The first kinetic enzymatic resolution of methyl ester of C75

Article abstract of DOI:10.2174/157017810791112405

Enantioselective hydrolysis of methyl ester of (±)-C75 was successfully accomplished by means of Acylase I from Aspergillus to afford (2,R,3S)-(+)-C75 with 96% e.e. The unreacted methyl ester was recovered with >99% e.e. This latter compound was either chemically or enzymatically hydrolyzed to furnish (2S,3,R)-(-)-C75 with >99% e.e.

Full text of DOI:10.2174/157017810791112405

Letters in Organic Chemistry, 2010, 7, 245-248
245
The First Kinetic Enzymatic Resolution of Methyl Ester of C75
Kuheli Chakrabarty, Cristina Forzato, Patrizia Nitti*, Giuliana Pitacco and Ennio Valentin
Dipartimento di Scienze Chimiche, Università di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy
Received October 23, 2009: Revised February 08, 2010: Accepted February 08, 2010
Abstract: Enantioselective hydrolysis of methyl ester of (±)-C75 was successfully accomplished by means of Acylase I
from Aspergillus to afford (2R,3S)-(+)-C75 with 96% e.e. The unreacted methyl ester was recovered with >99% e.e. This
latter compound was either chemically or enzymatically hydrolyzed to furnish (2S,3R)-(–)-C75 with >99% e.e.
Keywords: C75, enzymatic resolution, ꢀ-methylene-ꢁ-butyrolactone, paraconic acid, Acylase I.
INTRODUCTION
C75 is a fatty acid synthase (FAS) inhibitor and when
administered in racemic form, it causes anorexia and
reversible weight loss in rodents [14]. It also shows
significant in vivo antitumor activity in human cancer cells
[2a,15], suppresses DNA replication, induces apoptosis in
tumor cell lines [16] and it is active against mycobacteria of
the tuberculosis complex [17].
Tetrahydro-4-methylene-2-octyl-5-oxo-3-furancarboxylic
acid, designated as C75 [1] in the recent literature, is a
synthetic compound [2]. It belongs to the class of paraconic
acids which are characterized by the ꢁ-lactone moiety, an
alkyl chain at C-2, a carboxylic group at C-3, and a methyl
or a methylene group at C-4 [3]. Paraconic acids having a
methylene group at C-4 are in general biologically active
natural compounds Fig. (1) [4-13].
A considerable body of synthetic information exists in
the literature to prepare paraconic acids having a methylene
group at C-4 in both racemic and enantioselective version
[3,18]. Among these methods, an efficient route was
reported by Carlson and Oyler in 1976 [19]. In a slightly
modified version [2,20] of their procedure, the dianion of 4-
methoxybenzyl itaconate was condensed with aldehydes of
various chain lengths to give in one step, after rapid
exposure to strong acid, the desired ꢀ-methylene-ꢁ-
butyrolactone carboxylic acid. Both trans- and cis-isomer
were generated by this method and these diastereomers could
be separated by flash column chromatography. By this
procedure, compounds (1) [20], (2) [20], (3) [2], (4) [19] and
(5) [19] were synthesized in racemic form.
HOOC
3 4
2
5
R
O
O
(+)
1: R = n-C3H7
2: R = n-C5H11; Methylenolactocin
3: R = n-C8H17; C75
4: R = n-C11H23; Nephrosterinic acid
5: R = n-C13H27; Protolichesterinic acid
Fig. (1). Structure of paraconic acids 1-5.
Here we report the enzymatic resolution of racemic
methyl ester of C75 with Acylase I [21] as a preliminary
investigation on the use of enzymatic hydrolysis for the
obtainment of compounds (1)–(5) in enantiomerically pure
form. For our present investigation, the methyl ester of C75
was synthesized in racemic form by exploiting a modified
procedure with respect to that reported by Carlson and Oyler
[19].
