Enzymatic resolution of α-methyleneparaconic acids and evaluation of their biological activity

Article abstract of DOI:10.1002/chir.22419

Both enantiomers of three biologically relevant paraconic acids-MB-3, methylenolactocin, and C75-were obtained with enantioselectivities up to 99% by kinetic enzymatic resolutions. Good enantiomeric excesses were obtained for MB-3 and methylenolactocin, using α-chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was resolved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and antifungal activities, showing weak effects and practically no difference between enantiomers in each case. At high concentrations (16-64μg/mL), (-)- C75 acted as an antimicrobial agent against Gram-positive bacteria.

Full text of DOI:10.1002/chir.22419

CHIRALITY 27:239–246 (2015)
Enzymatic Resolution of α-Methyleneparaconic Acids and Evaluation
of their Biological Activity
KUHELI CHAKRABARTY,1 IVANA DEFRENZA,2 NUNZIO DENORA,2 SARA DRIOLI,3 CRISTINA FORZATO,3 MASSIMO FRANCO,2
2
3
3
*
GIOVANNI LENTINI, PATRIZIA NITTI, AND GIULIANA PITACCO
1Department of Chemistry, Visva-Bharati, Santiniketan, India
2Dipartimento di Farmacia – Scienze del Farmaco, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy
3Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Trieste, Italy
ABSTRACT
Both enantiomers of three biologically relevant paraconic acids—MB-3,
methylenolactocin, and C75—were obtained with enantioselectivities up to 99% by kinetic enzy-
matic resolutions. Good enantiomeric excesses were obtained for MB-3 and methylenolactocin,
using α-chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was re-
solved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and
antifungal activities, showing weak effects and practically no difference between enantiomers
in each case. At high concentrations (16–64 μg/mL), (–)- C75 acted as an antimicrobial agent
against Gram-positive bacteria. Chirality 27:239–246, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: α-methylene-γ-butyrolactones; MB-3, methylenolactocin; C75
INTRODUCTION
These findings stimulated interest in this class of com-
pounds and new syntheses of a variety of racemic and
The α-methylene-γ-butyrolactone moiety is part of a variety
of natural compounds, most of which belong to the sesquiter-
pene series and are associated with a wide spectrum of bio-
logical activities both in vitro and in vivo.1 γ-Butyrolactones
containing a β-carboxy group are classified as “paraconic
acids." They are generally lichen products and many of them
were isolated and characterized as antibiotic, antiviral, anti-
fungal, and antitumor agents.2–4
enantiopure paraconic acids are reported.47–56
As a part of our continuing study on enzymatic resolutions
of esters of α- methyleneparaconic acids,57 we have taken into
account compounds 1a–d (Fig. 1) with a twofold purpose: to
achieve their resolution by means of enzymatic methods and
to evaluate the biological activity of both enantiomers.
MATERIALS AND METHODS
A few structures of known α-methyleneparaconic acids are
reported in Figure 1, which also shows the correct configura-
tion for the enantiomer which is dextrorotatory in chloroform.
It must be underlined that the active diastereomer is that hav-
ing the substituents in trans relationship, namely 2R*,3S*.
Compound (+)-1a (indicated as MB-3 by Sigma-Aldrich in
the racemic form) is a toxin known to be responsible for the
black spots observed on fruit peel5and for its activity as inhib-
itor of acetyltransferase Gcn5 of human histone.6,7
Methylenolactocin (–)-1b, found in the culture filtrate of
Penicillium sp.,8 is active against some Gram-positive bacteria
and Ehrlich carcinoma.9 Compound C75 1c, although not
natural, is a fatty acid synthase (FAS) inhibitor,10–15 which
also shows antitumor,16–23 antiviral,24,25 and antiinflammatory
activity26 and it is active against mycobacteria of the tubercu-
losis complex.27 Nephrosterinic acid (+)-1d, isolated from the
lichen Nephromopsis endocrocea,28 showed antibacterial29 and
antitumor30 properties. Protolichesterinic acid (+)-1e, isolated
from various sources of lichens,31–37 exhibits in vitro and
in vivo antiinflammatory activity,38 in vitro antibacterial activity
against Helicobacter pylori,39 inhibited 5-lipoxygenase,40 and it
also showed in vitro antiproliferative effects on malignant cell
lines.41–43
IR spectra were recorded on a Thermo Nicolet AVATAR 320 FT/IR
spectrophotometer. 1H-NMR and 13C-NMR spectra were run on a Jeol
(Tokyo, Japan) EX-400 spectrometer (400 MHz for proton, and
100 MHz for carbon), and on a Jeol EX-270 spectrometer (270 MHz
for proton, 68 MHz for carbon) using deuteriochloroform as a solvent
and tetramethylsilane as the internal standard. Coupling constants are
given in Hz. Optical rotations at 589 nm were determined on
a
Perkin-Elmer (Boston, MA) Model 241 polarimeter; optical rotatory
power values are given in 10-1 deg cm2 g-1. CD spectra were obtained
on Jasco (Tokyo, Japan) J-710 spectropolarimeter (0.1 cm pathlength
cell); Δε values are given in L cm-1 mol-1. Capillary gas chromatographic
measurements were performed on a Carlo Erba (Milan, Italy) GC 8000
instrument and on a Shimadzu (Kyoto, Japan) GC-14B instrument,
equipped with a flame ionization detector, the capillary columns being
OV 1701 (25 m x 0.32 mm) (carrier gas He, 40 KPa, split 1:50) and a
Chiraldex type G-TA, trifluoroacetyl γ-cyclodextrin (γ-CDX) (40 m x
0.25 mm) (carrier gas He, 180 KPa, split 1:100, isotherm 150 °C) or
DiMePe ß-cyclodextrin (25 m x 0.25 mm) (ß-CDX) (carrier gas He,
110 KPa, split 1:50, isotherm 150 °C). High-performance liquid chroma-
tography (HPLC) measurements were performed on a Hewlett Packard
(Corvallis, OR) series 1100 instrument, the column being Lux 5 μ
Cellulose-2 Phenomenex, detector UV 220 nm, a mixture of hexane-
isopropanol in a 9:1 ratio as the eluent, flow 1 ml/min. Melting points
The cytotoxic and antitumor activity of these α-
methyleneparaconic acid derivatives is generally attributed
to the interaction between the external double bond and
nucleophilic sites of biologically important molecules.44–46
This mechanism was proposed for both molecules exhibiting
cytotoxic activity and those showing allergenic properties.
