Synthesis and application of enantioenriched functionalized α-tetrasubstituted α-amino acids from biocatalytic desymmetrization of prochiral α-aminomalonamides

Article abstract of DOI:10.1021/jo3007122

Catalyzed by Rhodococcus erythropolis AJ270, an amidase-containing microbial whole cell catalyst in neutral phosphate buffer at 30 °C, a number of prochiral α-substituted α-aminomalonamides underwent highly efficient and enantioselective hydrolytic desymm

Full text of DOI:10.1021/jo3007122

Article
pubs.acs.org/joc
Synthesis and Application of Enantioenriched Functionalized α-
Tetrasubstituted α-Amino Acids from Biocatalytic Desymmetrization
of Prochiral α-Aminomalonamides
Li-Bing Zhang,† De-Xian Wang,† Liang Zhao,‡ and Mei-Xiang Wang*,‡
†Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100190, China
‡MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University,
Beijing 100084, China
S
* Supporting Information
ABSTRACT: Catalyzed by Rhodococcus erythropolis AJ270, an
amidase-containing microbial whole cell catalyst in neutral
phosphate buffer at 30 °C, a number of prochiral α-substituted
α-aminomalonamides underwent highly efficient and enantiose-
lective hydrolytic desymmetrization to afford functionalized α-
tetrasubstituted α-amino acids in 74−98% chemical yields and
94.0 to >99.5% ee. The presence of a free α-amino (NH2)
substituent in the substrates was deemed important to ensure high
biocatalytic efficiency and enantioselectivity. The synthetic
application of biocatalytic desymmetrization was demonstrated by practical chemical transformations of (R)-2-amino-2-
carbamoylpent-4-enoic acid to α-substituted serine analogues and a bioactive diamino alcohol derivative.
rhodochrous IFO 15564,6 Rhodococcus sp. 361,7 Rhodococcus
INTRODUCTION
Biotransformations using either enzymes or whole cell catalysts
have become important and routinely applied preparative
methods in asymmetric synthesis owing to their high efficiency
■
erythropolis AJ270,8 and nitrilase III9 into 4-cyanobutyric acid
products with moderate to excellent enantioselectivity depend-
ing on the nature of 3-substituent, biocatalysts, and additives
used.
and enantioselectivity under eco-environmentally benign
conditions.1 Biocatalytic desymmetrization of prochiral sub-
Amidases [3.5.1.4] catalyze the conversion of primary amides
into carboxylic acids with the release of ammonia.10 A large
strates, in particular, constitutes an elegant and powerful
number of amidases and microorganisms that contain amidases
strategy for the generation of enantioenriched chiral com-
pounds.2 Unlike the kinetic resolution processes, desymmetri-
have been reported to date, and some of them have been
studied extensively in the kinetic resolution of racemic
zation provides the enabling technology to achieve a maximum
yield of 100% with excellent enantiocontrol. Furthermore, the
method, starting from readily available materials, generally
amides.11 Rhodococcus erythropolis AJ270, a soil-derived nitrile
hydratase and amidase-containing microbial strain,12 for
instance, has been shown to catalyze enantioselective
furnishes highly enantiopure chiral compounds that are hardly
biotransformation of a large number of structurally diverse
accessible by other synthetic means. Esterase-, lipase-, and
racemic amides including amino-,13 hydroxy- and alkoxy-,14
protease-catalyzed hydrolysis of prochiral malonates and 3-
alkenyl-,15 alkynyl-,16 allenyl-,16 and azido-bearing17 amides,
substituted glutarates, for example, leads to the formation of the
cyclopropanecarboxamide,18 and various heterocyclic
corresponding monoesters with varied enantiomeric excess (ee)
amides19−22 to produce highly enantiopure carboxylic acid
values, while enzymatic acylation of prochiral 2-substituted 1,3-
and amide products. Surprisingly, biocatalytic enantioselective
desymmetrization of prochiral dicarboxamides remains largely
alkanediols affords chiral alcohols in good yields with moderate
to good enantiocontrol.3 In addition to extensively used
unexplored. To the best of our knowledge, there are only two
biocatalytic hydrolysis and formation of C−O bonds,
literature reports.5a,23 In the study of desymmetrization of
desymmetric hydrolysis of prochiral dinitriles has also been
malononitriles,5b,c Wu and Li5a found in 2003 that α-methyl-α-
arylmethylmalonamides undergo biocatalytic hydrolysis to yield
chiral malonamic acids with high enantioselectivity. Since the
substrate structures are very simple, synthetic applications are
reported in the past two decades.4−8 In the presence of a nitrile-
hydrolyzing biocatalyst such as Rhodococcus rhodochrous IFO
155644 and Rhodococcus sp. CGMCC 0497,5 prochiral
malononitriles undergo hydrolysis to produce a mixture of
cyanoacetamides, cyanoacetic acids and carbamoylacetic acids
with different ee values, while prochiral glutaronitriles bearing a
3-substituent have been biotransformed by Rhodococcus
Received: April 7, 2012
Published: June 18, 2012
© 2012 American Chemical Society
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therefore limited.5a We have expanded very recently the scope
of the substrates, demonstrating a practical synthesis of
functionalized carbamoylacetic acids from Rhodococcus eryth-
ropolis AJ270-catalyzed desymmetrization of malonamides that
contain functional groups.23 On the basis of easy availability of
prochiral dicarboxamide compounds, high catalytic efficiency
and enantioselectivity of amidases, we envisioned that the
amidase-catalyzed desymmetrization of prochiral dicarboxa-
mides would provide a novel method for the synthesis of
enantioenriched amido-substituted carboxylic acid compounds,
invaluable nitrogen-containing chiral entities in organic syn-
thesis.
Scheme 1. Synthesis of Prochiral Malonamides 5
Nonproteinogenic amino acids are important entities in
organic and medicinal chemistry. α-Tetrasubstituted α-amino
acids or α,α-disubstituted α-amino acids, for instance, are often
used as building units in the construction of peptidomimetics of
pharmaceutical importance24 while incorporation of α-amino
acids functionalized with a terminal alkene moiety into peptides
enables preparation of stapled α-helical peptides.25 α-
Tetrasubstituted functional α-amino acids have also been
used extensively in the synthesis of natural and bioactive
products.26 Although a plethora of chemical and enzymatic
methods have been established for the synthesis of chiral
quaternary, carbon-centered carboxylic acids,26−28 surprisingly,
desymmetrization of prochiral α-amino-substituted malonate
diesters and their malonamide derivatives has never been
reported. Enzymatic preparation of functionalized and α-
tetrasubstituted α-amino acids documented in the literature
involves mainly kinetic resolution of racemic substrates using
various hydrolytic enzymes.27 Our continuous interest in
synthetic biocatalysis11a−c and in nonproteinogenic amino
acids13,19,20 led us to undertake the current study. We report
herein the first example of highly efficient, enantioselective, and
scalable amidase-catalyzed desymmetrization of α-substituted-
α-amino malonamides. The reaction produces various function-
alized α-tetrasubstituted α-amino acids in high yields with
excellent enantiomeric excesses. The synthetic versatility of
densely functional products resulting from biotransformation
has been demonstrated by practical and expedient synthesis of
enantiopure α-substituted serine analogues and diamino alcohol
products of pharmaceutical interest.
We initiated our biotransformation study with the examina-
tion of the reaction of prochiral α-allyl-α-aminomalonamide 5a.