For example, (2R,3S)-(+)-(1), a toxin that induces
formation of black spots on the peel of banana, was recently
isolated from the plant pathogen Lasiodiplodia theobromae
[4]. (–)-Methylenolactocin (2) isolated from the culture
filtrate of Penicillium sp. [5] is active against some Gram-
positive bacteria and it prolongs the life span of mice
inoculated with Ehrlich carcinoma [6]. (+)-Nephrosterinic
acid (4) was isolated from lichen Nephromopsis endocrocea
[7] and (+)-protolichesterinic acid (5), isolated from various
sources of Cetraria [8], from yellow Acarospora species of
subgenus Xantotallia [9] and from lichens of Parmelia
species [10], exhibiting in vitro anti-bacterial activity against
Helicobacter pylori [11], there by inhibiting 5-lipoxygenase
[12] and it also showed anti-proliferative effects on
malignant cell-lines and mitogen stimulated lymphocytes in
vitro [13].
RESULTS AND DISCUSSION
Synthesis of the Methyl Ester of (±)-C75
The synthesis of racemic trans methyl ester of C75 (12)
[17] (Scheme 1) started with the regioselective ring-opening
of itaconic anhydride (6) with methanol to give itaconic acid
methyl hemiester
7
[22]. Treatment with lithium
bis(trimethylsilyl)amide converted the hemiester into the
corresponding ester enolate (8), which was reacted with
nonanal. After acidification at –78 °C, a 3:2 mixture of the
corresponding syn and anti hydroxy acids (9) and (10) was
*Address correspondence to this author at the Dipartimento di Scienze
Chimiche, Università di Trieste, via L. Giorgieri 1, 34127 Trieste, Italy; Tel:
+39-040-5583923; Fax: +39-040-5583903; E-mail: pnitti@units.it
1
obtained. They were not isolated but only identified by H
NMR analysis of the crude reaction mixture. Their
1570-1786/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

246 Letters in Organic Chemistry, 2010, Vol. 7, No. 3
Chakrabarty et al.
O
OLi
2.5 eq. LiHMDS
THF/–78°C
OH
OLi
MeOH
MeO
MeO
reflux, 40 min
O
O
O
O
O
6
7
8
1) n-C8H17CHO
2) 6N H2SO4
O
MeOOC
MeOOC
OH
TFA/CH2Cl2
r.t.
MeO
n-C8H17
+
O
OH
n-C8H17
O
n-C8H17
O
O
O
[9, 10]
12
11
Scheme 1. Synthesis of racemic methyl ester of C75, (±)-(12) and its diastereomer (±)-(11).
lactonization into the corresponding cis and trans lactonic
esters (11) and (12) [23] respectively was accomplished with
TFA (0.5 eq) in CH2Cl2 at room temperature. Diastereomers
(11) and (12) were separated by flash chromatography (23%
and 18% total yield respectively).
On the contrary, hydrolysis proved enantioselective when
performed with two Acylases I. The results obtained using
Acylases I immobilized on Eupergit® C from Aspergillus sp.
and Amano Acylase from Aspergillus melleus are reported in
Table 1. Acylase I from porcine kidney, Grade I, was also
checked but without any result.
The reaction was scaled up using both Acylases. We
observed that while with Acylase I immobilized on
Eupergit® C, the enantiomeric ratio E decreased from 60 to
20, that of Amano Acylase increased from 22 to 41. In the
former case, the acid (+)-(3) was obtained with 86% e.e.
stopping the reaction at about 50% conversion [28]. After
recrystallization from light petroleum, its e.e. raised to 98%
(white solid, m.p. 88–90 °C [lit. [2a] m.p. 76–77 °C for the
Enzymatic Hydrolyses of the Methyl Ester of C75
Following the general criteria in developing the most
suitable procedure for kinetic enzymatic resolution [24] of
the trans lactone (±)-(12) (Scheme 2), several enzymes were
screened. Lipases such as Porcine pancreatic lipase (PPL)
and Lipase from Aspergillus niger (AP12), as well as a
protease such as ꢀ-chymotrypsin (ꢀ-CT) were inactive.
Esterases, such as horse liver acetone powder (HLAP) and
purified Pig liver esterase (PLE), hydrolyzed regioselectively
the methyl ester group of compound (±)-(12) but without any
enantioselection. The rate of hydrolysis was higher for PLE
than for HLAP.