*Correspondence to: Patrizia Nitti, Dipartimento di Scienze Chimiche e
Farmaceutiche, Università di Trieste, via Licio Giorgieri 1, I-34127 Trieste,
Italy. E-mail: pnitti@units.it
Received for publication 13 August 2014; Accepted 18 November 2014
DOI: 10.1002/chir.22419
Published online 9 January 2015 in Wiley Online Library
(wileyonlinelibrary.com).
© 2015 Wiley Periodicals, Inc.

240
CHAKRABARTY ET AL.
CH3); 13C NMR (68 MHz, CDCl3, δ): 169.7 (s), 168.3 (s), 133.0 (s),
125.15 (t), 78.8 (d), 52.9 (q), 49.8 (d), 37.8 (t), 18.1 (t), 13.6 (q); MS
(ESI, m/z): 221 (100) [M + Na]+.
Chiral HRGC: ß-CDX: retention time [tR] =11.7 min for (+)-(2R,3S)-5a,
tR =12.1 min for (–)-(2S,3R)-5a. Chiral HRGC, γ-CDX: tR =17.3 min for
(+)-(2R,3S)-5a, tR =18.1 min for (–)-(2S,3R)-5a.
Methyl (2R*,3R*)-4-methylene-5-oxo-2-propyltetrahydrofuran-3-
carboxylate 6a58
. Oil, 23% yield, after purification; IR (film): ν =1770,
1740, 1667 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.42 (d, J =2.2 Hz, 1H;
=CH), 5.83 (d, J =2.2 Hz, 1H; =CH), 4.64 (m, 1H; H2), 4.00 (dt,
J1 = J2 = 2.2 Hz, J3 = 7.7 Hz, 1H; H3), 3.76 (s, 3H,OCH3), 1.7–1.5 (m, 3H),
1.45 (m, 1H), 0.95 (t, J =7.0 Hz, 3H; CH3); 13C NMR (68 MHz, CDCl3,
δ): 169.5 (s), 169.0 (s), 133.6 (s), 125.0 (t), 77.9 (d), 52.4 (q), 49.1 (d),
33.5 (t), 18.9 (t), 13.7 (q); MS (ESI, m/z): 221 (100) [M + Na]+.
Fig. 1. Examples of α-methyleneparaconic acids.
were measured with a Büchi apparatus and were not corrected. Enzy-
matic hydrolyses were performed using a pH-stat Controller PHM290
Radiometer (Copenhagen, Denmark). Mass spectra were recorded on
an ion trap instrument Finningan (San Jose, CA) GCQ (70 eV) and on
an ESI-MS ion trap Bruker (Karlsruhe, Germany) Esquire 4000 instru-
ment. Thin-layer chromatographies (TLC’s) were performed on
Polygram Sil G/UV254 silica gel precoated plastic sheets (eluent: light
petroleum-ethyl acetate). Flash chromatography was run on silica gel
230–400 mesh ASTM (Kieselgel 60, Merck, Darmstadt, Germany).
Light petroleum refers to the fraction with b.p. 40–70 °C and ether to
diethyl ether. Anhydrous tetrahydrofuran (THF) was prepared by distil-
lation over sodium benzophenone ketyl. The following enzymes were
used in enzymatic hydrolyses of 5a–c: Acylase I from Aspergillus
immobilized on Eupergit C (Fluka), 102 U/g: 44 U/mmol substrate
in 1 mM CoCl2; Aminoacylase from Aspergillus melleus (Amano Acylase
from Sigma Aldrich, St. Louis, MO), >30,000 U/g: 1:1 w/w; Acylase I
porcine kidney (Sigma), 3442 U/mg: 78019 U/mmol; α-Chymotrypsin
(α-CT) from bovine pancreas (Fluka), 90 U/mg: 2400 U/mmol; Lipase
from Candida antarctica “Novozym 435” (Novo Nordisk A/S,
Bagsvaerd, Denmark), ≥10,000 U/g: 1:1 w/w; Pig pancreatic lipase
(PPL type II, Sigma, crude), 46 U/mg of protein 6133 U/mmol; Esterase
from hog liver (PLE, Fluka), 250 U/mg: 5000 U/mmol; Liver acetone
powder equine (HLAP, Sigma), 1:1 w/w; Porcine liver acetone powder
(PLAP, Sigma), 27 U/mg: 6230 U/mmol substrate; SPRIN Lipo CALB,
adsorbed immobilized preparation of CALB on polystyrene DVB cross-
linked (300–800 μm), >2000 U/g dry: 1:1 w/w; Lipase from Pseudomonas
cepacea immobilized by SPRIN: 1:1 w/w; Lipase from Pseudomonas
fluorescens (Fluka), 42.5 U/mg: 1:1 w/w; Protease from Bacillus subtilis,
11.6 U/mg: 1:1 w/w.
Methyl (2R*,3S*)-4-methylene-5-oxo-2-pentyltetrahydrofuran-3-
carboxylate 5b. Yellow oil, 22% yield, after purification. All spectro-
scopic data are in accordance with those reported in the literature.58–61
Chiral HRGC, ß-CDX: tR =28.9 min for (+)-(2R,3S)-5b, tR =30.2 min for
(–)-(2S,3R)-5b. Chiral HRGC, γ-CDX: tR =34.9 min for (+)-(2R,3S)-5b, tR
=37.0 min for (–)-(2S,3R)-5b.