Catalyzed by Rhodococcus erythropolis AJ270 whole cells in
neutral phosphate buffer at 30 °C, malonamide 5a underwent
very rapid and highly enantioselective hydrolysis to afford,
within 40 min, (R)-2-amino-2-carbamoylpent-4-enoic acid (R)-
6a in 87% yield with 97.0% ee (entry 1, Table 1). Encouraged
by the high efficiency and excellent enantioselectivity of the
amidase observed in desymmetrization reaction of 5a, a number
of prochiral α-aminomalonamides that contained various
substituents were subjected to biocatalysis. Gratifyingly, as
compiled in Table 1, under identical mild biocatalytic
conditions, almost all substrates tested were desymmetrically
hydrolyzed, producing highly enantioenriched (R)-α-amino
acids in good to excellent yields. For example, α-amino-
malonamides that have an α-(E)-but-2-enyl (5b) and an α-2-
methylallyl substituent (5c) were transformed almost quanti-
tatively within 1 h into the corresponding α-amino acids 6b and
6c with ee's of 97.4% and 98.6%, respectively (entries 2 and 3,
Table 1). Introduction of an (E)-cinnamyl group at the α-
position led to equally enantioselective hydrolysis of 5d, which
yielded 6d in 74% yield and 94.0% ee, albeit at a slower reaction
rate due to most likely the steric effect of the phenyl group
(entry 4, Table 1). Fast and almost complete conversion and
enantioselectivity was observed in the desymmetrization of α-
but-3-enyl-substituted α-aminomalonamide 5e, a substrate
contains a terminal olefin moiety γ-positioned to stereogenic
center. Enantiopure R-6e was produced in 96% yield from
incubation of 5e with microbial cells in less than 30 min (entry
5, Table 1). It is noteworthy that α-aminomalonamides bearing
substituents other than alkene were also excellent substrates
that were recognized nicely by the amidase. The biotransfor-
mation of alkyne-bearing substrates 5f and 5g, which contained,
respectively, a prop-2-ynyl and a but-2-ynyl group, thus
proceeded efficiently around 30 min to furnish an excellent
enantiomeric excess value (96.8%) of (R)-2-amino-2-carba-
moylpent-4-ynoic acid 6f in 80% yield and (R)-2-amino-2-
carbamoylhex-4-ynoic acid 6g in 94% yield (entries 6 and 7,
Table 1). Interestingly, the amidase within Rhodococcus
erythropolis AJ270 exhibited similarly excellent enantioselectiv-
ity against α-aminomalonamides that contain a simple alkyl
group. This has been exemplified by the hydrolytic desymmet-
RESULTS AND DISCUSSION
■
Starting prochiral α-substituted-α-amino malonamides 5 were
easily prepared from diethyl α-aminomalonate 1, a commer-
cially available compound (Scheme 1). According to a reported
method,29 diethyl α-aminomalonate 1 reacted with allyl
bromide, (E)-1-bromobut-2-ene, 3-bromo-2-methylprop-1-
ene, 1-bromobut-2-yne, and methyl iodide in the presence of
sodium ethoxide at 0 °C to produce the corresponding α-
substituted-α-aminomalonates 2a−c, 2g, and 2j. Other α-
substituted-α-aminomalonates such as 2d, 2e, 2f, and 2h were
obtained from the alkylation of imine of diethyl α-amino-
malonate 130 with (E)-(3-bromoprop-1-enyl)benzene, 4-
bromobut-1-ene, 3-bromoprop-1-yne, and benzyl bromide,
respectively, followed by hydrolysis of imines 4 under acidic
conditions.30 Diethyl α-amino-α-propylmalonate 2i was
synthesized from catalytic hydrogenation of diethyl α-allyl-α-
aminomalonate 2a. All malonates 2 were converted effectively
into prochiral α-substituted-α-aminomalonamides 5a−j by
means of ammonolysis with aqueous ammonia in methanol
at 0 °C.31
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a
rization of α-benzylated and α-propylated α-aminomalona-
mides 5h and 5i, which led to the high-yielding formation of
(R)-2-amino-2-carbamoyl-3-phenylpropanoic acid 6h and (R)-
2-amino-2-carbamoylpentanoic acid 6i in highly enantiomeri-
cally pure form (entries 8 and 9, Table 1). Only in the case of
biotransformation of α-amino-α-methylmalonamide 5j was low
enantioselectivity obtained (entry 10, Table 1).
Table 1. Biocatalytic Hydrolysis of Malonamides 5
It is very interesting to note that Rhodococcus erythropolis
AJ270-catalyzed desymmetrization reaction of prochiral α-
substituted α-aminomalonamides 5 proceeds much more
efficiently and enantioselectively than that of prochiral α-
substituted α-methylmalonamides and α,α-unsymmetrically
disubstituted malonamides.23 In other words, all prochiral α-
substituted α-aminomalonamides 5 tested in the current study
are excellent substrates for the amidase involved in microbial
whole cells. To shed light on the effect of amino group on the
biocatalytic desymmetrization, α-methylamino- and α-dimethy-
lamino-substituted malonamide substrates 5k and 5l were
incubated with biocatalyst under identical conditions. Intrigu-
ingly, biocatalytic hydrolysis of 5k proceeded very slowly, with
only about half of the substrate being converted into product
6k with 64.6% ee in 7 days (entry 11, Table 1). α-
Dimethylamino-α-allylmalonamide 5l was not accepted at all
by the amidase, and nearly quantitative reactant was recovered
after 7 days’ incubation with biocatalyst (entry 12, Table 1).
Although detailed mechanism of the amidase awaits further
investigation, hypothetically, there is most probably an
enantioselective pocket which recognizes the amino (NH2)
moiety of the substrates 5.
As a microbial whole catalyst, Rhodococcus erythropolis AJ270
has been found to be robust and tolerant to high concentrations
of many amide substrates and acid products, allowing the
scaled-up synthesis of desired chiral products.32 To demon-
strate the practical preparative biotransformation, immobilized
whole cell catalyst was employed and its recyclability was
examined. Using Rhodococcus erythropolis AJ270 whole cells
immobilized in alginate,33 reiterative incubation of biocatalyst
with 5a (10 mmol ×8) afforded (R)-6a (9.3 g, 74%) with 95.8%
ee.
Densely functionalized α-amino acids 6 from biocatalytic
desymmetrization are conceivably versatile organic entities in
synthesis based on functional group transformations. To
demonstrate their potential in organic synthesis and also to
obtain the enantiomeric excess values of products, all
compounds 6 were first of all derivatized into their benzyl
esters 7 (Scheme 2), which were successfully resolved on a
column coated with chiral stationary phase (see Supporting
Information). To further show synthetic application and to
determine its absolute configuration as well, (R)-6a was
converted conveniently into α-substituted serine analogues,
compounds that are useful in the study of peptide or protein
mimics.24,25 As illustrated in Scheme 2, treatment of (R)-6a
with diazomethane gives ester (R)-8 in 78% yield. Selective
reduction of the ester group of (R)-8 using a combination of
NaBH4 and LiCl·H2O34 followed by amide hydrolysis under
basic conditions results in the quantitative formation of (S)-
(−)-α-allylserine 9. Catalytic hydrogenation of (S)-(−)-9
affords (S)-(+)-α-propylserine 10 almost quantitatively.
Comparison of the optical rotation of 10 with that reported
in the literature35 allowed us to assign the absolute
configuration of (S)-10 and therefore of (R)-6a. The synthetic
versatility of (R)-6a was further demonstrated by the synthesis
of diamino alcohol (R)-(+)-12, a key precursor to chiral cyclic
a
Substrate 5 (2 mmol) was incubated with Rhodococcus erythropolis
AJ270 cells (2 g wet weight) in phosphate buffer (pH 7.0) at 30 °C.
b
c
Isolated yield. Determined by chiral HPLC analysis of their benzyl
d
ester derivatives 7 (see Scheme 2). Starting material 5k (42%) was
recovered. Starting material 5l (98%) was recovered.
e
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2.13 (s, 2H), 1.68 (t, d = 6.0 Hz, 3H), 1.29 (t, J = 7.2 Hz, 6H)], 2c
[0.38 g, 33%; H NMR (300 MHz, CDCl3) δ 4.94 (s, 1H), 4.84 (s,
Scheme 2. Synthesis of α-Substituted Serine and Diamino
Alcohol Derivatives
1
1H), 4.22 (q, J = 7.2 Hz, 4H), 2.78 (s, 2H), 2.08 (s, 2H), 1.69 (s, 3H),
1
1.28 (t, J = 7.2 Hz, 6H)], 2f [0.51 g, 48%; H NMR (300 MHz,
CDCl3) δ 4.30−4.20 (m, 4H), 2.92 (d, J = 2.4 Hz, 2H), 2.28 (s, 2H),
2.08 (t, J = 2.4 Hz, 1H), 1.28 (t, J = 7.2 Hz, 6H)], or 2j [0.30 g, 32%;
1H NMR (300 MHz, CDCl3) δ 4.22 (q, J = 7.2 Hz, 4H), 2.08 (s, 2H),
1.54 (s, 3H), 1.27 (t, J = 7.2 Hz, 6H)]. The products were used
directly for the next step without further characterization.
Preparation of α-Substituted α-Aminomalonates 2d, 2e, 2g,
and 2h (Method B).30 Synthesis of Imine 3. A mixture of the
hydrochloric acid salt of diethyl α-aminomalonate 1 (1.05 g, 5 mmol),
benzophenone (910 mg, 5 mmol), p-TSA (95 mg, 0.5 mmol), and
toluene (50 mL) was refluxed overnight using a Dean−Stark apparatus
to remove water. The solvent was then removed under vacuum. The
residue was chromatographed on a silica gel column using a mixture of
petroleum ether and ethyl acetate (15:1−10:1) as the mobile phase to
give imine intermediate 3 (900 mg, 53%): 1H NMR (300 MHz,
CDCl3) δ 7.69−7.18 (m, 10H), 4.85 (s, 1H), 4.30−4.20 (m, 4H), 1.28
(t, J = 7.2 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 173.9, 167.2, 139.0,
135.5, 130.9, 129.3, 129.1, 128.7, 127.8, 69.7, 61.9, 14.1.
