Chemoenzymatic synthesis of optically active α-methylene-γ-carboxy-γ-lactams and γ-lactones

Article abstract of DOI:10.1016/j.tetasy.2009.09.009

Three α-methylene-γ-carbomethoxy-γ-butyrolactams (methyl α-methylene-pyroglutamates) 11, 12 and 13, differing in the substitution at the heterocyclic nitrogen, as well as the structurally related γ-lactones 14 and 15 were synthesised and resolved enzymati

Full text of DOI:10.1016/j.tetasy.2009.09.009

Tetrahedron: Asymmetry 20 (2009) 2305–2310
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemoenzymatic synthesis of optically active
a-methylene-
c
-carboxy-
c
-lactams and c-lactones
*
Annalisa Bertoli, Lidia Fanfoni, Fulvia Felluga , Giuliana Pitacco, Ennio Valentin
Dipartimento di Scienze Chimiche, Università di Trieste, via L.Giorgieri, 1 I-34127 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Three
13, differing in the substitution at the heterocyclic nitrogen, as well as the structurally related
14 and 15 were synthesised and resolved enzymatically by hydrolysis of their ester function, mediated by
commercially available hydrolytic enzymes. In particular, the -chymotrypsin proved to be active to all
a
-methylene-
c
-carbomethoxy-
c
-butyrolactams (methyl
a
-methylene-pyroglutamates) 11, 12 and
Received 6 August 2009
Accepted 10 September 2009
Available online 14 October 2009
c
-lactones
a
the substrates examined, displaying a different degree of activity and enantioselectivity, this latter
increasing significantly towards the substrate with an aromatic substituent at the nitrogen.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
In addition to the classical a-methylenation of c
-lactones11 and
lactams,12 the use of nitrocompounds,13 as well as Baylis–Hillmann
chemistry,14 the method of most general applicability, leading to a
large variety of differently substituted derivatives, is the addition
of allylmetal compounds, in particular allyl boron,15 -zinc2b,10,16
and -indium17 reagents to aldehydes15a,c,d,f,g,17a–c,17e for the
a-exo-Methylene-c-butyrolactone and a-exo-methylene-c-bu-
tyrolactam1,2 rings are key structural units in many natural bioactive
compounds (Fig. 1). The former system occurs mainly in sesquiter-
pene lactones,1d,3 for example, 1 and 2, a large class of natural com-
pounds found almost exclusively in the family of Compositae, which
display strong cytotoxic, antiinflammatory, phytotoxic and antimi-
crobial properties.4 Similar activities are exhibited by methylenolac-
tocin 3 and protolichesterinic acid 4, both of which belong to the
class of paraconic acids.1a,b
construction of the
a-methylenated c-lactone skeleton, and to
imines10,15a–c,15e–g,16b–e,17d and oximes,18 for the nitrogen analogue.
To the best of our knowledge, the enzymatic kinetic resolution
of a c-lactam containing the a-exo-methylene functionality is so
far unexplored in the literature. As to the lactonic analogues, no
other examples have been described after the work of our group
published in 200019 on the PPL mediated resolution of ethyl 2-
methyl-4-methylene-tetrahydro-5-oxo-2-furancarboxylate 24.
The bioactivity4b,5 of these compounds towards many biological
targets has been ascribed to its exo C@C double bond, as demon-
strated by the complete loss of biological activity6 after reduction
of the exo double bond or its isomerisation to the endo position.
However, the application of these compounds for pharmaceuti-
cal purposes has been severely limited by their high toxicity exhib-
ited in vitro.1d,7
2. Results and discussion
In the frame of our research, focused on the asymmetric synthe-
sis of chiral compounds of biological interest by means of biotrans-
formation methods, we herein report the synthesis and resolution
The isosteric
a-methylene-c-lactam analogues are much less
present in nature than in the parent lactones. Examples are puke-
leimid E 5,8 isolated from cyanobacteria Lyngbyamajuscula, and
two imidazole alkaloids anantin 6 and isoanantin 79 found in the
leaf tissue of Cynometra.
However, these compounds have received considerable atten-
tion as a consequence of their proven biological properties,2b asso-
ciated with minor toxicity, when compared with the lactone
analogues.10
As a result of their biological relevance, synthetic routes to
these two classes of compounds have been explored, with particu-
lar attention to their asymmetric version accessing chiral non-
racemic derivatives.
of three
-methylene-pyroglutamates) 12, 13 and 14, differing in the sub-
stitution at the heterocyclic nitrogen, as well as the structurally re-
lated -lactones 15 and 16 (Scheme 2).
Incidentally,
-methylene pyroglutamic acid12a is a cyclic ana-
a-methylene-c-carbomethoxy-c-butyrolactams (methyl
a
c
a
logue of 4-methylene glutamic acid 10 (Scheme 1) which is in turn
a natural compound found in a variety of plants,20 and exhibiting a
potent central nervous system inhibitory action,21 due to activa-
tion of NMDA (N-methyl D
-aspartate) receptors.22
2.1. Synthesis of the racemic substrates
As a common starting material for the synthesis of the hetero-
cyclic racemic substrates, we synthesised the dimethyl ester of
* Corresponding author. Tel.: +39 040 5583924; fax: +39 040 5583023.