Methyl (2R*,3R*)-4-methylene-5-oxo-2-pentyltetrahydrofuran-3-
carboxylate 6b. Yellow oil, 32% yield, after purification. Spectroscopic
data are in accordance with those reported in the literature.58–60
Methyl (2R*,3S*)-4-methylene-5-oxo-2-octyltetrahydrofuran-3-car-
boxylate 5c. All spectroscopic data are in accordance with those
reported in the literature.57 Chiral HRGC, ß-CDX: tR =122.5 min for (+)-
(2R,3S)-5c, tR =126.9 min for (–)-(2S,3R)-5c. Chiral HRGC, γ-CDX: tR
=155.6 min for (+)-(2R,3S)-5c, tR =164.5 min for (–)-(2S,3R)-5c.
Methyl
(2R*,3R*)-4-methylene-5-oxo-2-octyltetrahydrofuran-3-
carboxylate 6c. All spectroscopic data are in accordance with those re-
ported in the literature.57
Methyl (2R*,3S*)-4-methylene-5-oxo-2-undecyltetrahydrofuran-3-
carboxylate 5d. Yellow solid, 23% yield, after purification, mp 38–39 °
C (lit.61 mp 38 °C). The other spectroscopic data are also in accordance
with those reported in the literature.61 Chiral HPLC: tR =10.0, 10.7 min
for ( )-5d.
Methyl (2R*,3R*)-4-methylene-5-oxo-2-undecyltetrahydrofuran-3-
carboxylate 6d. Yellow solid, 33% yield, after purification, mp 43–44
General Procedure for the Synthesis of Racemic Substrates
1
°C; IR (neat): ν =1760, 1732 cm-1; H NMR (400 MHz, CDCl3, δ): 6.41 (d,
To a solution of 17.35 ml of lithium bis(trimethylsilyl)amide (LiHMDS)
(1.0 M in THF), cooled to –78 °C, 1.0 g (6.94 mmol) of methyl hemi-ester
of itaconic acid in anhydrous THF (10.0 ml) was added dropwise. The
mixture was stirred at –78 °C for 1 h. The suitable aldehyde (9.7 mmol)
in 1.0 ml of anhydrous THF was then added and the mixture was stirred
for a further 5 h at –78 °C under argon atmosphere. The reaction was
quenched with 6 N H2SO4 (5 ml). It was then extracted with diethyl ether
and dried over anhydrous Na2SO4. The solvent was removed and the res-
idue was added with a solution of 250.0 μl of trifluoroacetic acid (TFA) in
dichloromethane (10.0 ml) and stirred overnight at room temperature.
The solvent was removed and traces of TFA were removed by
coevaporation with diethyl ether (three times). The residue was a mixture
of diastereomeric lactones which were separated by flash column chro-
matography (ethyl acetate-light petroleum, gradient from 2% up to 10%).
Before charging the column with the crude reaction mixture, the column
was eluted with 1% ethyl acetate in light petroleum with 1 ml of acetic acid
added to avoid isomerization of the α-methylene-γ-lactones into their α,ß-
butenolide isomers.
J =2.2 Hz, 1H; =CH2), 5.83 (d, J =2.2 Hz, 1H; =CH2), 4.60–4.64 (m, 1H; H2),
4.01 (dt, J1 = 2.2 Hz, J2 = 7.6 Hz, 1H; H3), 3.76 (s, 3H;OCH3), 1.53–1.63
(m, 2H; CH2), 1.21–1.39 (m, 18H; 9 CH2), 0.88 (t, J =7.2 Hz, 3H; CH3);
13C NMR (68 MHz, CDCl3, δ): 169.3 (s), 168.8 (s), 133.6 (s, C4), 125.0
(t, =CH2), 78.2 (d, C2), 52.3 (q, OCH3), 49.1 (d, C3), 31.9 (t), 31.5 (t),
29.6 (2 t), 29.4 (t), 29.34 (t), 29.30 (t), 29.2 (t), 25.5 (t), 22.6 (t), 14.1
(q). EIMS (m/z (%)): 310 (24) [M+•], 251 (100) [M+• – CO2CH3]+, 233
(46), 205 (36), 187 (49), 179 (39), 165 (35), 155 (35), 149 (42), 137
(39), 121 (44), 119 (45), 109 (36), 95 (45), 93 (45), 79 (52), 67 (56).
Enzymatic Hydrolyses: General Procedure for Small-Scale
Enzymatic Hydrolyses
Three slightly different procedures were used owing to the different
solubility of the substrates in diethyl ether. For compounds 5a and 5b,
the enzyme was added to the substrates (0.15 mmol) in phosphate buffer
at pH 7.4 (10 ml), the mixture was stirred for the time indicated in Table 1,
while maintaining the pH value constant by addition of 1 M NaOH, and
eventually extracted with ether. From the ethereal solution the unreacted
ester 5a (or 5b) was recovered after evaporation of the solvent, while the
acid 1a (or 1b) was isolated from the remaining buffer solution, by acid-
ification with 2 M HCl to pH 2 and extraction with ether. In order to mea-
sure the enantiomeric excess of the acids by chiral HRGC, they were
esterified with ethanol (2 ml) and Me3SiCl62 (20 μl) to give the corre-
sponding derivatives 5’a and 5’b.