Alkylation of Imine 3. To a solution of sodium hydride (0.65 g, 26
mmol) in dry THF (125 mL) and DMF (15 mL) or potassium
carbonate (3.59 g, 26 mmol) in acetone (125 mL) or acetonitrile (125
mL) was added imine intermediate 3 (8.5 g, 25 mmol). After the
mixture was stirred at room temperature for 1 h, RBr (30 mmol) was
added through a syringe. After the resulting mixture was stirred at
room temperature or at refluxing temperature for another 12 h, the
reaction was quenched by addition of water (300 mL) and then
extracted with ethyl acetate (3 × 150 mL). The organic layer was dried
with anhydrous MgSO4, and the solvent was removed under vacuum.
The residue was chromatographed on a silica gel column using a
mixture of petroleum ether and ethyl acetate (30:1−20:1) as the
mobile phase to give 4d (8.19 g, 72%), 4e (6.29 g, 64%), 4g (3.71 g,
38%), or 4 h (8.47 g, 79%). All intermediates were used directly for
the next step without characterization.
urea derivatives that are very potent and selective NK1 receptor
antagonists.36 Thus, interaction of (R)-6a with 2 equiv of
benzyl bromide gave rise to α-(benzylamino) ester intermediate
(R)-(+)-7a in a high yield. Serine amide analogue (S)-(+)-11
was obtained in a quantitative yield from selective reduction of
ester of (R)-(+)-7a by NaBH4 in the presence of LiCl·H2O.34
Reduction of amide by LiAlH4 furnished diamino alcohol
product (R)-(+)-12. Catalytic hydrogenation of (S)-(+)-11 led
simultaneously to the reduction of the carbon−carbon double
bond and the cleavage of the N-benzyl group to produce, after
hydrolysis, (S)-(+)-α-propylserine 10 in 91% yield (Scheme 2).
CONCLUSION
■
In conclusion, we have shown a highly efficient and
enantioselective biocatalytic desymmetrization reaction using
Rhodococcus erythropolis AJ270 whole cell catalyst under mild
conditions. The amidase-catalyzed hydrolysis of prochiral α-
substituted α-aminomalonamides provides a convenient and
straightforward synthetic route to densely functionalized α-
tetrasubstituted α-amino acids in high yields with excellent
enantiomeric excesses. The resulting enantioenriched products,
which are not readily available by other chemical and
biochemical means, are invaluable chiral building blocks in
organic synthesis, and their applications have been demon-
strated by the practical and expedient preparation of α-
substituted or functionalized serine analogues and diamino
alcohol.
Hydrolysis of Imine 4. To a mixture of ether (5 mL) and
hydrochloric acid (1 N, 1 mL) was added 4 (1 mmol). After the
mixture was stirred at room temperature overnight, a solution of
sodium hydroxide (2 N) was added to basify the mixture to pH = 8.
Water (10 mL) was added, and the mixture was then extracted with
ethyl acetate (3 × 20 mL). The organic layer was dried with anhydrous
MgSO4, and the solvent was removed under vacuum. The residue was
chromatographed on a silica gel column using a mixture of petroleum
ether and ethyl acetate (3:1−1:1) as the mobile phase to give product
2d (215 mg, 74%), 2e [199 mg, 87%; 1H NMR (300 MHz, CDCl3) δ
5.86−5.76 (m, 1H), 5.08−4.97 (m, 2H), 4.22 (q, J = 7.2 Hz, 4H),
1
2.07−2.05 (m, 6H), 1.27 (t, J = 7.2 Hz, 6H)], 2g [127 mg, 56%; H
NMR (300 MHz, CDCl3) δ 4.27−4.20 (m, 4H), 2.86 (q, J = 2.4 Hz,
2H), 2.26 (s, 2H), 1.77 (t, J = 2.4 Hz, 3H), 1.27 (t, J = 7.2 Hz, 6H)],
1
or 2h [209 mg, 79%; H NMR (300 MHz, CDCl3) δ 7.29−7.16 (m,
5H), 4.24 (q, J = 7.2 Hz, 4H), 3.32 (s, 2H), 1.94 (s, 2H), 1.28 (t, J =
7.2 Hz, 6H)]. Compounds were used directly without further
characterization.
EXPERIMENTAL SECTION
■
Preparation of α-Substituted α-Aminomalonates 2a−c, 2g,
and 2j (Method A).29 To a solution of sodium ethoxide (680 mg, 10
mmol) in EtOH (30 mL) was added the hydrochloric acid salt of
diethyl α-aminomalonate 1 (1.05 g, 5 mmol). After the solution was
stirred at 0 °C for 2 h, a solution of RBr or iodomethane (6 mmol) in
EtOH (3 mL) was added through a syringe. The resulting mixture was
stirred at 0 °C for another 3 h. The reaction was quenched by addition
of water (100 mL) at room temperature and then extracted with ethyl
acetate (3 × 50 mL). The organic layer was dried with anhydrous
MgSO4, and the solvent was removed under vacuum. The residue was
chromatographed on a silica gel column using a mixture of petroleum
ether and ethyl acetate (8:1−4:1) as the mobile phase to give product
2a [0.54 g, 50%; 1H NMR (300 MHz, CDCl3) δ 5.77−5.63 (m, 1H),
5.53−5.44 (m, 2H), 4.23 (q, J = 7.2 Hz, 4H), 3.19 (d, J = 7.2 Hz, 2H),
2.13 (s, 1H), 1.67 (d, J = 7.2 Hz, 6H), 1.67 (t, J = 7.2 Hz, 6H)], 2b
Preparation of α-Substituted α-Aminomalonates 2i (Meth-
od C). In the presence of Pd/C (10%, 200 mg), 2a (2.15 g, 10 mmol)
in methanol (50 mL) was hydrogenated using a hydrogen balloon.
When the starting material was consumed, the catalyst was filtered and
the filtrate was concentrated to give diethyl α-amino-α-propylmalonate
2i (2.15 g, 99%): 1H NMR (300 MHz, CDCl3) δ 4.22 (q, J = 7.2 Hz,
4H), 2.02 (s, 2H), 1.96−1.90 (m, 2H), 1.36−1.25 (m, 8H), 0.95 (t, J =
7.2 Hz, 3H). It was used directly for the next step without further
characterization.
Preparation of α-Substituted α-Aminomalonamides 5.31
A
mixture of α-substituted α-aminomalonates 2a−j (2 mmol), ammonia
solution (NH3·H2O, 25% w/w, 4 mL), and MeOH (4 mL) was stirred
at 0 °C until the reactant was consumed. The reaction mixture was
then neutralized with hydrochloric acid (1 N). After removal of solvent
under vacuum, the residue was chromatographed on a silica gel
column using ethyl acetate as the mobile phase to give product 5a−j.
1
[0.33 g, 29%; H NMR (300 MHz, CDCl3) δ 5.66−5.57 (m, 1H),
5.23−5.17 (m, 1H), 4.23 (q, J = 7.2 Hz, 4H), 3.19 (d, J = 7.2 Hz, 2H),
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Except for 5h, all products 5 are new compounds. Spectroscopic data
and microanalyses of 5a−j were listed as follows.
7.41 (s, 2H), 7.24 (s, 2H), 2.30 (s, 2H), 1.37 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 175.3, 61.3, 26.1; MS (ESI) m/z 154 [M + Na]+
(25), 132 [M + H]+ (100). Anal. Calcd for C4H9N3O2: C, 36.64; H,
6.92; N, 32.04. Found: C, 36.69; H, 6.95; N, 31.95.
Preparation of 5k and 5l. A mixture of 1a (157 mg, 1 mmol),
K2CO3 (35 mg, 0.25 mmol), CH3I (1 mL, 16 mmol), and DMF (1
mL) was stirred at room temperature for 2 h. After removal of solvent,
the residue was chromatographed on a silica gel column eluted with a
mixture of petroleum ether, ethyl acetate and methanol (1:2:0.05−
0:2:0.05) to give pure 5k and 5l along with the recovery of 1a (80 mg,
51%).
2-Allyl-2-(methylamino)malonamide (5k): 53 mg, 31%; 179 °C
sublimation; IR (KBr) ν, 3389, 1658 cm−1; 1H NMR (300 MHz,
CDCl3) δ 7.83 (s, 2H), 5.80−5.64 (m, 3H), 5.19−5.14 (m, 2H), 2.67
(d, J = 7.2 Hz, 2H), 2.35 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
173.5, 131.2, 119.7, 69.6, 42.3, 30.6; MS (ESI) m/z 194 [M + Na]+
(17), 172 [M + H]+ (100). Anal. Calcd for C7H13N3O2: C, 49.11; H,
7.65; N, 24.54. Found: C, 49.02; H, 7.65; N, 24.36.