E-mail address: ffelluga@units.it (F. Felluga).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.09.009

2306
A. Bertoli et al. / Tetrahedron: Asymmetry 20 (2009) 2305–2310
H
HO2C
O
H
O
O
O
R
O
O
O
O
HO
3: R=n-C5H11
4: R=n-C13H27
1
2
Me
N
N
N
Ph
Ph
N
O
H3CO
N
N
Me
Me
O
O
O
N
H
N
H
6
7
5
Figure 1. Selected examples of naturally occurring
E 5, anantine 6, isoanantine 7.
a-methylene-c-lactones and lactams: arglabin 1, helenalin 2, methylenolactocin 3, protolichesterinic acid 4, pukeleimide
4-methylene-glutamic acid in its hydrochloride salt form 11
(Scheme 1). Its preparation was carried out by a literature, three-
step procedure.23,24
On the contrary, a convenient route was found for the conver-
sion of 11 into the N-substituted compounds 13 and 14. Reductive
amination of benzaldehyde and 2,4-dimethoxybenzaldehyde
respectively with 11 as the amine partner furnished the desired
lactams in 75% yield. The reactions were performed in THF at room
temperature, in the presence of an equimolar amount of triethyl-
amine, using Na(AcO)3BH as the reducing agent.26 The reaction of
benzaldehyde proceeded smoothly to imine 17 (Scheme 2), which
was not reduced in situ, as expected, but isolated as a pure com-
pound. Its reduction, followed by spontaneous cyclisation to the
target compound 13 took place in MeOH and Na(AcO)3BH. On
the contrary, imine 18, derived from 2,4-dimethoxybenzaldehyde,
was not isolated, but only detected by 1H NMR in the reaction mix-
ture after 2 h, and underwent complete reduction/cyclisation
in situ to the desired lactam 14.
from 8
1. AcNHCH(CO2Et)2
CO2R
CO2R
NH3+Cl-
EtO-Na+/EtOH
Br
RO2C
10 R = H
11 R = Me
2. 6M HCl, 100°C
ref. 23
MeOH, SOCl2,
-30°C
ref. 24
8 R = Et
9 R = H
Scheme 1.
The transformations of the parent molecule 11 into the com-
pounds of interest 12, 13, 14 and 15 occurred with yields ranging
from 10% to 75% (Scheme 2). In particular, cyclisation of 11 in
refluxing methanol saturated with ammonia, gave 12 in very poor
yield.25
It is important to note that the reductive amination of 2,4-dime-
thoxybenzaldehyde with amine 11 offers an alternative, efficient
route to
a
-methylenepyroglutamate 12. In fact the benzylic pro-
NH3(g)
MeOH
10%
MeO2C
O
N
H
12
CO2Me
TFA
CH2Cl2, rt
98%
from 14
MeO2C
NH3+Cl-
11
ArCHO
NaNO2
MeO2C
O
0°C
10%
Na(AcO)3BH
75%
N
CH2Ar
13 Ar = Ph
14 Ar = 2,4-(MeO)2C6H3
RO2C
O
O
15 R = Me
16 R = Et
CO2Me
CO2Et
OH
In
MeO2C
N
Ar
HO2C
THF, H2O
50%
17: Ar = Ph
19
18: Ar = 2,4-(MeO)2C6H3
8 + HCOCO2Et
Scheme 2.

A. Bertoli et al. / Tetrahedron: Asymmetry 20 (2009) 2305–2310
2307
tecting group at the nitrogen atom could be easily removed from
14 by treatment with TFA at room temperature, to give 12 in quan-
titative yield.
chromatography. Due to the low enantioselectivity observed and
the disadvantageous work-up, we did not investigate this reaction
any further.
On the contrary, transformation of 11 into the corresponding
lactone 15 by nitrosation, following the procedure described for
the cyclisation of
-glutamic acid,11b gave very small amounts of
c
-
The insertion of a benzyl-type substituent at the heterocyclic
nitrogen, in addition to increasing the overall yield of the racemic
synthesis, markedly improved the enantioselectivity of the enzy-
L
the desired product, which formed in an admixture with unidenti-
fied compounds.
Yields in the lactonic product increased to an acceptable value
(50%) by the indium-promoted allylation of ethylglyoxylate with
2-(bromomethyl)acrylic acid 9 (Scheme 2),19 followed by cyclisa-
tion of the hydroxy hemiester intermediate 19, to give lactone 16.
matic resolution. In fact, a-chymotrypsin hydrolysed both lactams
13 and 14 with very high stereoselectivity (E >100 in both cases),
thus allowing the obtainment of the carboxylic acids (R)-(ꢀ)-21
and (R)-(ꢀ)-22 with excellent enantiomeric excesses and recovery
of their respective unreacted esters (S)-(+)-13 and (S)-(+)-14 as
enantiomerically pure forms (Table 1, entries 2 and 3). This result
is in accordance with the well-known affinity of a-Ct for substrates
2.2. Enzymatic resolution
carrying a hydrophobic aromatic substituent near the reaction
centre.27,31
The resolution of compounds 12–15 was accomplished by enzy-
matic hydrolysis of their respective racemic esters using a-chymo-
trypsin, while PPL was the preferred enzyme for the hydrolysis of
lactone 16.