25
racemic mixture], [ꢀ]D = +8.4 (c 0.15, MeOH), CD in
MeOH: ꢁꢂ258 +0.26, ꢁꢂ225 –10.58). The unreacted ester (–)-
(12) was recovered with 77% e.e.
With Amano Acylase at 56% conversion value, [29] the
25
recovered unreacted ester (–)-(12) had >99% e.e. ([ꢀ]D = –
MeOOC
MeOOC
HOOC
6N HCl
(or PLE)
Enzyme
pH 7.4
+
(–)-3
n-C8H17
O
n-C8H17
O
n-C8H17
O
O
O
O
(±)-12
(+)-3
(–)-12
Scheme 2. Enzymatic resolution of the methyl ester of C75.
Table 1. Enantioselective Hydrolyses of (±)-(12) with Acylases I
(+)-(3)
(–)-(12)
Entry
Enzyme
Time
Conv.a (%)
Ed
e.e. (%) b
e.e. (%)c
Acylases I,
immobilized on Eupergit® Ce
Amano Acylasef
1
5h
4h
18
17
96
90
21
18
60
22
2
aCalculated values, Ref. [25].
bDetermined by chiral HRGC on a ChiraldexTM type G-TA, trifluoroacetyl ꢃ-cyclodextrin column (40 m x 0.25 mm) (carrier gas He, 180 KPa, split 1:100) of its ethyl ester [26] Rt
183.6 min for (2R,3S)-(+)-enantiomer and Rt 201.7 min for (2S,3R)-(–)-enantiomer (150°C).
cDetermined by chiral HRGC on a ChiraldexTM type G-TA, trifluoroacetyl ꢃ-cyclodextrin column (40 m x 0.25 mm) (carrier gas He, 180 KPa, split 1:100) Rt 155.6 min for (2R,3S)-
(+)-enantiomer and Rt 164.5 min for (2S,3R)-(–)-enantiomer (150 °C).
dRef. [27].
eConditions: compound (±)-(12) (0.034 g, 0.12 mmol), 0.1 M phosphate buffer (10 mL) at pH 7.4, 1 mM CoCl2, 0.1 g of Acylase I immobilized on Eupergit® C 102 U/g, r.t.
fConditions: compound (±)-(12) (0.050 g, 0.19 mmol), 0.1 M phosphate buffer (10 mL) at pH 7.4, 0.05 g of Amano Acylase 30000 U/g, r.t.

The First Kinetic Enzymatic Resolution of Methyl Ester of C75
Letters in Organic Chemistry, 2010, Vol. 7, No. 3
247
Valentin, E. In Targets in Heterocyclic Systems; Attanasi, O.A.;
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7.1 (c 0.42, MeOH), CD in MeOH: ꢀꢁ258 –0.30, ꢀꢁ223 +9.3),
while the acid (+)-(3) was isolated with 78% e.e.
[4]
[5]
[6]
[7]
[8]
He, G.; Matsuura, H.; Yoshihara, T. Isolation of an ꢀ-methylene-ꢁ-
With the purpose of obtaining the enantiomeric lactonic
acid (–)-(3), the enantiopure lactonic ester (–)-(12) having
>99% e.e. was subjected to both chemical and enzymatic
hydrolyses. Chemical hydrolysis was performed with 6N
HCl [18k,30], while Pig liver esterase (PLE) was used for
enzymatic hydrolysis. [31] In both cases the enantiomeric
excess of the ester was retained in the resulting acid and
optically pure (–)-C75 was obtained with >99% e.e. (m.p.
88–90 °C, ([ꢂ]D25 = –9.5 (c 0.49, MeOH)).
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The (2R,3S) absolute configuration was assigned to the
dextrorotatory enantiomer of C75 by comparison of its CD
spectrum (ꢀꢁ258 +0.26, ꢀꢁ225 –10.58) with that of (2R,3S)-
(+)-protolichesterinic acid (5) (ꢀꢁ257 +0.32, ꢀꢁ220 –9.62)
[32], whose absolute configuration is known. On the other
hand, the positive sign of its specific optical rotation was
already indicative that the absolute configuration of (+)-C75
was the same as that of (+)-methylenolactocin (2), (+)-
nephrosterinic acid (4) and (+)-protolichesterinic acid (5),
namely (2R,3S) [18e].