Methyl (2R*,3S*)-4-methylene-5-oxo-2-propyltetrahydrofuran-3-
carboxylate 5a58
. Oil, 18% yield, after purification; IR (neat): ν =1769,
1742, 1663 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.42 (d, J =2.9 Hz, 1H;
=CH), 5.93 (d, J =2.9 Hz, 1H; =CH), 4.82 (dt, J1 = J2 = 5.6 Hz, J3 = 7.5 Hz,
1H; H2), 3.80 (s, 3H; OCH3), 3.58 (dt, J1 = J2 = 2.8 Hz, J3 = 5.7 Hz, 1H; H3),
1.80–1.63 (m, 2H; CH2), 1.60–1.40 (m, 2H, CH2), 0.97 (t, J =7.3 Hz, 3H;
Chirality DOI 10.1002/chir

ENZYMATIC RESOLUTION OF α-METHYLENEPARACONIC ACIDS
241
Ethyl (2R*,3S*)-4-methylene-5-oxo-2-propyltetrahydrofuran-3-
carboxylate 5’a. HRGC, ß-CDX: tR =14.4 min for (+)-(2R,3S)-5’a,
tR =14.8 min for (–)-(2S,3R)-5’a. HRGC, γ-CDX: tR =19.9 min for (+)-(2R,3S)-
5’a, tR =20.4 min for (–)-(2S,3R)-5’a.
Ethyl
(2R*,3S*)-4-methylene-5-oxo-2-pentyltetrahydrofuran-3-
carboxylate 5’b. HRGC, ß-CDX: tR =36.3 min for (+)-(2R,3S)-5’b,
tR =37.5 min for (–)-(2S,3R)-5’b.HRGC, γ-CDX: tR =44.1 min for (+)-(2R,3S)-
5’b, tR =47.6 min for (–)-(2S,3R)-5’b.
As for compound 5c, at the end of the procedure described above for
5a and 5b, the unreacted ester could not be separated from its hydroly-
sis product 1c since both were soluble in ether. Therefore, compound 1c
was separated from its ester 5c treating the ethereal phase with 5% aque-
ous solution of NaHCO3. The organic phase containing the ester was
dried on anhydrous Na2SO4 and, after elimination of the solvent under
vacuum, the unreacted ester 5c was obtained. The basic aqueous phase
was acidified with 2 M HCl to pH 2, extracted with ether and dried on an-
hydrous Na2SO4. Evaporation of the solvent afforded the acid 1c which
was esterified with ethanol and Me3SiCl62 to give 5’c, in order to mea-
sure its enantiomeric excess (ee) by chiral HRGC.
Ethyl (2R*,3S*)-4-methylene-5-oxo-2-octyltetrahydrofuran-3-car-
boxylate 5’c. HRGC, ß-CDX: tR =153.0 min for (+)-(2R,3S)-5’c,
tR =159.0 min for (–)-(2S,3R)-5’c. HRGC, γ-CDX: tR =183.6 min for
(+)-(2R,3S)-5’c, tR =201.7 min for (–)-(2S,3R)-5’c.
Similarly, compound 1d was recovered in admixture with its ester 5d
but it could not be separated from it following the procedure used for 1c
and 5c, and therefore the two compounds were separated by flash chro-
matography (eluent: gradient from 7.5% ethyl acetate, 92% light petro-
leum, 0.5% acetic acid up to 35.5% ethyl acetate, 64% light petroleum,
0.5% acetic acid). The ee’s of 1d and 5d were determined by chiral
HPLC. During HPLC elution both compounds largely isomerized into
their α,ß-butenolide isomers.
Large-Scale Enzymatic Hydrolyses
Hydrolysis of 5a with α-CT. To an emulsion of 5a (565 mg,
2.85 mmol) in phosphate buffer (40 ml) α-CT (74 mg) was added under
vigorous stirring. After 10 min, following the above-described procedure,
the unreacted ester (+)-5a with 11% ee was isolated (390 mg, 69% yield),
together with (–)-1a with 74% ee (76 mg, 14% yield). (–)-(2S,3R)-1a: white
solid, mp 77–80 °C (Lit.5 mp 77–80 °C); [α]2D5 = –2.9 (c =0.25 in = –2.9
(c =0.25 in chloroform); CD: Δε263 = –0.2, Δε225 = +6.1 (MeOH).
The unreacted ester (+)-5a (390 mg, 1.97 mmol) having 11% ee was hy-
drolyzed in phosphate buffer (36 ml) with α-CT (51 mg) for 73 min. After
the usual workup, (+)-5a with 68% ee was isolated in admixture with 33%
of its α,ß-butenolide isomer (144 mg, 37% yield), together with (–)-1a
with 39% ee (163 mg, 45% yield).
(+)-(2R,3S)-5a: [α]2D5 = +15.1 (c =0.24 in methanol) in admixture with
33% of its α,ß-butenolide isomer.
Hydrolysis of 5a with Aminoacylase. The unreacted ester (+)-5a
(144 mg with 33% of its α,ß-butenolide isomer) having 68% ee was hydro-
lyzed with aminoacylase (130 mg) in phosphate buffer (30 ml) for 95 min.
After the usual workup, a mixture of 22% of (–)-5a with 26% ee and 78% of
its α,ß-butenolide isomer with 30% ee was isolated in 55% yield, together
with (+)-1a with 81% ee (49 mg, 37% yield).
(+)-(2R,3S)-1a: white solid, mp 69–73 °C; [α]2D5 = +3.7 (c =0.35 in chloro-
form); [α]2D5 = –5.3(c =0.15 in methanol) {Lit.5 mp 77–80 °C; [α]2D5 = +18.2
(c =0.22 in chloroform for the (2R,3S) enantiomer}; CD: Δε267 = +0.1,
Δε225 = –4.8 (MeOH).