A further important consequence was the opportunity to obtain
lactam (S)-(+)-12 in good yield and high enantiomeric excess, via
acidic removal of the dimethoxybenzyl protecting group from
(S)-(+)-14, as seen in Scheme 2. This indirect route gave (S)-(+)-
12 with >99% ee and 21% overall yield, starting from ( )-11. More-
over, a stereochemical correlation could be established between
(S)-(+)-14, so far unknown in the literature, and (S)-(+)-12, whose
The choice of
carbomethoxy-
a
-chymotrypsin for the resolution of the three
c-
a
-methylene- -lactams 12–14 was based on the al-
c
ready known ability of this enzyme to hydrolyse the laevo enantio-
mer of unsubstituted methyl pyroglutamate and some other 5-
substituted derivatives.27
The results found are summarised in Table 1, which lists the ee
values of the isolated lactam and lactone acids 20–23, those of
their recovered unreacted esters 12–15 and yields. After determin-
ing the enantiomeric ratio E28 at approximately 50% conversion,
resolutions were run up to the conversions indicated in the table
in order to isolate the carboxylic acid products and the unreacted
ester in the highest possible ee.
As expected, enzymatic hydrolyses of heterocycles bearing no
benzyl-type substituent at position 1 of the ring were poorly enan-
tioselective (Table 1, entries 1, 6 and 7), as indicated by the low
values of the enantiomeric ratios E. Enzymatic hydrolysis of 12 (en-
try 1) gave the corresponding carboxylic acid (R)-(ꢀ)-20 with low
ee, while the corresponding unreacted ester (S)-(+)-1218,29 had 72%
ee. Furthermore, this reaction was difficult as far as work-up is
concerned. As a consequence of the high water solubility of the es-
ter, its separation from the acid had to be performed by column
absolute configuration had already been reported.18,29 All
c-lactam
esters isolated from the enzymatic hydrolyses, namely (S)-(+)-12,
(S)-(+)-13 and (S)-(+)-14, exhibited the same positive Cotton effect
at approximately the same wavelength in their CD spectra in
MeOH, (D233 +1.4; D239 +1.1; D240 +1.3, respectively), which al-
lowed the configurational assignment to also be made for the un-
known N-benzylsubstituted derivative (S)-(+)-13.
As for the enzymatic resolutions of lactones ( )-15 and ( )-16,
with
lectivity. This had already been observed for the
mediated hydrolysis of the homologous -carboethoxy-
lene- -unsubstitut-
-valerolactone 24 (Fig. 2),19 as well as for the
ed derivative 25.27
a
-chymotrypsin they occurred with unsatisfactory enantiose-
-chymotrypsin
-methy-
a
c
a
c
a
However, the enantioselectivity of the reaction increased to a
satisfactory degree using PPL, especially when acetone was added
to the reaction medium as the cosolvent (E = 19). In this latter case,
acid (R)-(ꢀ)-23 was obtained in a 81% ee, at low conversion, while
the unreacted ester (S)-(+)-16 could be isolated as an enantiomeri-
Table 1
Enzymatic hydrolysis of substrates 12–16a
Entry
Substrate
Enzyme
E
Conv. values (time)
(R)-(ꢀ)-Acid, ee %b,c
Conv. values (time)
(S)-(+)-Ester ee %b
(yield %)d
(yield %)d
1
2
3
4
5
6
7
( )-12
X = NH, R = Me
( )-13
X = NBn, R = Me
( )-13
X = NBn, R = Me
( )-14
X = N-DMBn, R = Me
( )-14
X = N-DMBn, R = Me
( )-15
X = O, R = Me
( )-16
X = O, R = Et
a
a
a
a
a
a
-Ct
-Ct
-Ct
-Ct
-Ct
-Ct
7
57e (3 h)
35 (6 h)
20
57e (3 h)
64 (18 h)
52 (8 h)
12
55 (16)f
21
72b (28)f
13
>200
>200
140
140
4
99 (25)
21
>99 (30)
13
52 (8 h)
97 (38)
22
98 (39)
14
28 (2 h)
65 (14 h)
54 (4 h)
98c,g (22)
22
>99g (27)
14
51 (4 h)
95 (37)
23
96 (41)
15
79 (16)
16
53 (15 min)
20 (15 min)
80 (2 h)
44 (32)
23
PPL in 10% acetone/buffer
19
71 (30 min)
81h (13)
99 (22)
a
b
c
Reaction conditions: 1.0 g substrate, 0.1 g enzyme, 0.1 M phosphate buffer at pH 7.4 (5 ml/mmol), room temperature.
Determined by HRGC (b-cyclodextrin).