[9]
[10]
CONCLUSION
[11]
Two cases are reported in the literature on enzymatic
resolution of ꢂ-methylene-ꢃ-lactones [33], both bearing the
alkoxycarbonyl group at the ꢃ-position. It is known that in
the presence of a carboxy group the ꢄ-position greatly favors
the exo-endo equilibration of the double bond [19], thus
preventing any chemical resolution. The enzymatic
resolution reported here is of particular interest.
[12]
[13]
[14]
Furthermore, this work represents another interesting
example of the ability of acylases to hydrolyze the ester
group [34].
ACKNOWLEDGEMENTS
We are grateful to the Ministero dell’Università e della
Ricerca (PRIN2007) and to the University of Trieste
(Finanziamento Ricerca d’Ateneo) for financial supports.
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of Acylase I immobilized on Eupergit® C (1.5 g, 153 U) in a 1 mM
solution of CoCl2 (150 mL of phosphate buffer at pH 7.4). After
stirring for 24 h, maintaining the pH value constant by addition of
1N NaOH, the mixture was extracted with ether at pH 7.4. The
solvent extracted both the acid and the unreacted ester, so they
needed to be separated. The organic phase was then treated with a
5% NaHCO3 solution. The organic phase containing the ester was
dried on anhydrous Na2SO4 and, after elimination of the solvent
under vacuum, compound (-)-12 was obtained with 77% e.e. (0.190
g, 42% yield). The basic aqueous phase was acidified to pH 2 with
HCl, extracted with ether and dried on anhydrous Na2SO4.
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yield, 86% e.e.) as a white solid. The solid was refluxed in
petroleum ether, the acid (+)-3, recovered by decantation of the hot
solvent, had 98% e.e, m.p. 88-90°C, [ꢂ]D25 = +8.4 (c 0.15, MeOH),
CD in MeOH ꢃꢄ258 +0.26, ꢃꢄ225 -10.58.
[29]
Compound (±)-12 (0.239 g, 0.9 mmol) was added to a suspension
of Amano Acylase (0.239 g, 7170 U) in phosphate buffer at pH 7.4
(40 ml), the mixture was stirred for 48 h, while maintaining the pH
value constant by addition of 1N NaOH, and extracted with ether.
The acidic compound (+)-3 with 78% e.e. (28% yield) was
separated and the recovered ester (-)-12 was found to have >99%
e.e., 34% yield, [ꢂ]D25 = -7.1 (c 0.42, MeOH), CD in MeOH: ꢃꢄ258
0.30, ꢃꢄ223 +9.3.
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Baker, B.R.; Schaub, R.E.; Williams, J.H. An antimalarial alkaloid
from hydrangea. X. Synthesis of 3-[ꢂ-keto-ꢁ-(4-methyl-2-
pyrrolidyl)propyl]-4-quinazolone J. Org. Chem. 1952, 17, 116-131.
[30]
[31]
Compound (-)-12 (0.085 g, 0.32 mmol) with 99% e.e. was refluxed
in 2-butanone with 12 drops of 6N HCl. After 4 hours the solvent
was evaporated and the solid mass was extracted with 5% NaHCO3
solution to separate the acid from the unreacted ester. The aqueous
layer, acidified to pH 2, was then extracted with diethyl ether, the
organic layer was dried over anhydrous Na2SO4. Evaporation of the
solvent under vacuum furnished the acidic lactone (-)-3 (0.068 g,
[21]
82% yield), m.p. 88-90oC, [ꢂ]D25 = -9.5 (c 0.49, MeOH), >99% e.e.
