H NMR (300 MHz, D2O) δ 7.22-
7.19 (m, 3H), 7.10-7.07 (m, 2H), 3.11 (d, 1H, J ) 14.4 Hz), 2.79
(d, 1H, J ) 14.4 Hz), 1.37 (s, 3H); IR (KBr) 2500-3300, 1650
(CdO); MS (ESI) 202 ([M + Na]+), 180 ([M + H]+).
evaporated for flash chromatograph to afford (R)-9 (94% yield):
white solid, lit.14 mp 81 °C; [R]22 -46.6 (c 0.798, CHCl3) {lit.14
D
[R]D -46.1 (c 2, CHCl3), S}; the enantiomeric excess was
determined by HPLC on a Chiralcel OJ column with hexane/2-
propanol mixtures 9:1; 1H NMR (300 MHz, CDCl3) δ 7.40-7.34
(m, 3H), 7.30-7.27 (m, 2H), 4.94 (brs, 1H), 3.74 (s, 3H), 3.29 (d,
1H, J ) 13.8 Hz), 3.19 (d, 1H, J ) 13.5 Hz), 1.66 (s, 3H); IR
(KBr) ν 3325 (NH), 2240 (CN), 1699 (CdO); MS m/z (%) 219 (M+
+ 1, 8), 218 (M+, 15), 192 (M+ - CN, 99), 91 (100).
Ack n ow led gm en t. We are grateful to the National
Science Foundation of China (Grant No. 20032020) for
financial support. We also thank Prof. Lijun Xia and
Miss Zuoding Ding for technical assistance.
(S) An d (R)-r-Meth ylp h en yla la n in e 10. (S)-2-Ethoxycar-
bonylamino-2-methyl-3-phenylpropanoic acid ethyl ester 7 or (R)-
2-methoxycarbonylamino-2-methyl-3-phenylpropanonitrile 9 (0.13
mmol) was hydrolyzed by refluxing for 3 h with 20% aqueous
hydrochloric acid (3 mL). The solution was evaporated under
Su p p or tin g In for m a tion Ava ila ble: Experiment proce-
dures and full characterization for compounds 1-3, 4a -4m ,
5b-5j, 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
(14) Cativiela, C.; Diaz-de-Villegas, M. D.; Galvez, J . A. Tetrahe-
dron: Asymmetry 1994, 5, 261.
(15) Kruizinga, W. H.; Bolster, J .; Kellogg, R. M.; Kampuis, J .;
Boesten, W. H. J .; Meijer E. M.; Schoemaker, H. E. J . Org. Chem. 1988,
53, 1826.
J O026691U
2482 J . Org. Chem., Vol. 68, No. 6, 2003