All the other spectroscopic data are in accordance with those found in
the literature.5,6
Hydrolysis of 5b with α-CT. To an emulsion of 5b (370 mg,
1.64 mmol) in phosphate buffer (90 ml), α-CT (36 mg) was added under
vigorous stirring. After 7 h, following the above-described procedure,
the unreacted ester (+)-5b with 79% ee was isolated (143 mg, 39% yield)
together with (–)-methylenolactocin 1b with 73% ee (63 mg, 18% yield).
Chirality DOI 10.1002/chir

242
CHAKRABARTY ET AL.
After recrystallization from ethyl acetate-light petroleum, the ee of
Biological Assays
(–)-1b increased to 98%. (–)-(2S,3R)-1b: white solid, mp 81–83 °C; [α]
Cytotoxicity assays. Cytotoxicity assays were carried out against hu-
man MCF-7 breast carcinoma and rat C6 glioma cells. MCF-7 cell lines
were maintained at 37 °C in a humidified incubator containing 5% CO2
in Dulbecco’s Modified Eagle’s medium (DMEM) (Lonza) nutrient sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-
glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The per-
centage of DMSO, the organic solvent in which the tested compounds
were dissolved, never exceeded 1% (v/v) in the samples. In preliminary
experiments, we verified that this amount did not affect cell viability. Cy-
totoxicity of the tested compounds is expressed as IC50 values—the con-
25 = –7.5 (c =0.71 in methanol); {Lit.9mp 82.5–83.5 °C; [α]2D6 = –2.37 (c
D
=3.00 in methanol); Lit.48 mp 81–83 °C; [α]2D5 = –7.0 (c =0.12 in metha-
nol); Lit.50 mp 82–84 °C; [α]2D4 = –17.13 (c =1.96 in chloroform); Lit.51
[α]2D3 = –6.46 (c =0.5 in methanol); Lit.52 [α]2D0 = –10 (c =0.5 in methanol);
Lit.53 mp 83–84 °C; [α]2D5 = –2.23 (c = 1.56 in methanol); Lit.63 mp
82–83 °C; [α]2D5 = –12.4 (c =0.5 in methanol); Lit.59 mp 82.5 °C;[α]3D2 = –18.8
(c =0.31 in chloroform); Lit.64mp 82–83 °C; [α]3D2 = –8.5 (c =0.31 in meth-
anol),[α]2D9 = –11.6 (c =0.31 in chloroform); Lit.65 [α]2D5 = –6.77 (c =0.52 in
methanol); Lit.66mp 82–84 °C; [α]2D6 = –6.7 (c =0.5 in methanol)}; CD:
Δε261 = –0.27, Δε224 = +11.1 (MeOH).
centrations that cause 50% growth inhibition.
A compound was
considered inactive when its IC50 value was >100 μM. The results were
determined using the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) assay. Cells were dispensed into 96-well microtiter
plates at a density of 5000 cells/well. Following overnight incuba-
tion, cells were treated with a range of compound concentrations
(0.5–100 μM). Then the plates were incubated at 37 °C for 72 h. An
amount of 10 μL of 0.5% w/v MTT was further added to each well
and the plates were incubated for an additional 4 h at 37 °C. Finally,
the cells were lysed by addition of 100 μL of 1:1 v/v DMSO:EtOH
solution. The absorbance at 570 nm was determined using a Perkin-Elmer
2030 multilabel reader Victor TM X3.
Hydrolysis of 5b with Aminoacylase. The unreacted ester (+)-5b
(143 mg, 0.63 mmol) recovered from the previous reaction and having
79% ee was hydrolyzed with aminoacylase (143 mg) in phosphate buffer
(20 ml) for 30 min. After the usual workup, (+)- methylenolactocin 1b
with 99% ee was isolated (20 mg, 15% yield).
(+)-(2R,3S)-1b: white solid, mp 80–81 °C; [α]2D5 =+ 6.8 (c =0.28 in meth-
anol) {Lit.51 [α]D23 = +6.5 (c =1.5 in methanol), Lit.53 mp 82 °C; [α]2D5 = +2.25
(c =1.46 in methanol), Lit.64 mp 82–83 °C [α] 32 = +7.4 (c =0.33 in
D
methanol), [α]3D0 = +16.6 (c =0.33 in chloroform)}; CD: Δε262 = +0.16,
Δε224 = –8.62 (MeOH).
Hydrolysis of 5b with Aminoacylase IPE. To a solution of 5b (40 mg,
0.18 mmol) in isopropyl ether (2 ml), aminoacylase (40 mg) in phosphate
buffer (2 ml) was added under vigorous stirring. After 4 days, following
the above-described procedure, the unreacted ester (–)-5b with 96% ee
was isolated (10 mg, 24% yield) together with (+)-methylenolactocin 1b
with 70% ee (5 mg, 12% yield).
Antimicrobial Studies
Antibacterial studies. The in vitro minimum inhibitory concentrations
(MICs, μg/mL) were assessed by the broth microdilution method, using
96-well plates, according to CLSI guidelines.68,69 Stock solutions of the
tested compounds were obtained in DMSO. Then twofold serial dilutions
in the suitable test medium between 512 and 0.03 μg/mL were plated. To
be sure that the solvent had no adverse effect on bacterial growth, a con-
trol test was carried out using DMSO at its maximum concentration along
with the medium. Bacteria strains available as freeze-dried discs, belong-
ing to the ATCC (American Type Culture Collection, Manassas, VA),
were used: S. aureus 29213, E. faecalis 29212 as the Gram-positive strains,
and E. coli 25922 as the Gram-negative one. To preserve the purity of cul-
tures and to allow reproducibility, a series of cryovials of all microbial
strains in glycerolic medium were set up and stored at –80 °C.
