Regio- and enantioselectivity of the enzyme-catalyzed hydrolysis of citric acid derivatives

Article abstract of DOI:10.1016/S0957-4166(98)00448-0

The hydrolysis of triethyl citrate in the presence of three serine proteases (chymotrypsin, subtilisin BPN', subtilisin carlsberg) is highly regioselective and gives the symmetric diester. Several lipases and proteases have the complementary regioselectivity and give the chiral diester. Pig liver esterase, Aspergillus niger lipase and Candida antarctica lipase give the chiral (R)-diester with good regio- and enantioselectivity. The stereoselective hydrolysis of the meso citric derivatives 7a,b in the presence of Candida antarctica lipase gives the corresponding (R)-monoester.

Full text of DOI:10.1016/S0957-4166(98)00448-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 4325–4329
Regio- and enantioselectivity of the enzyme-catalyzed hydrolysis
of citric acid derivatives
Robert Chênevert,∗ Béatrice Tchédam Ngatcha, Yannick Stéphane Rose and Daniel Goupil
Département de chimie, Faculté des sciences et de génie, Université Laval, Québec, G1K 7P4 Canada
Received 17 September 1998; accepted 22 October 1998
Abstract
The hydrolysis of triethyl citrate in the presence of three serine proteases (chymotrypsin, subtilisin BPN0,
subtilisin carlsberg) is highly regioselective and gives the symmetric diester. Several lipases and proteases have
the complementary regioselectivity and give the chiral diester. Pig liver esterase, Aspergillus niger lipase and
Candida antarctica lipase give the chiral (R)-diester with good regio- and enantioselectivity. The stereoselective
hydrolysis of the meso citric derivatives 7a,b in the presence of Candida antarctica lipase gives the corresponding
(R)-monoester. © 1998 Elsevier Science Ltd. All rights reserved.
The citric sub-unit is found in various natural products1–8 or synthetic bioactive compounds.9,10 For
instance, rhizobactin 10211 (1) and rhizoferrin,2,3 siderophores isolated from fungi, are chiral citrate
derivatives. Viridiofungins4 (2) and squalestatins5–7 (also called zaragozic acids) are members of two
families of squalene synthase inhibitors and antifungal natural products incorporating the citrate moiety.
Recently, Bergeron et al.11 reported the resolution of 1,2-dimethyl citrate by fractional crystallization of
the corresponding (−)-brucine salts, and determined the absolute configuration by single crystal X-ray
diffraction. Also, the citric acid derivative 8a was resolved via fractional crystallization of the (R)- or
(S)-α-methylbenzylamine salts.12 We report here a study on the regio- and enantioselectivity of enzyme-
catalyzed hydrolysis of citric acid derivatives.
First, we found that the hydrolysis of triethyl citrate 3 in the presence of three serine proteases (α-
chymotrypsin, subtilisin BPN0 and subtilisin carlsberg) was highly regioselective, and that the symmetric
∗
Corresponding author. E-mail: robert.chenevert@chm.ulaval.ca
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00448-0

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diester 4 was obtained as the sole product (Scheme 1). Anthonsen et al.13 reported the regioselective
hydrolysis of citrates by a subtilisin of unspecified origin.
Scheme 1. Reagents: (i) enzyme, r.t., (see Table 1)
Next, we did some screening to find enzymes which were able to distinguish the enantiotopic groups
of triethyl citrate. Many lipases and proteases showed the complementary regioselectivity and gave chiral
diester 5 as the sole or the major product, but the enantioselectivities were low (0–50%).14
Hydrolysis of 3 in the presence of pig liver esterase (PLE) rapidly gave (2 h) the chiral diester 5 with
fair regioselectivity (4/1) and good enantioselectivity (ee 90%). Lipase from Aspergillus niger gave 5 with
high regio- (19/1) and enantioselectivity (ee 90%), but the reaction was very slow. Candida antarctica
lipase produced 5 as the sole product with good enantioselectivity (ee 90%) in a short reaction time (3
1
h) (Table 1). The isomer ratio 5:4 was measured by H NMR analysis and the diesters 4 and 5 were
easily separated by standard flash chromatography. The enantiomeric composition of 5 was determined
by reaction with (S)-(−)-(1-naphthyl)ethylamine in the presence of 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide (EDC), followed by NMR analysis of the resulting diastereomeric amides.
Following from this, we investigated the desymmetrization of the citric acid derivative 7. The diacid
610 was esterified with methanol or ethanol in the presence of the corresponding 2,2-dialkoxypropane
and an acidic resin to give esters 7a,b (Scheme 2). Hydrolysis of 7a,b in the presence of pig liver
esterase in phosphate buffer provided monoesters 8a,b in good yield (80–85%) and high enantio-
selectivity (ee 90–92%). The enantiomeric composition of 8a,b was determined by reaction with (−)-
(naphthyl)ethylamine in the presence of EDC, followed by NMR analysis of the resulting rearranged
amides. The initially formed amides 9a,b were rearranged to cyclic imides 10a,b by intramolecular ring-
opening of the 1,3-dioxolan-4-one.15 A similar reaction was observed by Gilbert et al.10
The absolute configurations of 5 and 8a,b were determined by comparison or correlation with (R)-
(+)-11 and (R)-(+)-8a of known absolute configurations11,12 (Scheme 3). Ring-opening of the 1,3-
dioxolan-4-one ring by refluxing 8a,b with the appropriate alcohol and triethylamine gave (R)-11 and
Table 1
Enzymatic hydrolysis of triethyl citrate