2-Allyl-2-aminomalonamide (5a). 160 mg, 51%; mp 106−107 °C;
IR (KBr) ν 3430, 3339, 1667 cm−1; 1H NMR (300 MHz, CDCl3) 7.82
(s, 2H), 5.96 (s, 2H), 5.75−5.61 (m, 1H), 5.25−5.17 (m, 2H), 70 (d, J
= 7.2 Hz, 2H), 2.16 (s, 2H); 13C NMR (75 MHz, CDCl3) 174.8,
131.4, 121.0, 63.8, 45.4; MS (ESI) m/z 180 [M + Na]+ (67), 158 [M +
H]+ (100). Anal. Calcd for C6H11N3O2: C, 45.85; H, 7.05; N, 26.74.
Found: C, 45.68; H, 7.12; N, 26.66.
(E)-2-Amino-2-(but-2-en-1-yl)malonamide (5b). 161 mg, 47%; mp
135−136 °C; IR (KBr) ν 3395, 3187, 1687, 1352 cm−1; 1H NMR (300
MHz, DMSO-d6) 7.46 (s, 2H), 7.32 (s, 2H) 5.56−5.48 (m, 1H),
5.31−5.23 (m, 1H), 2.47 (d, J = 7.2 Hz, 2H), 2.12 (s, 2 H), 1.62 (d, J =
6.0 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) 174.0, 129.3, 125.3,
64.1, 42.1, 17.9; MS (ESI) m/z 194 [M + Na]+ (20), 172 [M + H]+
(100). Anal. Calcd for C7H13N3O2: C, 49.11; H, 7.65; N, 24.54.
Found: C, 49.29; H, 7.63; N, 24.94.
2-Amino-2-(2-methylallyl)malonamide (5c). 154 mg, 45%; mp
133−134 °C; IR (KBr) ν 3409, 3269, 1670, 1573 cm−1; 1H NMR (300
MHz, DMSO-d6) 7.52 (s, 2H), 7.38 (s, 2H) 4.85 (s, 1H), 4.77 (s, 1H),
2.57 (s, 2H), 2.14 (s, 2H), 1.66 (s, 3H); 13C NMR (75 MHz,
DMSODMSO-d6) 174.2, 141.0, 115.7, 63.3, 46.4, 22.7; MS (ESI) m/z
194 [M + Na]+ (20), 172 [M + H]+ (100). Anal. Calcd for
C7H13N3O2: C, 49.11; H, 7.65; N, 24.54. Found: C, 49.16 7.63; N,
24.69.
2-Allyl-2-(dimethylamino)malonamide (5l): 32 mg, 17%; mp
1
101−102 °C; IR (KBr) ν, 3432, 3279, 1658 cm−1; H NMR (300
MHz, CDCl3) δ 7.66 (s, 2H), 7.33 (s, 2H), 5.77−5.67 (m, 1H), 5.09−
4.97 (m, 2H), 2.61 (d, J = 7.2 Hz, 2H), 2.32 (s, 6H); 13C NMR (75
MHz, DMSO-d6) δ 172.4, 134.0, 117.4, 72.7, 40.3, 38.7; MS (ESI) m/
z 208 [M + Na]+ (43), 286 [M + H]+ (100). Anal. Calcd for
C8H15N3O2: C, 51.88; H, 8.16; N, 22.69. Found: C, 51.63; H, 7.92; N,
22.62.
2-Amino-2-cinnamylmalonamide (5d). 224 mg, 48%; mp 120−
1
121 °C; IR (KBr) ν 3430, 3252, 1683 cm−1; H NMR (300 MHz,
General Procedure for the Biocatalytic Desymmetrization of
Prochiral Dicarboxamides 5. To an Erlenmeyer flask (150 mL)
with a screw cap were added Rhodococcus erythropolis AJ270 cells12a,14
(2 g wet weight) and potassium phosphate buffer (0.1 M, pH 7.0, 50
mL), and the resting cells were activated at 30 °C for 0.5 h with orbital
shaking. A prochiral diamide substrate 5a−l (2 mmol) was added in
one portion to the flask, and the mixture was incubated at 30 °C using
an orbital shaker (200 rpm). The reaction, monitored by TLC and
GC, was quenched after a specified period of time (Table 1) by
filtration of the mixture through a Celite pad. The aqueous filtrate was
freeze-dried, and the residue was chromatographed on a reversed-
phase silica gel column (SP-120-50-ODS-A) using water as mobile
phase or on an ion-exchange resin column (Dowex50wx8) using
aqueous ammonium solution (10%) as mobile phase to give product
6a−k. Except for 6j, products 6 are new compounds. All products were
fully characterized by means of spectroscopic data and microanalyses.
(R)-2-Amino-2-carbamoylpent-4-enoic acid (6a): 275 mg, 87%;
mp 145−146 °C; [α]25D −22.4 (c 1.70, H2O); IR (KBr) ν 3397, 1668,
DMSO-d6) 7.36 (s, 2H), 7.34−7.28 (m, 6H), 7.2−7.19 (m, 1), 6.49
(d, J = 15.0 Hz, 1H), 6.17−6.07 (m, 1H), 2.71 (d, J = 7.3, 2H), 2.27 (s,
2H); 13C NMR (75 MHz, DMSO-d6) 173.9, 136.9, 133.3, 128.5,
127.2, 126.0, 124.8, 64.5, 42.3; MS (ESI) m/z 256 [M + Na]+ (19),
234 [M + H]+ (100). Anal. Calcd for C12H15N3O2: C, 61.79; H, 6.48
N, 18.01. Found: C, 62.10; H, 6.54; N, 17.85.
2-Amino-2-(but-3-en-1-yl)malonamide (5e): 109 mg, 32%; mp
1
133−134 °C; IR (KBr) ν 3416, 1657 cm−1; H NMR (300 MHz,
DMSO-d6) 7.82 (s, 2H), 5.91 (s, 2H), 5.85−5.72 (m, 1H), 5.08−4.97
(m, 2H), 2.14−2.01 (m, 6H); 13C NMR (75 MHz, DMSO-d6) 172.4,
134.2, 113.1, 62.2, 37.2, 25.5; MS (ESI) m/z 194 [M + Na]+ (38), 172
[M + H]+ (100). Anal. Calcd for C7H13N3O2: C, 49.11; H, 7.65; N,
24.54. Found: C, 48.91; H, 7.36; N, 24.41.
2-Amino-2-(prop-2-yn-1-yl)malonamide (5f): 167 mg, 54%; mp
1
122−123 °C; IR (KBr) ν 3400, 1689 cm−1; H NMR (300 MHz,
DMSO-d6) 7.42 (s, 2H), 7.38 (s, 2H), 2.87(s, 2H), 2.73 (d, J = 2.2,
2H), 2.33 (s, 2H); 13C NMR (75 MHz, DMSO-d6) 172.5, 80.5, 73.7,
63.9, 28.6; MS (ESI) m/z 156 [M + H]+ (100). Anal. Calcd for
C6H9N3O2: C, 46.45; H, 5.85; N, 27.08. Found: C, 46.43; H, 5.87; N,
26.78.
1
1516 cm−1; H NMR (300 MHz, D2O) 5.67−5.53 (m, 1H), 5.25−
5.19 (m, 2H), 2.84−2.69 (m, 2H); 13C NMR (75 MHz, D2O) 170.9,
170.5, 129.3, 122.2, 67.0, 40.3; MS (ESI) m/z 157 [M − H]− (100),
calcd for C6H10N2O3 159.0764 [M + H]+, found (P-SIMS) 159.0765
[M + H]+.
2-Amino-2-(but-2-yn-1-yl)malonamide (5g): 176 mg, 52%; mp
1
168−169 °C; IR (KBr) ν 3394, 3181, 1680 cm−1; H NMR (300
MHz, DMSO-d6) 7.40 (s, 2H), 7.34 (s, 2H), 2.69 (d, J = 2.4 Hz, 2H),
2.29 (s, 2H), 1.73 (s, 3H); 13C NMR (75 MHz, DMSO-d6) 172.8,
78.5, 75.0, 64.0, 29.2, 3.2; MS (ESI) m/z 192 [M + Na]+ (68), 170 [M
+ H]+ (100). Anal. Calcd for C7H11N3O2: C, 49.70; H, 6.55; N, 24.84.
Found: C, 49.75; H, 6.43; N, 24.70.