Precultures of each bacterial strain were prepared in Cation Adjusted
Mueller–-Hinton broth (CAMHB) and incubated at 37 °C until the growth
ceased. The turbidity of bacterial cell suspension was calibrated to 0.5
McFarland Standard by the spectrophotometric method (625 nm, range
0.08–0.10), and further the standardized suspension was diluted 1:100
with CAMHB to have 1–2 x 106 CFU/mL. All wells were seeded with
100 μL of inoculum. A number of wells containing only inoculated broth
as control growth were prepared. The plates were incubated at 37 °C
for 24 h, and the MIC values were recorded as the last well containing
no bacterial growth. Each assay was repeated twice in triplicate.
Norfloxacin was used as the reference drug. A compound was considered
inactive when its MIC was >64 μg/mL.
(–)-(2S,3R)-5b: oil; [α]D25 = –4.7 (c =0.42 in methanol) [Lit.63 [α]D25 = –5.3
(c =0.956 in chloroform)]; CD: Δε258 = –0.27, Δε223 = +7.98 (MeOH).
All spectroscopic data are in accordance with those reported in the
literature.63
Hydrolysis of 5c with Aminoacylase57
. To an emulsion of 5c
(362 mg, 1.35 mmol) in phosphate buffer (61.2 ml) and acetone (2 ml)
aminoacylase (242 mg) was added under vigorous stirring. After 4 h
30 min, following the above-described procedure, the unreacted ester
(–)-5c with 32% ee was isolated (159 mg, 44% yield), together with (+)-
C75 1c with 91% ee (38 mg, 11% yield). After recrystallization from light
petroleum the ee of (+)-1c increased to 98%.
(+)-(2R,3S)-1c: white solid, mp 88-90 °C; [α]2D5 = +8.4 (c =0.15 in metha-
nol), {Lit.10 mp 88-89 °C; [α]2D5 = +9.5 (c =0.15 in methanol), [α]2D5 = +11.4
(c =1.0 in chloroform)}; CD: Δε258 = +0.26, Δε225 = –10.58 (MeOH).
The unreacted ester (–)-5c (158 mg, 0.59 mmol) having 32% ee was hy-
drolyzed with aminoacylase (158 mg), in phosphate buffer (26.5 ml) and
acetone (4 ml) added for 24 h. After the usual workup, (–)-5c with 94%
ee was isolated (87 mg, 55% yield), together with (+)-C75 1c with 89% ee
(12 mg, 8% yield).
Antifungal Studies
Hydrolysis of 5c with HLAP. To a suspension of (–)-5c (86 mg,
0.32 mmol) having 94% ee in phosphate buffer (27.3 ml) and acetone
(2 ml) HLAP (86 mg) was added under vigorous stirring. After 22 h, fol-
lowing the above-described procedure, (–)-C75 1c with 94% ee was iso-
lated (24 mg, 29% yield). After recrystallization from light petroleum, the
ee of (–)-1c increased to 99%.
Antifungal studies68,70 were carried out against C. albicans 10231, C.
parapsilosis 22019, C. tropicalis 750, C. krusei 6258, belonging to the
ATCC collection. Preparation of stock solutions and purity of cultures
preservation were obtained as above described for antibacterial studies.
Precultures of each yeast strain were prepared in Sabouraud broth 2% glu-
cose (SAB), and incubated at 37 °C until the growth ceased. The turbidity
of yeast stock suspension was calibrated to 0.5 McFarland Standard by
the spectrophotometric method (530 nm, range 0.12–0.15), and further
the standardized suspension was diluted first 1:50 with SAB and then
1:20 in the same medium to have 1–5 x 106 CFU/mL. All wells were
seeded with 100 μL of inoculum. A number of wells containing only inoc-
ulated broth as control growth were prepared. The plates were incubated
at 37 °C for 24–48 h, and the MIC values were recorded as the last well
containing no fungal growth. Each assay was repeated twice in triplicate.
Fluconazole was used as the reference drug. A compound was consid-
ered inactive when its MIC was >64 μg/mL.
(–)-(2S,3R)-1c: white solid, mp 88–90 °C; [α]2D5 = –9.5 (c =0.49 in metha-
nol) {Lit.10 mp 88–89 °C; [α]2D5 = –11.4 (c =1.0 in chloroform)}.
All spectroscopic data are in accordance with those reported in the
literature.10,14,57,67
Hydrolysis of 5d with PLAP. To a solution of 5d (82 mg, 0.26 mmol)
in phosphate buffer (8 ml) and acetone (2 ml) PLAP (61 mg) was added
under vigorous stirring. After 75 min, following the above-described pro-
cedure, the unreacted ester 5d with 8% ee was isolated (22 mg, 27% yield),
together with nephrosterinic acid 1d47,49,61,64 with 11% ee (4 mg, 5%
yield). Chiral HPLC: tR =7.7, 8.8 min for ( )-1d.
Chirality DOI 10.1002/chir

ENZYMATIC RESOLUTION OF α-METHYLENEPARACONIC ACIDS
243
RESULTS AND DISCUSSION
Synthesis of Racemic Substrates
was isolated with ee’s ranging from 90 to 96% using acylases
from Aspergillus sp.57 (Entries 1–3). Novozym 435 was
enantiocomplementary to the previous enzymes, although
with poor enantioselectivity (Entry 6), and Acylase I from
porcine kidney, α-CT and PPL were ineffective. The best
enzymes for lactones 5a and 5b were acylases from Aspergil-
lus sp. and α-CT (Entries 1–3, 5), although with less
enantioselectivity, as indicated by the ee’s of their respective
acids 1a and 1b that ranged from 70% to 87%. Interestingly,
however, acylases and α-CT were enantiocomplementary.