After esterification with MeOH, Me3SiCl.30
d
e
f
Yield in isolated product.
Resolution at low conversion values was not carried out, because of work-up problems (see text).
After chromatographic separation.
g
h
After removal of the protecting group with TFA.
After esterification with EtOH, Me3SiCl.30

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A. Bertoli et al. / Tetrahedron: Asymmetry 20 (2009) 2305–2310
4.2. Synthesis of the substrates
4.2.1. Ethyl 2-(bromomethyl)acrylate 8
O
O
Compound 8 was synthesised as described in the literature.32
O
O
EtO2C
EtO2C
24
25
4.2.2. ( )-4-Methyleneglutamic acid, hydrochloride 10
Compound 10 was synthesised as described in the literature23
yield 96%. Mp 179–180 °C; IR (Nujol) 3405, 1737, 1678, 1626,
Figure 2.
1597 cmꢀ1 1H NMR (D2O) d 6.19 (s, 1H, @CH), 5.73 (s, 1H, @CH),
;
cally pure compound at 71% conversion, with a 22% yield in the iso-
lated product. In analogy to what was observed in the case of 24,19
other hydrolases tested (esterases and lipases) proved inactive or
less enantioselective than PPL, while acetone was found to be the
only co-solvent with any effect on the stereoselectivity of the
resolution.
4.06 (dd, J 8.0, 6.1, 1H, H-2), 2.79 (dd, J 14.6, 6.1, 1H, H-3), 2.64
(dd, J 14.6, 8.0, 1H, H-3); 13C NMR (D2O) d 172.0, 170.3, 134.3,
133.0, 53.0, 33.3; ESI-MS m/z 160.0 [M+].
4.2.3. Dimethyl ( )-4-methyleneglutamate, hydrochloride 11
Esterification of 10 was run as described in the literature24 to
give 11 quantitatively. IR (film) 3391, 3006, 1750, 1720; 1H NMR
d 6.46 (s, 1H, @CH), 5.97 (s, 1H, @CH), 4.38 (dd, J 7.5, 6.3, 1H, H-
2), 3.84, 3.80 (2s, 6H, OCH3), 3.07 (dd, J 14.5, 6.3, 1H, H-3), 2.93
(dd, J 14.5, 7.5, 1H, H-3). 13C NMR d 172.8, 171.2, 135.9, 135.4,
56.3, 55.5, 54.8, 35.2; IR (Nujol) 1633, 1594 cmꢀ1; ESI-MS m/z
188.0 [M+], 210.0 [M+Na+].
The absolute configuration was assigned to (+)-16 by comparison
with the literature data.11b In fact, compound (S)-(+)-16 had been al-
ready described as being derived from
sation/cyclisation reaction, which is known to occur with retention
of configuration, followed by direct -methylenation. Further con-
L
-glutamic acid,11b by a nitro-
a
firmation of the stereochemical assignment came from the CD spec-
trum of (S)-(+)-16 which showed a positive Cotton effect at 220 nm,
(CD: D221 +1.1 (MeOH)) as reported for the spectrum of the (S)-enan-
tiomer of lactone 24, [CD: D226 +3.2 (MeOH)].19
4.2.4. Methyl ( )-4-methylene 5-oxo-pyrrolidin-2-carboxylate
1225
Interestingly, this result suggests that the enantiopreference of
PPL towards the substrate 16 is opposite with respect to the case of
24. In fact, in its hydrolysis with lactone 24, PPL showed an (S)-
enantiopreference,19 whereas with lactone 16, it exhibited an (R)-
enantiopreference, with the methyl group at C-2 in 24 playing an
important role in the approach of the molecule to the active site
of the enzyme.
A methanolic solution (20 ml) of 11 (1.20 g, 7.70 mmol) was
saturated with NH3(g), then refluxed for 4 h. Evaporation of the sol-
vent left an oily residue which was chromatographed on column
(eluent: ethyl acetate) to give 12 as a white solid, mp 94–96 °C
(10%), HRGC (b-CDX) tR = 34.18 and 41.68 min (150 °C isoth). IR
(film) 1728, 1698, 1659, cmꢀ1 1H NMR d 6.55 (s, 1H, NH), 6.06
;
(t, J 2.4, 1H, @CH), 5.43 (br s, 1H, @CH), 4.29 (dd, J 9.2, 4.3, 1H,
H-2), 3.79 (s, 3H, OCH3), 3.19 (tdd, J 17.3, 9.2, 2.4, 1H, H-3), 2.98
(tdd, J 17.3, 4.4, 2.4, 1H, H-3); 13C NMR d 172.4, 170.9, 137.4,
117.0, 52.4, 30.1; ESI-MS m/z 156.0 [M+H+], 178.0 [M+Na+].
3. Conclusion
In conclusion, the first example of a kinetic resolution of
a-exo-
4.2.5. Methyl ( )-1-(methylphenyl)-4-methylene-5-oxo-
pyrrolidin-2-carboxylate 13
methylene- -lactams was achieved. The preferred enzyme was a-
c
chymotrypsin when a benzyl-protecting group was present at the
nitrogen atom, thus reflecting the high tendency of this enzyme
to interact with substrates bearing aromatic groups near the reac-
tion centre.