(R,E)-2-Amino-2-carbamoylhex-4-enoic acid (6b): 320 mg, 93%;
mp 148−149 °C; [α]25D −18.7 (c 1.50, H2O); IR (KBr) ν 3326, 1685,
1
1508 cm−1; H NMR (300 MHz, D2O) 5.78−5.66 (m, 1H), 5.32−
5.22 (m, 1H), 2.87−2.66 (m, 2H), 1.61 (d, J = 6.0 Hz, 3H); 13C NMR
(75 MHz, D2O) 170.9, 170.6, 134.1, 121.2, 67.3, 39.4, 17.3; MS (ESI)
m/z 195 [M + Na]+ (33), 173 [M + H]+ (100); calcd for C7H12N2O3
173.0921 [M + H]+, found (P-SIMS) 173.0922 [M + H]+.
2-Amino-2-benzylmalonamide (5h): 207 mg, 50%; mp 175−176
°C; IR (KBr) ν 3421, 3206, 1687 cm−1; 1H NMR (300 MHz, DMSO-
d6) 7.49 (s, 2H), 7.41 (s, 2H), 7.29−7.21 (m, 5H), 3.11 (s, 2H), 2.06
(s, 2H); 13C NMR (75 MHz, DMSO-d6) 173.7, 136.3, 130.2, 127.9,
126.6, 65.3, 43.8; MS (ESI) m/z 230 [M + Na]+ (100), 208 [M + H]+
(90). Anal. Calcd for C10H13N3O2: C, 57.96; H, 6.32; N, 20.28. Found:
C, 57.79; H, 6.40; N, 20.49.
(R)-2-Amino-2-carbamoyl-4-methylpent-4-enoic acid (6c): 337
mg, 98%; mp 139−140 °C; [α]25 −20.6 (c 1.55, H2O); IR (KBr) ν
D
3128, 1685, 1501 cm−1; 1H NMR (300 MHz, D2O) 5.01 (s, 1H), 4.87
(s, 1H), 4.87 (s, 1H), 2.91 (d, J = 14.4 Hz, 1H), 2.78 (d, J = 14.4 Hz,
1H), 1.69 (s, 3H); 13C NMR (75 MHz, D2O) 171.4, 170.9, 138.7,
117.9, 66.3, 43.7, 21.8; MS (ESI) m/z 171 [M − H]− (100), calcd for
C7H12N2O3 173.0921 [M + H]+, found (P-SIMS) 173.0923 [M + H]+.
(R,E)-2-Amino-2-carbamoyl-5-phenylpent-4-enoic acid (6d): 346
2-Amino-2-propylmalonamide (5i): 108 mg, 34%; mp 168−169
1
°C; IR (KBr) ν 3374, 1678 cm−1; H NMR (300 MHz, DMSO-d6)
7.49 (s, 2H), 7.33 (s, 2H), 2.24 (s, 2H), 1.76−1.70 (m, 2H), 1.28−
1.16 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, DMSO-
d6) 177.5, 66.3, 43.2, 18.2, 14.4; MS (ESI) m/z 160 [M + H]+ (100).
Anal. Calcd for C6H13N3O2: C, 45.27; H, 8.23; N, 26.40. Found: C,
45.30; H, 8.18; N, 26.18.
mg, 74%; mp 162−163 °C; [α]25 +11.8 (c 0.85, DMF); IR (KBr) ν
D
3140, 1682, 1507 cm−1; 1H NMR (300 MHz, DMSO-d6) 7.76 (s, 2H),
7.40−7.20 (m, 5H), 6.47 (d, J = 12.8 Hz, 1H), 6.21−6.11 (m, 1H),
2.85−2.81 (m, 2H); 13C NMR (75 MHz, MeOD/DMSO-d6) 170.4,
166.9, 136.9, 133.4, 128.4, 127.3, 126.2, 123.5, 66.4, 40.2; MS (ESI)
2-Amino-2-methylmalonamide (5j): 145 mg, 57%; mp 218−219
°C; IR (KBr) ν, 3396, 1673 cm−1; 1H NMR (300 MHz, DMSO-d6) δ
5588
dx.doi.org/10.1021/jo3007122 | J. Org. Chem. 2012, 77, 5584−5591

The Journal of Organic Chemistry
Article
m/z 233 [M − H]− (100); cCalcd for C12H14N2O3 235.1077 [M +
cm−1; H NMR (300 MHz, CDCl3) 7.36−7.20 (m, 10H), 7.11 (s,
1H), 5.75 (s, 1H), 5.62−5.50 (m, 1H), 5.36−5.15 (m, 3H), 3.63 (d, J
= 12.3 Hz, 1H), 3.56 (d, J = 12.0 Hz, 1H), 2.91−2.76 (m, 2H), 2.31 (s,
1H), 1.63 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) 172.3,
170.8, 139.0, 135.4, 130.5, 128.6, 128.55, 128.47, 128.2, 127.4, 123.7,
70.6, 67.4, 47.6, 35.6, 18.1; MS (ESI) m/z 375 [M + Na]+ (17), 353
[M + H]+ (100). Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86; N,
7.95. Found: C, 71.50; H, 6.87; N, 8.02.
1
H]+, found (P-SIMS) 235.1079 [M + H]+.
(R)-2-Amino-2-carbamoylhex-5-enoic acid (6e): 330 mg, 96%; mp
158−159 °C; [α]25 −28.1 (c 1.85, H2O); IR (KBr) ν 3332, 1682,
D
1
1508 cm−1; H NMR (300 MHz, D2O) 5.86−5.72 (m, 1H), 5.09−
4.98 (m, 2H), 2.18−2.14 (m, 2H), 2.08−2.05 (m, 2H); 13C NMR (75
MHz, D2O) 171.2, 170.7, 136.3, 116.1, 67.6, 35.2, 27.6; MS (ESI) m/z
171 [M − H]− (100); calcd for C7H12N2O3 173.0921 [M + H]+,
found (P-SIMS) 173.0922 [M + H]+.
Benzyl (R)-2-(benzylamino)-2-carbamoyl-4-methylpent-4-enoate
(7c): 137 mg, 67%; mp 139−140 °C; [α]25D +7.62 (c 1.45, CHCl3); ee
= 98.6% (chiral HPLC analysis); IR (KBr) ν 3382, 1740, 1680, 1213
(R)-2-Amino-2-carbamoylpent-4-ynoic acid (6f): 251 mg, 80%;
mp 125−126 °C; [α]25 −5.7 (c 1.75, H2O); IR (KBr) ν 3395, 1671,
D
1
cm−1; H NMR (300 MHz, CDCl3) 7.35−7.21 (m, 11H), 5.67 (s,
1513 cm−1; 1H NMR (300 MHz, D2O) 2.98 (s, 2H), 2.49 (s, 1H); 13
C
1H), 5.26 (d, J = 12.3 Hz, 1H), 5.19 (d, J = 12.3 Hz, 1H), 4.87 (d, J =
17.4 Hz, 2H), 3.67 (d, J = 12.0 Hz, 1H), 3.61 (d, J = 12.0 Hz, 1H),
2.97 (d, J = 12.0 Hz, 1H), 2.91 (d, J = 12.0 Hz, 1H), 2.34 (s, 1H), 1.76
(s, 3H); 13C NMR (75 MHz, CDCl3) 172.3, 170.9, 140.3, 139.0,
135.2, 128.60, 128.55, 128.47, 128.4, 128.2, 127.4, 115.6, 70.6, 67.5,
47.9, 39.7, 23.7; MS (ESI) m/z 375 [M + Na]+ (17), 353 [M + H]+
(100). Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86; N, 7.95. Found:
C, 71.59; H, 6.78; N, 7.81.
NMR (75 MHz, D2O) 171.4, 170.9, 77.1, 74.0, 66.0, 26.5; MS (ESI)
m/z 155 [M − H]− (100); calcd for C6H8N2O3 157.0608 [M + H]+,
found (P-SIMS) 157.0609 [M + H]+.
(R)-2-Amino-2-carbamoylhex-4-ynoic acid (6g): 320 mg, 94%; mp
129−130 °C; [α]25 −27.7 (c 1.56, H2O); IR (KBr) ν 3382, 1697,
D
1335 cm−1; 1H NMR (300 MHz, D2O) 2.91 (s, 2H), 1.69 (s, 3H); 13
C
NMR (75 MHz, D2O) 171.0, 170.6, 82.4, 71.1, 66.5, 26.9, 2.5; MS
(ESI) m/z 169 [M − H]− (100); calcd for C7H10N2O3 171.0764 [M +
H]+, found (P-SIMS) 171.0762 [M + H]+.