On the contrary, Acylase I from porcine kidney, Novozym
435 and PPL were found poorly enantioselective (Entries 4,
6, 7). As is evident from Table 1 (Entry 8), PLE and HLAP
were able to hydrolyze all substrates; however, with no
enantioselectivity, and therefore they were used as alterna-
tives to chemical hydrolysis63 of enantiopure methyl
α-methyleneparaconates. Attempts to increase the enan-
tioselectivity of these hydrolyses by adding acetone or
ethanol as cosolvents were unsuccessful, while addition of
isopropyl ether resulted in a slight increase of the E values
(Entry 3).
In scaling up the enzymatic hydrolyses, both enantiomers
of 1a and 1b were obtained from the corresponding methyl
esters 5a and 5b using α-CT and aminoacylase, which were
enantiocomplementary. However, as their efficiencies were
not high, as demonstrated by their low E values, a particular
procedure was followed to take advantage of both enzymes.
For compound 5b, in a first run, performed with α-CT, the hy-
drolysis reaction was stopped at about 50% conversion. The
acid (–)-1b was isolated with 73% ee in 18% yield and the
unreacted ester (+)-5b was isolated with 79% ee in 39% yield.
To improve the optical yield of both compounds, the acid
(–)-1b was fractionally crystallized, thus increasing its ee to
98%, while the ester (+)-5b was subjected to a further resolu-
tion with aminoacylase that allowed the isolation of (+)-1b
with 99% ee (Scheme 2). However, since optical yield optimi-
zation was detrimental to chemical yield, the biological assays
were performed with (+)-1b and (–)- 1b having 92 and 93% ee,
respectively.
The methyl esters of the target compounds 1a–d to be sub-
jected to enzymatic kinetic resolutions, namely 5a-d, were syn-
thesized using a slightly modified version of the procedure
reported by Carlson and Oyler47 (Scheme 1). Methyl hemi-
ester of itaconic acid 2,71 prepared by nucleophilic ring fission
of itaconic anhydride, was converted into its lithium enolate
and reacted with the suitable aldehyde (RCHO= butanal, hexa-
nal, nonanal, and dodecanal). Acidification of the resulting re-
action mixture carried out at –78°C gave the corresponding
anti and syn hydroxy hemi-esters 3a–d and 4a–d in the ratio
of 2:3, identified only by 1H NMR analysis. Their lactonization,
carried out with TFA in CH2Cl2, furnished the corresponding
(2R*,3S*) and (2R*,3R*) lactonic esters 5a-d and 6a-d, re-
spectively, in about 50% total yield. Compounds 5a-d were sep-
arated from 6a-d by flash chromatography using as stationary
phase a silica gel pretreated with acetic acid to avoid the dou-
ble bond isomerization.
Attempts were made to isomerise compounds 6a-d into
their respective diastereomers 5a- d; however, unsuccessfully.
The first strategy involved protection of the double bond via a
Michael addition of a thiol (ethanethiol and dodecanethiol),72
equilibration, and deprotection via a retro-Michael reaction.
This reaction sequence was attempted on 6d, but in the first
step, conversion into its α,ß-butenolide isomer occurred, prob-
ably because of the slightly basic conditions required by the
procedure. Following a second strategy proposed in the litera-
ture for the transformation of 6b,58,59,63 compound 6a was
treated with 6 M HCl in butanone under reflux for 2 h, leading
to a complex inseparable mixture of compounds.
Enzymatic Hydrolyses of Lactonic Esters 5a-d
Prior to enzymatic hydrolysis, it was important to deter-
mine whether the lactonic esters 5a-d underwent chemical
hydrolysis under the conditions used. This was tested on
compound 5b. On standing in phosphate buffer at pH 7.4 for
3.5 h, 5b was recovered unchanged, except for the presence
of less than 5% of its α,ß-butenolide isomer. Incidentally, un-
der the same conditions, the diastereomer 6b underwent
more important modifications not including hydrolysis.
Enzymatic hydrolyses were carried out using a series of com-
mercially available enzymes, namely, Acylase I on Eupergit,
Aminoacylase, Acylase I from porcine kidney, α-chymotrypsin
(α-CT), Candida antarctica lipase (Novozym 435), Porcine
pancreatic lipase (PPL), Esterase from hog liver (PLE), and
Liver acetone powder equine (HLAP). A preliminary evaluation
of enzyme selectivity was performed on 30–50mg of substrates
5a–d in 10mL of phosphate buffer at pH7.4. The results
obtained for compounds 5a-c are listed in Table 1. Compound
5d is not included in the table because it was hydrolyzed only
by porcine liver acetone powder (PLAP, 20% conversion, 2h,
The strategy adopted for obtaining (+)-1b and (–)-1b in
pure enantiomeric forms was not as satisfactory when applied
to 1a, as fractional crystallization of enantioenriched acids did
not improve their respective ee’s significantly; (+)- and (–)-1a
(optical rotations determined for chloroform solutions) were
obtained with 81% and 74% ee, respectively.
As to the lactonic acid 1c, both enantiomers were obtained
with excellent ee [(+)-1c, 98% ee, (–)-1c, 99% ee], as reported
in the literature.57
E
73 =1.3); however, with negligible enantioselection, whereas
the other enzymes checked, namely, Acylase I on Eupergit C,
Aminoacylase, α-CT, Lipase from Candida antarctica (CAL-B),
Lipase from Candida antarctica (Novozym 435), PLE, Lipase
from Pseudomonas cepacea, Lipase from Pseudomonas fluo-
rescens, Proteases from Bacillus subtilis and PPL, were
ineffective.