A mixture of 11 (1.05 g, 4.80 mmol) and benzaldehyde (freshly
distiled, 0.45 g, 4.2 mmol) in THF was added of Et3N (0.25 ml,
5.1 mmol) and sodium triacetoxyborohydride, Na(AcO)3BH
(1.12 g, 4.8 mmol). The mixture was left to stir overnight at room
temperature, then washed with NaHCO3 (aq, 5% w/v), water and
brine. The organic phase was dried and evaporated to give 0.31 g
of dimethyl N-benzal-4-methyleneglutamate 17 as an essentially
pure compound.
4. Experimental part
4.1. General
IR (film) 2952, 1739, 1641 cmꢀ1 1H NMR d 8.21 (s, 1H, CH@N),
;
IR spectra were recorded on an AVATAR 320 FT/IR spectrometer.
1H NMR spectra were run on a Jeol EX-400 (400 MHz), 13C NMR on
a JEOL 270 (67.5 MHz for carbon) using deuterochloroform as a sol-
vent and tetramethylsilane as an internal standard, unless other-
wise stated. Optical rotations were determined on a Perkin–
Elmer Model 241 polarimeter, at 25 °C. CD spectra were recorded
on a JASCO J-710 spectropolarimeter. SI-MS were run on a Bruker
Esquire 4000 instrument. Enzymatic hydrolyses were performed
using a pH-stat Controller PHM290 Radiometer, Copenhagen. Chi-
ral High Resolution GLC analyses were run on a Shimadzu GC-14B
7.75 (m, 2H, ArH), 7.42 (m, 3H, ArH), 6.22 (s, 1H, @CH), 5.63 (s, 1H,
@CH), 4.26 (dd, J 8.3, 5.7, 1H, H-2), 3.76, 3.75 (2s, 6H, CO2CH3), 3.12
(dd, J 13.8, 5.7, 1H, H-3), 2.82 (dd, J 13.8, 8.3, 1H, H-3); 13C NMR d
171.7, 167.1, 164.1, 135.8, 135.4, 131.2, 129.1, 128.6, 128.5, 71.7,
52.2, 51.9, 36.2; ESI-MS (m/z) 276.1 [MH+].
Imine 17 (0.6 g, 2.2 mmol) was dissolved in MeOH and a further
amount of Na(AcOEt)3BH (0.32 g, 1.5 mmol) added. After 12 h stir-
ring, the solvent was removed, the residue dissolved in diethyl
ether and washed with a saturated solution of NaHCO3 The organic
phase was evaporated to dryness leaving a residue which was
chromatographed on a column (eluent: petroleum ether/ethyl ace-
tate, gradient) to give compound 13 (0.4 g, 75% yield); HRGC (b-
CDX) tR = 156.1 and 159.4 (150 °C isoth). IR (film) 3030, 1743,
instrument, the capillary columns being ChiraldexTM type G-TA,
c-
cyclodextrin (40 m ꢁ 0.25 mm) (carrier gas helium, 180 KPa, split
1:100), or DiMePe b-cyclodextrin (25 m ꢁ 0.25 mm) (carrier gas
He, 110 Kpa, split 1:50); TLC’s were performed on PolygramÒ Sil
G/UV254 silica gel pre-coated plastic sheets (eluent: light petro-
leum/ethyl acetate). Flash chromatography was run on silica gel,
230–400 mesh ASTM (Kieselgel 60, Merck), using mixtures of light
petroleum 40–70 °C and ethyl acetate as the eluent. a-Chymotryp-
sin was purchased from Fluka, PPL (Porcine Pancreatic Lipase) from
Sigma.
1697, 1662, 754.1, 703 cmꢀ1 1H NMR d 7.29 (m, 3H, Ph), 7.21 (d,
;
2H, Ph), 6.11 (t, J 2.7, 1H, @CH), 5.42 (t, J 2.2 Hz, 1H, @CH), 5.14
(d, J 14.8, 1H, CH2Ph), 4.12 (d, J 14.8, 1H, CH2Ph), 4.03 (dd, J 9.3,
3.3, H-2), 3.68 (s, 3H, OCH3), 3.00 (tdd, J 17.2, 9.3, 2.7, 1H, H-3),
2.77 (tdd, J 17.2, 3.3, 2.7, 1H, H-3); 13C NMR d 171.6, 167.9, 136.9
135.5, 128.8, 128.7, 127.8, 117.0, 55.7, 52.4, 46.0, 29.0; ESI-MS m/

A. Bertoli et al. / Tetrahedron: Asymmetry 20 (2009) 2305–2310
2309
z 246.0 [M+H+], 268.0 [M+Na+]. Anal. Calcd for C14H15NO3: C,
68.56; H, 6.16; N, 5.71. Found: C, 68.66; H, 6.10; N, 5.78.