Benzyl (R,E)-2-(benzylamino)-2-carbamoyl-5-phenylpent-4-
enoate (7d): 131 mg, 74%; mp 125−126 °C; [α]25 +16.9 (c 1.30,
(R)-2-Amino-2-carbamoyl-3-phenylpropanoic acid (6h): 347 mg,
D
83%; mp 148−149 °C; [α]25D −23.3 (c 0.60, DMF); IR (KBr) ν 3377,
CHCl3); ee = 94.0% (chiral HPLC analysis); IR (KBr) ν 3423, 1727,
1696, 1190 cm−1; 1H NMR (300 MHz, CDCl3) 7.37−7.22 (m, 15H),
7.10 (s, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.11−6.00 (m, 1H), 5.76 (s,
1H), 5.26 (d, J = 12.0 Hz, 1H), 5.19 (d, J = 12.0 Hz, 1H), 3.70 (d, J =
12.3 Hz, 2H), 3.62 (d, J = 12.3 Hz, 1H), 3.13−2.97 (m, 2H), 2.36 (s,
1H); 13C NMR (75 MHz, CDCl3) 172.1, 170.7, 138.9, 137.0, 135.4,
134.5, 128.7, 128.60, 128.58, 128.52, 128.2, 127.52, 127.49, 126.3,
122.9, 70.9, 67.6, 47.8, 36.0; MS (ESI) m/z 415 [M + H]+ (100). Anal.
Calcd for C26H26N2O3: C, 75.34; H, 6.32; N, 6.76. Found: C, 75.14;
H, 6.29; N, 6.83.
1
1682, 1503 cm−1; H NMR (300 MHz, D2O) 7.34−7.22 (m, 5H),
3.44 (d, J = 14.4 Hz, 1H), 3.33 (d, J = 14.4 Hz, 1H); 13C NMR (75
MHz, D2O) 171.0, 170.6, 132.9, 129.9, 129.1, 128.3, 68.3, 41.6; MS
(ESI) m/z 207 [M − H]− (100); calcd for C10H12N2O3 209.0921 [M
+ H]+, found (P-SIMS) 209.0921 [M + H]+.
(R)-2-Amino-2-carbamoylpentanoic acid (6i): 269 mg, 84%; mp
163−164 °C; [α]25 −22.1 (c 1.90, H2O); IR (KBr) ν 3441, 1681,
D
1
1508 cm−1; H NMR (300 MHz, D2O) 2.05−1.88 (m, 2H), 1.32−
1.08 (m, 2H), 0.81 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, D2O)
171.5, 171.1, 67.8, 38.1, 16.9, 12.9; MS (ESI) m/z 159 [M − H]−
(100); calcd for C6H12N2O3 161.0921 [M + H]+, found (P-SIMS)
161.0921 [M + H]+.
Benzyl (R)-2-(benzylamino)-2-carbamoylhex-5-enoate (7e): 126
mg, 61%; mp 78−79 °C; [α]25 +12.4 (c 1.45, CHCl3); ee >99.5%
D
(chiral HPLC analysis); IR (KBr) ν 3439, 1729, 1644, 1221 cm−1; 1H
NMR (300 MHz, CDCl3) 7.64−7.19 (m, 10H), 7.09 (s, 1H), 5.83−
5.72 (m, 2H), 5.27 (d, J = 12.0 Hz, 1H), 5.21 (d, J = 12.0 Hz, 1H),
5.15−4.95 (m, 2H), 3.59 (d, J = 12.0 Hz, 1H), 3.48 (d, J = 12.0 Hz,
1H), 2.23−2.02 (m, 5H); 13C NMR (75 MHz, CDCl3) 172.4, 171.1,
138.8, 137.2, 135.4, 128.63, 128.59, 128.50, 128.46, 128.2, 127.5,
115.3, 70.5, 67.4, 47.8, 31.2, 27.7; MS (ESI) m/z 375 [M + Na]+ (12),
353 [M + H]+ (100). Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86;
N, 7.95. Found: C, 71.56; H, 6.77; N, 8.02.
(R)-2-Amino-2-carbamoylpropanoic acid (6j): 211 mg, 80%; mp
216−217 °C; [α]25D +6.1 (c 1.65, H2O); IR (KBr) ν 3370, 1634, 1097
cm−1; 1H NMR (300 MHz, D2O) δ 1.62 (s, 3H); 13C NMR (75 MHz,
D2O) δ 172.0, 171.7, 63.8, 21.8; MS (ESI) m/z 133 [M + 1]+ (100);
calcd for C4H8N2O3 133.0608 [M + 1]+, found (P-SIMS) 133.0608
[M + 1]+.
(R)-2-Carbamoyl-2-(methylamino)pent-4-enoic acid (6k): 182
mg, 53%; mp 177−178 °C; [α]25 −10.0 (c 0.60, H2O); IR (KBr)
D
ν 3352, 1676, 1399 cm−1; 1H NMR (300 MHz, D2O) δ 5.69−5.55 (m,
1H), 5.32−5.23 (m, 2H), 2.93−2.80 (m, 2H), 2.59 (s, 3H); 13C NMR
(75 MHz, D2O) δ 170.2, 169.5, 129.0, 122.0, 72.6, 37.4, 29.0; MS
(ESI) m/z 171 [M + H]+ (100); calcd for C7H10N2O3 173.0921 [M +
1]+, found (P-SIMS) 173.0921 [M + 1]+.
Benzyl (R)-2-(benzylamino)-2-carbamoylpent-4-ynoate (7f): 127
mg, 59%; mp 113−114 °C; [α]25D +24.6 (c 1.30, CHCl3); ee = 96.8%
(chiral HPLC analysis); IR (KBr) ν 3430, 1729, 1701, 1026 cm−1; 1H
NMR (300 MHz, CDCl3) 7.35−7.26 (m, 10H), 7.21 (s, 1H), 5.76 (s,
1H), 5.25 (d, J = 12.3 Hz, 1H), 5.20 (d, J = 12.3 Hz, 1H), 3.67 (d, J =
12.0 Hz, 1H), 3.61 (d, J = 12.0 Hz, 1H), 3.22 (dd, J = 17.7, 2.4 Hz,
1H), 3.06 (dd, J = 17.7, 2.4 Hz, 1H), 2.65 (s, 1H), 2.07 (t, J = 2.7 Hz,
1H); 13C NMR (75 MHz, CDCl3) 170.9, 169.3, 138.7, 135.1, 128.6,
128.5, 128.3, 128.2, 127.5, 78.3, 72.4, 69.6, 67.9, 47.3, 22.5; MS (ESI)
m/z 359 [M + Na]+ (40), 337 [M + H]+ (100). Anal. Calcd for
C20H20N2O3: C, 71.41; H,5.99; N, 8.33. Found: C, 71.62; H, 6.07; N,
8.25.
Benzyl (R)-2-(benzylamino)-2-carbamoylhex-4-ynoate (7g): 121
mg, 59%; mp 95−96 °C; [α]25D: +31.1° (c 1.80, CHCl3); ee = 96.8%
(chiral HPLC analysis); IR (KBr) ν 3430, 3252, 1702, 1185 cm−1; 1H
NMR (300 MHz, CDCl3) δ 7.33−7.21 (m, 11H), 6.11 (s, 1H), 5.21
(s, 2H), 3.67 (d, J = 12.0 Hz, 1H), 3.59 (d, J = 12.0 Hz, 1H), 3.16 (dd,
J = 17.3, 1.8 Hz, 1H), 2.89 (dd, J = 17.3, 1.8 Hz, 1H), 2.59 (s, 1H),
1.73 (s, 3H); 13C NMR (75 MHz, CDCl3) 171.5, 169.7, 139.1, 135.3,
128.5, 128.3, 128.2, 128.1, 127.4, 79.8, 72.8, 69.9, 67.6, 47.2, 23.0, 3.6;
MS (ESI) m/z 373 [M + Na]+ (22), 351 [M + H]+ (100). Anal. Calcd
for C21H22N2O3: C, 71.98; H, 6.33; N, 7.99. Found: C, 71.98; H, 6.18;
N, 8.15.
General Procedure for the Conversion of Acid Products 6
into Esters 7. To a solution of 6 (100 mg) in DMF (3 mL) were
added K2CO3 (200 mg) and benzyl bromide (0.2 mL). The resulting
mixture was allowed to stir at room temperature overnight. Water (25
mL) was added, and the mixture was extracted with ethyl acetate (3 ×
25 mL). The organic phase was dried with anhydrous MgSO4. After
removal of the solvent under vacuum, the residue was chromato-
graphed on a silica gel column with a mixture of petroleum ether and
ethyl acetate (3:1) as the mobile phase to give products 7.