As it is evident from the E values listed in Table 1 for the
various hydrolyses, enantioselectivity was good only for 5c.
In fact, at low conversion values the corresponding acid 1c
Scheme 1. Synthesis of racemic substrates.
Chirality DOI 10.1002/chir

244
CHAKRABARTY ET AL.
Scheme 2. Enzymatic resolution of lactonic esters 5a,b.
The resolution outcome depends on the rate at which the α-
lines but (+)- and (–)-1c displayed moderate cytotoxic effects at
the middle micromolar level. Both enantiomers were about
four-fold higher on C6 glioma cells (IC50 =12–18 μM) but still
less active than cisplatin (0.6 0.2 μM), taken as a reference com-
pound. The observed antiproliferative activity was characterized
by low, significant (P <0.01) stereoselectivity in the MCF-7
assay, (–)-1c being more active than its enantiomer (IC50 values
68.4 0.3 vs. 84.5 2.3μM). This observation was in agreement
with previously reported results.10 No stereoselectivity was
observed when the activity on G6 cells was evaluated.
methylene-γ-lactone turns into its α,ß-butenolide isomer and
this is a function of the chain length, being higher for C3 than
for C8, under the experimental conditions used. In spite of the
presence of this competitive reaction, α-methyleneparaconic
acids can be obtained in the pure state because their α,ß-
butenolide isomers do not undergo hydrolysis within the
enzymatic reaction times, and thus they can be easily sepa-
rated from the esters.
The (2R,3S) absolute configuration was assigned to the
enantiomers which in chloroform were dextrorotatory, by
comparison with the signs of their optical rotation values with
those reported in the literature (see Materials and Method)
and by comparison with their CD spectra with that of
Antimicrobial Studies
Antibacterial studies. These studies were performed in agree-
ment with a previously reported protocol.68 According to the
Clinical Laboratory Standards Institute (CLSI) guidelines,69
compounds 1a–c were tested against Gram-positive and
Gram-negative bacteria belonging to the ATTC collection
(Staphylococcus aureus, S. aureus 29213; Enterococcus faecalis,
E. faecalis 29212; Escherichia coli, E. coli 25922) using
norfloxacin as a reference compound.
All tested compounds displayed no antibacterial activity
against E. coli, while moderate activity was observed against
Gram-positive bacteria. (–)-1c was the most active compound
(MIC: 16 μg/mL; 60 μM) about three times less potent than
norfloxacin on E. faecalis
(2R,3S)-(+)-protolichesterinic acid 1e (Δε257 = + 0.32, Δε220
–9.62),75 whose absolute configuration is known (compound
1a: Δε267 = +0.1, Δε225 = –4.8 (MeOH); compound 1b: Δε262
=
=
+0.16, Δε224 = –8.62 (MeOH); compound 1c: Δε258 = + 0.26,
Δε225 = –10.58 (MeOH)).
The optical rotation values for the α-methylene paraconic acid
derivatives deserve a comment. The values reported in the liter-
ature for compounds having approximately the same high en-
antiomeric excess, at comparable concentrations, are rather
variable. If one compares literature data for methylenolactocin
(–)-1b optical rotation values obtained for methanolic solutions
range from –6.46 (23°C) to –12.4 (temperature not given),
while those obtained for chloroform solutions, vary from –11.6
to –18.8. The syntheses reported are multistep and either the
chirality is already present in the parent molecule or it is intro-
duced in a stereochemically controlled step of the synthesis. In
no case, however, was the enantiomeric excess of the target
molecule measured because it is assumed to be the same as
in the parent molecule. On the contrary, in the present study
the enantiomeric excess is measured on the final esterified
products.
Antifungal studies. These studies were performed in agreement
with a previously reported protocol.68 According to the CLSI
guidelines,70 compounds 1a–c were tested against a panel of
fungi strains belonging to the ATCC collection (Candida
albicans, C. albicans 10231; Candida tropicalis, C. tropicalis 750;
Candida parapsilosis, C. parapsilosis 22019; Candida krusei, C.
krusei 6258) using fluconazole as a reference compound.
All tested compounds were inactive as antifungal agents.
CONCLUSION
Finally, it is interesting to note that 1a showed a solvent
dependency of the optical rotation, which changed its sign,
replacing methanol for chloroform, different from what was
observed for 1b (see Materials and Methods), 1c10 and 1d.64
Optically pure paraconic acids can be obtained by enzymatic
resolutions of their methyl esters. High enantioselectivity for hy-
drolyses of 5a and 5b could be achieved taking advantage of
enantiocomplementary enzymes. Thus, the unreacted esters,
isolated from the reaction with α-CT, could be resolved success-
fully with aminoacylase.
As to the biological results, in agreement with previously
reported results,10 C75 displayed moderate cytotoxic effects
on human breast carcinoma and rat glioma cells. Both C75
enantiomers displayed higher cytotoxic effects on C6 glioma
Biological Assays
Cytotoxicity assays. Antiproliferative activities of both enantio-
mers of lactonic acids 1a–c were evaluated in both human breast
carcinoma and rat glioma cells (MCF-7 and C6, respectively) by
the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. All tested compounds were inactive on both cell
Chirality DOI 10.1002/chir

ENZYMATIC RESOLUTION OF α-METHYLENEPARACONIC ACIDS
245
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cells. This observation casts doubt on the possibility to
develop antibacterials using C75 as the lead compound.
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C75, a covalent bioactive compound with potential anticancer activities.
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We thank the Ministero dell’Istruzione, dell’Università e della
Ricerca (MIUR) (Project PRIN 2010FPTBSH_005 “NANO
Molecular tEchnologies for Drug delivery – NANOMED”) and
to the University of Trieste (Fondo per la Ricerca d’Ateneo,
FRA 2012) for financial support.
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