Compound (-)-12 (0.083 g, 0.31 mmol) with 99% e.e. in acetone (3
mL) and phosphate buffer, pH 7.4 (27 mL) was stirred with 6 mg
PLE (4839 U/mmol) at room temperature for 23 hr maintaining the
pH value constant by adding 1N NaOH solution. After the reaction
was complete the reaction mixture was extracted with ether at pH
7.4. The organic layer was dried over anhydrous Na2SO4, the
solvent was removed and (-)-3 was recovered (>99% e.e., 0.056 g,
71% yield).
Huneck, S.; Schreiber, K.; Höfle, G.; Snatzke G. Neodihydromurol
and murolic acid, two new ꢁ-lactonecarboxylic acids from
Lecanora muralis. J. Hattori Bot. Lab. 1979, 45, 1-23.
a) Bertoli, A.; Fanfoni, L.; Felluga, F.; Pitacco, G.; Valentin, E.
Chemoenzymatic synthesis of optically active ꢀ-methylene-ꢁ-
carboxy-ꢁ-lactams and ꢁ-lactone. Tetrahedron: Asymmetry, 2009,
20, 2305-2310; b) Pitacco, G.; Sessanta o Santi, A.; Valentin, E.
Enzymic resolution of an ꢀ-methylene-ꢁ-lactonic ester.
Tetrahedron: Asymmetry, 2000, 11, 3263-3267.
[22]
[23]
Cis lactone 11: 1H NMR (ꢀ ppm, 400 MHz): 6.40 (1H, d, J=2.2 Hz,
C=CH2), 5.83 (1H, d, J=2.2 Hz, C=CH2), 4.63 (1H, ddd, J1=4.2,
J2=7.9, J3=9.0 Hz, H-2), 4.01 (1H, dt, J1=J2=2.2, J3=7.7 Hz, H-3),
3.76 (3H, s, OCH3), 1.60 (2H, m), 1.4-1.2 (12H, m), 0.88 (3H, t,
J=6.8, CH3); 13C NMR (ꢀ ppm, 67.80 MHz) 169.3 (s), 168.8 (s),
133.6 (s, C-4), 124.9 (t, C=CH2), 79.0 (d, C-2), 52.3 (q, OCH3),
49.1 (d, C-3), 31.7 (t), 31.5 (t), 29.3 (t), 29.2 (t), 29.1 (t), 25.5 (t),
[32]
[33]
1
22.6 (t), 14.0 (q). Trans lactone 12: H NMR (ꢀ ppm, 400 MHz):
6.40 (1H, d, J=2.8 Hz, C=CH2), 5.91 (1H, d, J=2.8 Hz, C=CH2),
4.80 (1H, q, J=6.2 Hz, H-2), 3.80 (3H, s, OCH3), 3.59 (1H, dt,
J1=J2=2.8, J3=6.2 Hz, H-3), 1.72 (2H, m), 1.5-1.2 (12H, m), 0.88
(3H, t, J=6.5, CH3); 13C NMR (ꢀ ppm, 67.80 MHz) 169.7 (s), 168.3
(s), 133.0 (s, C-4), 125.1 (t, C=CH2), 79.0 (d, C-2), 52.9 (q, OCH3),
49.7 (d, C-3), 35.7 (t), 31.7 (t), 29.3 (t), 29.1 (2t), 24.7 (t), 22.6 (t),
14.0 (q).
[34]
a) Youshko, M.I.; van Langen, L.M.; Sheldon, R.A.; ꢃvedas, V.K.
Application of aminoacylase I to the enantioselective resolution of
ꢀ-amino acid esters and amides. Tetrahedron: Asymmetry, 2004,
15, 1933-1936; b) Liljeblad, A.; Aksela, R.; Kanerva, L.T. Use of
enantio-, chemo- and regioselectivity of acylase I: resolution of
polycarboxylic acid esters. Tetrahedron: Asymmetry, 2001, 12,
2059-2066.
[24]
a) Bornscheuer, U.T.; Kazlauskas, R.J. Hydrolases in Organic
Synthesis: Regio-and Stereoselective Biotransformations, 2nd ed.;
Wiley-VCH: Weinheim, Germany, 2005; b) Faber, K.;
Biotransformations in Organic Chemistry, 5th ed., Springer-Verlag,