Benzyl (R)-2-(benzylamino)-2-carbamoylpent-4-enoate (7a): 196
mg, 91%; mp 145−146 °C; [α]25D +17.9 (c 1.90, CHCl3); ee = 97.0%
(chiral HPLC analysis); IR (KBr) ν 3437, 1742, 1689, 1202 cm−1; 1H
NMR (300 MHz, CDCl3) 7.32−7.19 (m, 10H), 7.08 (s, 1H), 5.80−
5.66 (m, 2H), 5.29−5.13 (m, 4H), 3.64 (d, J = 12.0 Hz, 1H), 3.56 (d, J
= 12.0 Hz, 1H), 2.97−2.84 (m, 2H), 2.31 (s, 1H); 13C NMR (75
MHz, CDCl3) 172.2, 170.7, 135.4, 131.5, 128.63, 128.58, 128.51,
128.47, 128.2, 128.1, 127.5, 119.7, 70.5, 67.5, 47.6, 36.7; MS (ESI) m/
z 339 [M + H]+ (100). Anal. Calcd.for C20H22N2O3: C, 70.99; H, 6.55;
N, 8.28. Found: C, 70.81; H, 6.57; N, 8.25.
Benzyl (R)-2-(benzylamino)-2-carbamoyl-3-phenylpropanoate
Benzyl (R,E)-2-(benzylamino)-2-carbamoylhex-4-enoate (7b):
(7h): 104 mg, 56%; mp 81−82 °C; [α]25 +16.0 (c 2.00, CHCl3);
D
127 mg, 62%; mp 88−89 °C; [α]25 +17.5° (c 1.60 CHCl3); ee =
ee >99.5% (chiral HPLC analysis); IR (KBr) ν 3440, 1714, 1680, 1186
D
1
97.4% (chiral HPLC analysis); IR (KBr) ν 3433, 1745, 1683, 1202
cm−1; H NMR (300 MHz, CDCl3) 7.35−7.14 (m, 15H), 6.89 (s,
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1H), 5.45 (s, 1H), 5.25 (d, J = 12.3 Hz, 1H), 5.16 (d, J = 12.3 Hz, 1H),
3.79 (d, J = 12.2 Hz, 1H), 3.69 (d, J = 12.2 Hz, 1H), 3.47 (s, 2H), 2.19
(s, 1H); 13C NMR (75 MHz, CDCl3) 171.9, 170.6, 135.2, 130.1,
128.64, 128.63, 128.56, 128.4, 128.1, 127.5, 127.1, 72.0, 67.6, 48.1,
38.0; MS (ESI) m/z 389 [M + H]+ (100). Anal. Calcd. for
C24H24N2O3: C, 74.21; H, 6.23; N, 7.21. Found: C, 74.17; H, 6.26;
N, 7.10.
vacuum. The residue was chromatographed on an ion-exchange resin
(Dowex 50WX8, 200−400 mesh) column with aqueous ammonia
(10%) as mobile phase to give products (S)-(−)-9 (46 mg, 99%): mp
178−179 °C (lit.37 mp 215−220 °C); [α]25D = −34.7° (c 0.75, H2O);
1
IR (KBr) ν 3080, 1620 cm−1; H NMR (300 MHz, D2O) 5.72−5.58
(m, 1H), 5.23−5.18 (m, 2H), 3.87 (d, J = 12.3 Hz, 1H), 3.64 (d, J =
12.3 Hz, 1H), 2.53 (dd, J = 14.4, 6.3 Hz, 1H), 2.37 (dd, J = 14.4, 8.4
Hz, 1H); 13C NMR (75 MHz, D2O) 173.8, 129.9, 1214, 65.7, 63.9,
36.7; MS (ESI) m/z 168 [M + Na]+ (23), 146 [M + H]+ (100).
Synthesis of (S)-2-Amino-2-(hydroxymethyl)pentanoic Acid (10).
In the presence of Pd/C catalyst (10%, 10 mg), compound (S)-(−)-9
(30 mg, 0.2 mmol) in methanol (2 mL) was hydrogenated using a
hydrogen balloon. After the starting material was consumed, the
catalyst was filtered, and the filtrate was concentrated to give (S)-
(+)-10 (30 mg, 99%). Alternatively, compound (S)-(+)-10 was
obtained from (S)-(+)-11. Thus, in the presence of Pd/C catalyst
(10%, 20 mg), compound (S)-(+)-11 (234 mg, 1 mmol) in methanol
(5 mL) was hydrogenated using a hydrogen balloon. After the starting
material was consumed, the catalyst was removed by filtration, and the
filtrate was concentrated. The residue was mixed with aqueous NaOH
(2 N, 1 mL) and MeOH (3 mL). After being heated at 60 °C for 5 h,
the reaction mixture was then neutralized with hydrochloric acid (1 N)
at room temperature. The solvent was removed under vacuum. The
residue was chromatographed on an ion-exchange resin (Dow-
ex50wx8, 200−400 mesh) column using aqueous ammonia (10%) as
the mobile phase to give products S-(+)-10 (133 mg, 91%): mp 188−
Benzyl (R)-2-(benzylamino)-2-carbamoylpentanoate (7i): 123
mg, 56%; mp 163−164 °C; [α]25 +16.5 (c 1.70, CHCl3); ee =
D
97.8% (chiral HPLC analysis); IR (KBr) ν 3440, 1728, 1642, 1218
1
cm−1; H NMR (300 MHz, CDCl3) 7.33−7.16 (m, 10H), 7.12 (s,
1H), 5.93 (s, 1H), 5.27 (d, J = 12.3 Hz, 1H), 5.16 (d, J = 12.3 Hz, 1H),
3.57 (d, J = 12.0 Hz, 1H), 3.47 (d, J = 12.0 Hz, 1H), 2.28 (s, 1H),
2.10−2.05 (m, 2H), 1.34−1.25 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13C
NMR (75 MHz, CDCl3) 172.8, 171.4, 135.5, 128.61, 128.56, 128.5,
128.4, 128.2, 127.5, 70.8, 67.3, 47.8, 34.2, 16.8, 14.1; MS (ESI) m/z
341 [M + H]+ (100); calcd for C20H24N2O3 341.1860 [M + 1]+, found
(P-SIMS) 341.1857 [M + 1]+.
Benzyl (R)-2-(benzylamino)-2-carbamoylpropanoate (7j): 121
mg, 51%; mp 207−208 °C; [α]25 +10.0 (c 0.60, CHCl3); ee =
D
24.8% (chiral HPLC analysis); IR (KBr) ν 3325, 1690, 1261 cm−1; 1H
NMR (300 MHz, CDCl3) δ 7.27−7.11 (m, 10H), 7.02 (s, 1H), 5.55
(s, 1H), 5.16 (dd, J = 36.3, 12.3 Hz, 2H), 3.70−3.33 (m, 2H), 2.30 (s,
1H), 1.59 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 174.0, 171.7, 139.1,
135.5, 128.62, 128.57, 128.4, 128.3, 128.2, 127.4, 67.9, 67.4, 48.3, 20.5;
MS (ESI) m/z 335 [M + Na]+ (55), 313 [M + H]+ (100); calcd for
C18H20N2O3 313.1547 [M + 1]+, found (P-SIMS) 313.1547 [M + 1]+.
Benzyl (R)-2-(benzyl(methyl)amino)-2-carbamoylpent-4-enoate
189 °C (lit.38 mp 279 °C); [α]25 = +9.5 (c 1.05, 1 N HCl) (lit.35
D
[α]25D = +10 (c 0.160, 1 N HCl)); IR (KBr) ν 3140, 3044, 1405 cm−1;
(7k): 133 mg, 65%; oil; [α]25 +23.3 (c 2.15, CHCl3); ee = 64.6%
1H NMR (300 MHz, D2O) 3.78 (d, J = 12.0 Hz, 1H), 3.53 (d, J = 12.0
D
(chiral HPLC analysis); IR (KBr) ν 3460, 2924, 1690, 1215 cm−1; 1H
NMR (300 MHz, CDCl3) δ 7.35−7.23 (m, 10H), 6.84 (s, 1H), 5.98−
5.86 (m, 2H), 5.32−5.13 (m, 4H), 3.85 (d, J = 14.2, Hz, 1H), 3.63 (d,
J = 14.2, Hz, 1H), 3.00−2.85 (m, 2H) 2.24 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 171.8, 169.0, 138.5, 135.3, 132.1, 128.3, 128.1, 127.4,
126.9, 76.0, 66.8, 57.2, 38.5, 36.3; MS (ESI) m/z (%) 357 [M + Na]+
(45), 353 [M + H]+ (100); calcd for C21H24N2O3 353.1860 [M + 1]+,
found (P-SIMS) 353.1852 [M + 1]+.
Hz, 1H), 1.67−1.46 (m, 2H), 1.30−1.04 (m, 2H), 0.77 (t, J = 7.5 Hz,
3H); 13C NMR (75 MHz, D2O) 174.5, 66.4, 64.3, 34.3, 16.5, 13.4; MS
(ESI) m/z 148 [M + H]+ (100).