with CHCl3 (3 ꢁ 10 ml). In the case of enzymatic hydrolysis of 12, a
chromatographic separation was necessary to separate the ester
from the carboxylic acid. Determination of the enantiomeric ex-
cesses of the acidic products was made after esterification with
methanol and trimethylchlorosilane.30
4.2.6. Methyl ( )-1-(2,4-dimethoxyphenyl)methyl-4-
methylene-5-oxo-2-pyrrolidinecarboxylate 15
Compound 11 (1.05 g, 4.8 mmol) was reacted with 2,4-dime-
thoxybenzaldehyde (0.69 g, 4.2 mmol) by the above-described pro-
cedure, to give the lactam 14, 75% yield after purification on a
column (eluent: petroleum ether/ethyl acetate gradient). Oil, IR
(film) 3000, 1746, 1695, 1663, 835, 807 cmꢀ1 1H NMR d 7.20 (1H,
d, ArH), 6.45 (m, 2H, ArH), 6.04 (t, J 2.8, 1H, @CH), 5.34 (t, J
2.3 Hz, 1H, @CH), 4.94 (d, J 14.5, 1H, CH2Ar), 4.22 (d, J 14.5, 1H,
CH2Ar), 4.07 (dd, J 9.5, 3.4, 1H, H-2), 3.79, 3.76, (2s, 6H, OCH3),
3.72 (3H, s, OCH3), 2.96 (tdd, J 17.3, 9.5, 2.8, 1H, H-3), 2.68 (tdd, J
17.3, 3.4, 2.3, 1H, H-3); 13C NMR d 172.5, 168.2, 161.1, 159.1,
137.7, 132.2, 116.5, 104.6, 98.4, 56.2, 55.5, 55.4, 52.4, 40.4,
29.4 ppm; ESI-MS m/z 328.1 [M+Na+]. Anal. Calcd for C16H19NO5:
C, 62.94; H, 6.27; N, 4.59. Found: C, 62.82; H, 6.20; N, 4.73.
Lactam 14 was stirred in TFA for 12 h. Evaporation of the sol-
vent under reduced pressure gave a residue which was purified
on a short SiO2 column using ethyl acetate as the eluent, to give
12 in a quantitative yield.
4.3.1. Methyl (S)-(+)-4-methylene 5-oxo-pyrrolidin-2-
carboxylate 12
Compound 12 was obtained in a 28% yield (purification by flash
chromatography, eluent: ethyl acetate) by stopping the enzymatic
hydrolysis at 57% conversion, 72% ee, ½a 25
¼ þ8:6 (c 1.3, AcOEt),
ꢂ
lit.18
½
a 2D5
ꢂ
¼ þ15:6 (c 0.34, AcOEt), D208 ꢀD0.4, D233 +1.4 (MeOH).
4.3.2. (R)-(ꢀ)-4-Methylene-5-oxo-pyrrolidine-2-carboxylic acid
20
Compound 20 was obtained in 16% yield (purification by flash
chromatography, eluent: ethyl acetate) by stopping the enzymatic
hydrolysis at 57% conversion, 55% ee; mp >200 °C (dec.); 1H NMR
(D2O) d 5.97 (t, J 2.8, 1H, @CH), 5.64 (t, J 2.5, 1H, @CH), 4.30 (dd,
J 9.4, 4.2, 1H, H-2), 3.33 (tdd, J 17.7, 9.4, 2.8, 1H, H-3), 2.85 (tdd, J
17.7, 4.2, 2.5, 1H, H-3); 13C NMR (D2O) d 179.9, 172.9, 138.6,
117.1, 55.2, 31.2; IR (Nujol) 3284, 1702, 1678, 1650, 1633,
1584 cmꢀ1; ESI-MS m/z 139.9 [M+H+].
4.2.7. Ethyl 4-methylene-tetrahydro-5-oxo-2-furancarboxylate
16
4.3.3. Methyl (S)-(+)-1-(methylphenyl)-4-methylene-5-oxo-
pyrrolidin-2-carboxylate 13
2-(Bromoethyl)acrylic acid 9 (1.68 g, 10 mmol), ethyl glyoxylate
(10 mmol, 50% solution in toluene) and indium powder (1.12 g,
10 mmol) were mixed in a 1:1 mixture of THF and H2O (10 ml).
An exothermic reaction took place, with the formation of a turbid
suspension from which a metallic indium conglomerate separated.
Stirring was continued until the disappearance of the starting
material (TLC, ethyl acetate/petroleum ether 1:4), then ethyl ace-
tate was added and the suspension was centrifuged to separate
the white precipitate formed. The organic phase was dried over
anhydrous Na2SO4 to give, after evaporation, a colourless oil con-
taining essentially pure 2-hydroxy-4-carboethoxy-1-pentenoic
acid 19 (8.6 g 78% yield); IR (film) cmꢀ1 3600–2950, 480, 1740,
1633; 1H NMR d 6.30 (s, 1H, C@CH), 5.60, (br, 2H), 5.74 (s, 1H,
C@CH), 4.40 (dd, J 7.8, 4.4, H-2), 4.14 (q, 2H, OCH2CH3), 2.86 (dd,
J 14.3, 4.4, 1H, H-3), 2.65 (dd, J 14.2, 7.8, 1H, H-3), 1.22 (t, 3H,
CH3CH2O); 13C NMR (67.5 MHz) d 174.6, 167.5, 135.4, 128.8, 69.5,
60.5, 37.2; ESI-MS m/z 211.0 [M+Na+].