R. Chênevert et al. / Tetrahedron: Asymmetry 9 (1998) 4325–4329
4327
Scheme 2. Reagents: (i) ROH, H+, (CH3)2C(OR)2, reflux, 85%; (ii) pig liver esterase, phosphate buffer, r.t., 80%; (iii) NEA,
EDC, DMAP, r.t.
(R)-5, respectively. Treatment of 8a with sodium ethoxide in ethanol yielded 5 (ring opening and
transesterification). These correlations proved that compounds 5, 8a,b have the R configuration.
Scheme 3. Reagents: (i) MeOH, Et3N, reflux, 60%; (ii) EtONa, EtOH, r.t., 50%; (iii) EtOH, Et3N, reflux, 60%.
1. Experimental
NMR spectra were recorded using a Bruker AC-300 instrument. Optical rotations were measured on
a JASCO DIP-360 digital polarimeter (c as g of compound per 100 ml). All proteases were obtained
from Sigma. Lipases were purchased from Amano International Enzyme Co. except for porcine pancreas
lipase (Sigma) and Pseudomonas fluorescens lipase (Fluka). Pig liver esterase was from Sigma or Amano.
Candida Antarctica lipase (novozym 435) was a gift from Novo Nordisk.
1.1. Enzymatic hydrolysis of triethyl citrate — general procedure
In a typical experiment, a suspension of triethyl citrate (150 mg, 1.84 mmol) was hydrolyzed with
PLE (50 mg) in a phosphate buffer (pH 7) at room temperature. The enzymatic reaction was indicated by
the decrease of pH, which was maintained at its initial value by the addition of a 0.1 M NaOH solution.
The reaction was monitored by the consumption of base, and terminated when one ester equivalent was
hydrolyzed. The aqueous solution was extracted with ether to remove any remaining starting material.
The aqueous solution was acidified with 5 N HCl to pH 2, and the hydrolysis products were extracted
with ethyl acetate. The isomeric diesters 4 and 5 were separated by flash chromatography on silica gel
using hexane:ethyl acetate 4.5:5.5 as eluant. Compound 4: IR (neat) 3600–3040, 2980–2860, 1750,
1370, 1190, 1020, 850, 780 cm−1. 1H NMR (CDCl3) 1.25 (t, J=7.1 Hz, 6H), 2.91 (m, 4H), 4.15 (q, J=7.1
Hz, 4H). 13C NMR (CDCl3) 177.13, 170.09, 73.16, 61.36, 42.76, 14.01. Compound 5: [α]D +3.8 (c
25
1.84, MeOH); IR (neat) 3600–3040, 2980–2860, 1750, 1370, 1120, 1020, 830 cm−1. 1H NMR (CDCl3)