Synthesis of (S)-2-(Benzylamino)-2-(hydroxymethyl)pent-4-en-
amide (11). To a mixture of compound (R)-(+)-7a (170 mg, 0.5
mmol) in THF (5 mL) and EtOH (5 mL) were added LiCl·H2O (30
mg, 0.5 mmol) and NaBH4 (38 mg, 1 mmol), and the resulting
mixture was kept stirring at room temperature. When the starting
material was consumed, water (20 mL) was added, and the mixture
was extracted with ethyl acetate (3 × 20 mL). The organic layer was
dried with anhydrous MgSO4. After removal of the solvent under
vacuum, the residue was chromatographed on a silica gel column with
a mixture of petroleum ether and ethyl acetate (1:1−1:2) as the mobile
phase to give product (S)-(+)-11 (116 mg, 99%): mp 95−96 °C;
Procedure for Biotransformation of 5a Using an Immobi-
lized Whole Cell Catalyst. The immobilized Rhodococcus erythropolis
AJ270 cells (4 g wet weight) in alginate capsules33 were activated in
sodium bicarbonate buffer solution (pH 7.25, 100 mL) at 30 °C for 30
min. A solution of 5a (1.57 g, 10 mmol) in bicarbonate buffer solution
(pH 7.25, 50 mL) was added. The resulting mixture was incubated at
30 °C with orbital shaking for 30 min. The immobilized catalyst was
filtered and reused for another seven times. The combined aqueous
solution was worked up, and (R)-6a (9.3 g, 74% yield, 95.8% ee) was
obtained.
[α]25 = +2.05 (c 1.95, CHCl3); IR (KBr) ν 3443, 3285, 1659 cm−1;
D
1H NMR (300 MHz, CDCl3) 77.36−7.24 (m, 6H), 6.00 (s, 1H),
5.92−5.78 (m, 1H), 5.20−5.15 (m, 2H), 3.84 (s, 2H), 3.79 (d, J = 12.3
Hz, 1H), 3.72 (d, J = 12.3 Hz, 1H), 2.58−2.41 (m, 2H); 13C NMR (75
MHz, CDCl3) 178.1, 139.9, 132.4, 128.6, 128.0, 127.3, 119.4, 65.1,
64.1, 46.9, 38.2; MS (ESI) m/z 257 [M + Na]+ (28), 235 [M + H]+
(100). Anal. Calcd for C13H18N2O2: C, 66.64; H, 7.74; N, 11.96.
Found: C, 66.72; H, 7.67; N, 11.92.
Synthesis of (R)-Methyl 2-Amino-2-carbamoylpent-4-enoate (8).
To a solution of R-(−)-6a (316 mg, 2 mmol) in methanol (20 mL)
was added an ether solution of CH2N2 (2 M, 10 mL) at 0 °C for 0.5 h.
The mixture was stirred at room temperature overnight. The solvent
was removed under vacuum. The residue was chromatographed on a
silica gel column using a mixture of petroleum ether and ethyl acetate
(1:1−1:2) as the mobile phase to give (R)-(+)-8 (269 mg, 78%): mp
Synthesis of (R)-2-(Aminomethyl)-2-(benzylamino)pent-4-en-1-ol
(12). To a solution of (S)-(+)-11 (117 mg, 0.5 mmol) in dry THF (10
mL) under argon protection was added through a syringe a solution of
LiAlH4 in THF (1 N, 5 mL). The resulting mixture was refluxed for 48
h. The reaction was quenched by addition of EtOH (2 mL) and
NaOH (2 N, 3 mL) at room temperature, and then the mixture was
filtered and washed with EtOH (3 × 10 mL). The filtrate was
concentrated under vacuum, and the residue was chromatographed on
a silica gel column with a mixture of ethyl acetate, ethanol, and
ammonia (25% w/w) (100:5:1) as the mobile phase to give products
66−67 °C; [α]25 = +5.0 (c 2.00, CHCl3); IR (KBr) ν 3407, 1730,
D
1
1633 cm−1; H NMR (300 MHz, CDCl3) 7.42 (s, 1H), 6.27 (s, 1H),
5.79−5.71 (m, 1H), 5.23−5.18 (m, 2H), 3.81 (s, 3H), 2.85−2.78 (m,
1H), 2.69−2.62 (m, 1H), 2.10 (s, 2H); 13C NMR (75 MHz, CDCl3)
173.7, 172.4, 131.9, 120.8, 65.0, 53.2, 41.7; MS (ESI) m/z 173 [M +
H]+ (100); calcd for C7H12N2O3 173.0921 [M + 1]+, found (P-SIMS)
173.0915 [M + 1]+.
Synthesis of (S)-2-Amino-2-(hydroxymethyl)pent-4-enoic Acid
(9). To a solution of (R)-(+)-8 (50 mg, 0.3 mmol) in THF (4 mL)
and EtOH (4 mL) were added LiCl·H2O (30 mg, 0.5 mmol) and
NaBH4 (38 mg, 1 mmol), and the resulting mixture was stirred at
room temperature. When the starting material was consumed, the
solvent was removed under vacuum. The residue was mixed with
MeOH (1 mL) and aqueous NaOH (2 N, 1 mL). After being heated
at 60 °C for 5 h, the mixture was then neutralized with hydrochloric
acid (1 N) at room temperature. The solvent was removed under
(R)-(+)-12 (72 mg, 65%): mp 49−50 °C; [α]25 = +10.0 (c 1.60,
D
1
MeOH); IR (KBr) ν 3359, 2864, 1639 cm−1; H NMR (300 MHz,
CDCl3) δ 7.38 (d, J = 5.7 Hz, 2H), 7.31 (t, J = 5.7 Hz, 2H), 7.24 (q, J
= 5.7 Hz, 1H), 5.97−5.86 (m, 1H), 5.21−5.13 (m, 2H), 3.72 (d, J =
9.0 Hz, 1H), 3.68 (d, J = 9.0 Hz, 1H), 3.55 (d, J = 8.4 Hz, 1H), 3.51
(d, J = 8.4 Hz, 1H), 2.72 (d, J = 9.9 Hz, 1H), 2.68 (d, J = 9.9 Hz, 1H),
2.28 (d, J = 5.4 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 140.4, 133.3,
128.11, 128.06, 126.7, 117.5, 63.0, 58.5, 45.1, 43.6, 36.1; MS (ESI) m/
5590
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z 221 [M + H]+ (100); calcd for C13H20N2O: 221.1648 [M + 1]+,
(15) Gao, M.; Wang, D.-X.; Zheng, Q.-Y.; Wang, M.-X. J. Org. Chem.
2006, 71, 9532 and ferences cited therein.
found (P-SIMS) 221.1656 [M + 1]+.
(16) Gao, M.; Wang, D.-X.; Zheng, Q.-Y.; Huang, Z.-T.; Wang, M.-X.
J. Org. Chem. 2007, 72, 6060.
(17) Ma, D.-Y.; Wang, D.-X.; Zheng, Q.-Y.; Wang, M.-X.
Tetrahedron: Asymmetry 2006, 17, 2366.
(18) Wang, M.-X.; Feng, G.-Q. Adv. Synth. Catal. 2003, 345, 695 and
references cited therein..
(19) Wang, M.-X.; Deng, G.; Wang, D.-X.; Zheng, Q.-Y. J. Org. Chem.
2005, 70, 2439 and references cited therein.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Chiral HPLC analysis and H and 13C spectra of all products.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(20) Wang, J.-Y.; Wang, D.-X.; Pan, J.; Huang, Z.-T.; Wang, M.-X. J.
Org. Chem. 2007, 72, 9391 and references cited therein..
(21) (a) Leng, D.-H.; Wang, D.-X.; Pan, J.; Huang, Z.-T.; Wang, M.-
X. J. Org. Chem. 2009, 74, 6077. (b) Leng, D.-H.; Wang, D.-X.; Huang,
Z.-T.; Wang, M.-X. Org. Biomol. Chem. 2010, 8, 4736.
(22) Liu, J.; Wang, D.-X.; Zheng, Q.-Y.; Wang, M.-X. Chem. J. Chem
2006, 24, 1665.
AUTHOR INFORMATION
Corresponding Author
*E-mail: wangmx@mail.tsinghua.edu.cn.
Notes
■
The authors declare no competing financial interest.
(23) Zhang, L.-B.; Wang, D.-X.; Wang., M.-X. Tetrahedron 2011, 67,
5604.
(24) Liskamp, R. M. J.; Rijkers, D. T. S.; Kruijtzer, J. A. W.;
Kemmink, J. ChemBioChem 2011, 12, 1626 and references cited
therein..
(25) (a) Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.;
Barbuto, S.; Wright, R.; Wanger, G.; Verdine, G. L. Science 2004, 305,
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ACKNOWLEDGMENTS
■
We thank the Ministry of Science and Technology
(2009CB724704), National Science Foundation of China
(20772132), Chinese Academy of Sciences, and Tsinghua
University for financial support.
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