Compound 13 was obtained in a 30% yield at 64% conversion
with >99% ee ½a 2D5
¼ þ36 (c 0.95, AcOEt), D213 ꢀ0.4 (MeOH), D239
ꢂ
+1.1 (MeOH).
4.3.4. (R)-(ꢀ)-1-(Methylphenyl)-4-methylene-5-oxo-pyrrolidin-
2-carboxylic acid 21
Compound 21 was obtained in 25% yield, at 42% conversion,
99% ee, ½a 2D5
ꢂ
¼ ꢀ127:3 (c 1.0, MeOH); 1H NMR d 8.40 (br, 1H,
COOH), 7.25 (5H, Ph), 6.10 (t, J 2.4, 1H, @CH), 5.44 (br s, 1H,
@CH), 5.26 (d, J 14.8, 1H, CH2Ph), 4.06 (d, J 14.8, 1H, CH2Ph), 4.04
(1H, dd, J 9.7, 3.6, 1H, H-2), 3.01 (m, 1H, H-3), 2.83 (m, 1H, H-3);
13C NMR d 174.3, 168.4, 136.7, 135.1, 129.2, 129.0, 128.3, 118.1,
55.7, 46.2, 29.1; IR (Nujol) 1741, 1673, 741, 697 cmꢀ1; ESI-MS m/
z 232.0 [M+H+], 254.0 [M+Na+]. Anal. Calcd for C13H13NO3: C,
67.52; H, 5.67; N, 6.06. Found: C, 67.68; H 5.50; N, 6.04.
Refluxing the crude hydroxy hemiester 19 in toluene in the
presence of a catalytic amount of p-toluensulfonic acid led to com-
pound 16 in 70% overall yield after flash chromatography. HRGC
(b-CDX) tR = 9.42 and 9.72 (150 °C isoth.); IR (film) 1774, 1744,
4.3.5. Methyl (S)-(+)-1-[methyl(2,4-dimethoxyphenyl)]-4-
methylene-5-oxo-pyrrolidin-2-carboxylate 14
Compound 14 was obtained at 70% conversion in a 21% yield,
with >99% ee ½a 2D5
¼ þ88:0 (c 1, AcOEt); D221 ꢀ0.5; D240 +1.3;
ꢂ
1667 cmꢀ1
;
1H NMR d 6.29 (t, J 2.9, 1H, @CH), 5.70 (t, J 2.5, 1H,
D279 +0.4 (MeOH).
@CH), 4.99 (dd, J 9.5, 2.9, 1H, H-2), 4.21 (q, 2H, OCH2CH3), 3.25
(tdd, J 17.3, 9.5, 2.9, 1H, H-3), 2.99 (tdd, J 17.3, 2.5, 1H, H-3), 1.25
(t, 3H, OCH2CH3).13C NMR d 169.6, 169.0, 131.6, 123.4, 72.8, 62.1,
31.2, 14.0; ESI-MS m/z 193.0 [M+Na+]. Anal. Calcd for C8H10O4: C,
56.47; H, 5.92; O, 37.61. Found: C, 56.6; H, 5.9; O, 37.5.
4.3.6. (R)-(ꢀ)-1-[Methyl(2,4-dimethoxyphenyl)]-4-methylene-
5-oxo-pyrrolidin-2-carboxylic acid 22
Compound 22 was obtained in 22% yield, at 28% conversion,
98% ee, ½a 2D5
ꢂ
¼ ꢀ177:1 (c 1.35, MeOH); mp 157–159 °C; IR (film)
3006, 1742–1614, 933.2–754.6 cmꢀ1
;
1H NMR d 7.22 (d, 1H,
4.3. General procedure for enzymatic hydrolyses
ArH), 6.43 (m, 2H, ArH), 6.07 (br s, 1H, @CH), 5.38 (br s, 1H,
@CH), 4.99 (d, J 14.4, 1H, CH2Ar,), 4.29 (d, J 14.4, 1H, CH2Ar), 4.15
(dd, J 9.8, 3.2, 1H, H-2), 3.78, 3.76 (2s, 6H, OCH3), 3.03 (dd, J 17.2,
9.6, 1H, H-2), 2.79 (bd, J 17.2, 1H, H-3); 13C NMR d 175.8, 168.8,
161.3, 159.1, 137.5, 132.5, 117.2, 116.2, 104.7, 98.5, 59.1, 55.6,
55.3, 40.7, 29.3; ESI-MS m/z (negative-ion polarity) 290.0 [Mꢀ1].
Anal. Calcd for C15H17NO5: C, 61.85; H, 5.88; N, 4.81. Found: C,
61.63; H, 5.80; N, 4.78.