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1.25 (t, J=9.1 Hz, 6H), 2.86 (m, 4H), 4.12 (q, J=9.1 Hz, 2H), 4.28 (q, J=9.1 Hz, 2H). 13C NMR (CDCl3)
174.76, 174.03, 169.64, 73.12, 62.39, 60.86, 42.88, 13.89.
1.2. Preparation of 7a,b
Diacid 610 (1.0 g, 4.90 mmol) was dissolved in the selected alcohol (methanol or ethanol, 20 ml), then
the corresponding acetal (dimethoxypropane or diethoxypropane, 4 ml) and an acidic ion-exchange resin
(Dowex 50WX4, 0.5 g) were added. The mixture was heated to reflux for 24 h. The reaction mixture was
filtered to remove the resin and evaporated. The crude product was dissolved in ethyl acetate (100 ml)
and washed with aq. Na2CO3 (2×20 ml) and brine (2×20 ml). The organic phase was dried (MgSO4)
and evaporated. The crude product was purified by flash chromatography (AcOEt:hexane 3:10) to give
corresponding diester. Diester 7a: yield 86%, m.p. 60–63°C; IR (KBr) 3000, 2960, 1800, 1740, 1440,
1
1370, 1240, 1180, 1060, 990 cm−1; H NMR (acetone-d6) 2.97 (s, 4H), 3.66 (s, 6H), 5.50 (s, 2H); 13C
NMR (acetone-d6) 174.9, 171.0, 96.3, 77.1, 52.3, 41.8. Diester 7b: yield 85%, oil; IR (KBr) 2980, 2930,
1800, 1740, 1375, 1180, 1060, 1020 cm−1; 1H NMR (acetone-d6) 1.21 (t, J=7.0 Hz, 6H), 2.94 (s, 4H),
4.11 (q, J=7.0 Hz, 4H), 5.51 (s, 2H); 13C NMR (acetone-d6) 173.3, 169.4, 95.4, 76.3, 61.4, 41.6, 14.2.
1.3. Enzymatic hydrolysis of 7a,b
Preparation followed the general procedure described above: 7a,b (0.43 mmol), pig liver esterase (50
mg), reaction time: 2 h. Compound 8a: [α]D25 +8.4 (c 2.46, MeOH); IR (KBr) 3500–2500, 1790, 1720,
1
1180, 1145, 1000 cm−1; H NMR (CDCl3) 2.90 and 2.95 (2s, 4H), 3.71 (s, 3H), 5.55 (s, 2H), 7.97 (s,
1H); 13C NMR (CDCl3) 173.4, 172.8, 168.9, 95,0, 75.3, 52.2, 40.8. Compound 8b: [α]D25 +10.2 (c 1.84,
MeOH); IR (KBr) 3500–2500, 1790, 1720, 1180, 1145, 1000 cm−1; 1H NMR (CDCl3) 1.20 (t, J=7.0 Hz,
3H), 2.83 and 2.89 (2s, 4H), 4.10 (q, J=7,0 Hz, 2H), 5.49 (s, 2H), 8.92 (s, 1H); 13C NMR (CDCl3) 173.6,
172.8, 168.4, 75.3, 61.3, 41.1, 40.8, 13.9.
1.4. Ring opening of compound 8a,b
Compound 8a or 8b (0.85 mmol) was dissolved in dry methanol or ethanol (20 ml). Freshly distilled
triethylamine (236 µl, 1.70 mmol) was added and the solution was heated at reflux for 24 h. After
allowing to cool to room temperature, the solution was evaporated to dryness. The excess of amine was
neutralized with 1 N HCl. The aqueous solution was extracted with ethyl acetate (4×15 ml). The organic
solution was washed with brine (2×20 ml), dried (MgSO4) and evaporated to yield 5 or 11. Compound
5: [α]D25 +3.8 (c 1.84, MeOH), data described earlier. Compound 11: [α]D25 +3.9 (c 1, MeOH); lit.11,12
25
[α]D +4.0 (c 1, MeOH). Spectral data are in agreement with those reported in the literature.10,11
1.5. Ring opening and transesterification of compound 8a
Sodium (47 mg, 2.06 mmol) was added to dry ethanol cooled to 0°C. A solution of 8a (180 mg,
0.825 mmol) in dry ethanol was added and the mixture was allowed to warm to room temperature and
stirred overnight. The solution was evaporated to dryness and the residue was dissolved in ethyl acetate.
The organic layer was neutralized with 1 N HCl and washed with brine. The organic phase was dried
(MgSO4) and evaporated to give 5 ([α]D25 +3.8 (c 1.84, MeOH)).

R. Chênevert et al. / Tetrahedron: Asymmetry 9 (1998) 4325–4329
4329
Acknowledgements
We acknowledge the financial support of this work by the Natural Sciences and Engineering Research
Council of Canada (NSERC). B.T.N. thanks ‘Agence Canadienne de Développement International’, D.G.
thanks NSERC and Y.S.R. thanks ‘le Fonds pour la Formation de Chercheurs et l’Aide à la Recherche,
Québec (FCAR)’ for postgraduate scholarships.
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