A suspension of the racemic substrates 12–15 (0.2 g), in 20 ml
of 0.1 N KH2PO4/Na3PO4 buffer (pH 7.4) was hydrolysed with
a-
chymotrypsin (100 mg/mmol substrate), at room temperature un-
der vigorous stirring. Hydrolysis of 16 was also performed with PPL
(100 mg/mmol substrate) in a 10% acetone/phosphate buffer solu-
tion. The pH was kept at the initial value by the continuous addi-
tion of 1 M NaOH. At the desired conversion value, the unreacted
ester was extracted from the suspension with ethyl acetate
(3 ꢁ 10 ml) at pH 7.8 (5% NaHCO3) using a centrifuge for the sepa-
ration of the layers. For the isolation of the acids 20, 21, 22 and 23
the aqueous layer was acidified to pH 2 with 2 M HCl and extracted
4.3.7. Ethyl (S)-(+)-4-methylene-tetrahydro-5-oxo-2-
furancarboxylate 16
Compound 16 was isolated from the hydrolysis carried out with
PPL, using 10% acetone/phosphate buffer as the solvent, 99% ee at

2310
A. Bertoli et al. / Tetrahedron: Asymmetry 20 (2009) 2305–2310
71% conversion ½a D25
ꢂ
¼ þ19:5 (c 0.45, EtOH); (lit.11b
½
a 2D5
ꢂ
¼ þ12:7 (c
Drioli, S.; Felluga, F.; Forzato, C.; Pitacco, G.; Valentin, E. J.Chem. Soc., Perkin
Trans. 1 2000, 16, 2839–2842.
12. (a) Dieterich, P.; Young, D. W. Org. Biomol. Chem. 2006, 4, 1492–1496; (b)
Moody, C. M.; Young, D. W. J. Chem. Soc., Perkin Trans. 1 1997, 3519–3530; (c)
Ezquerra, J.; Pedregal, C.; Micó, I.; Nájera, C. Tetrahedron: Asymmetry 1994, 5,
921–926.
2.2, EtOH), D221 +1.1 (MeOH).
4.3.8. (R)-(ꢀ)-4-Methylene-tetrahydro-5-oxo-2-furancarboxylic
acid 23
13. (a) Krawczyk, H.; Albrecht, L.; Wojciechowski, J.; Wolf, W. M.; Krajewska, U.;
Rozalski, M. Tetrahedron 2008, 42, 6307–6314; (b) Béji, F.; Lebreton, J.; Villiéras,
J.; Amri, H. Tetrahedron 2001, 57, 9959–9962; (c) Janecki, T.; Blaszcyk, E.;
Studzian, K.; Janecka, A.; Krajewska, U.; Rozalski, M. J. Med. Chem. 2005, 48,
3516–3521; (d) Ballini, R.; Bosica, G.; Livi, D. Synthesis 2001, 10, 1519–1522; (e)
Ballini, R.; Marcantoni, E.; Perella, S. J .Org. Chem. 1999, 64, 2954–2957.
14. (a) Lee, K. Y.; Lee, H. S.; Kim, J. N. Tetrahedron Lett. 2007, 48, 2007–2011; (b) Le
Lamer, A. C.; Gouault, N.; David, M. J. Comb. Chem. 2006, 8, 643–645; (c)
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15. (a) Elford, T. G.; Ulaczyk-Lesanko, A.; De Pascale, G.; Wright, G. D.; Hall, D. G. J.
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49, 6995–6998; (c) Chataigner, I.; Zammattio, F.; Lebreton, J.; Villiéras, J.
Tetrahedron 2008, 64, 2441–2455; (d) Yu, S. H.; Ferguson, M. J.; McDonald, R.;
Hall, D. G. J. Am. Chem. Soc. 2005, 127, 12808–12809; (e) Chataigner, I.;
Zammattio, F.; Lebreton, J.; Villiéras, J. Synlett 1998, 275–276; (f) Nyzam, V.;
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Compound 23 was isolated at 20% conversion, 81% ee,
½
a 2D5
ꢂ
¼ ꢀ11:7 (c 2.5, EtOH) [lit.11b+15.5 (c 2.1, EtOH) for the enan-
tiopure (S)-enantiomer]. IR (film) cmꢀ1 3200 (br), 1775, 1736; 1H
NMR d 6.33 (t, J 2.9, 1H, @CH), 5.78 (t, J 2.5, 1H, @CH), 5.02 (dd, J
9.6, 4.8, 1H, H-2), 3.35 (tdd, J 17.5, 9.6, 2.9, 1H, H-3), 3.08 (tdd, J
17.5, 4.8, 2.5, 1H, H-3); 13C NMR d 174.1, 169.2, 131.3, 124.5,
72.3, 31.2; ESI-MS m/z (negative-ion polarity) 141.0 [Mꢀ1].
Acknowledgments
We are grateful to the Ministero dell’Università e della Ricerca
(PRIN 2007) and to the University of Trieste for their financial
support.
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