Chemo-enzymatic preparation from renewable resources of enantiopure 1,3- oxazolidine-2-thiones

Article abstract of DOI:10.1016/S0957-4166(99)00574-1

Chiral 1,3-oxazolidine-2-thiones were prepared in enantiopure form from renewable resources through an enzymatic process involving immobilized myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.1). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(99)00574-1

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 4775–4780
Chemo-enzymatic preparation from renewable resources of
enantiopure 1,3-oxazolidine-2-thiones
Onofrio Leoni,a Roberta Bernardi,a David Gueyrard,b Patrick Rollin b,∗ and Sandro Palmieri a
aIstituto Sperimentale per le Colture Industriali, Italian Ministry of Agricultural and Forestry Politics, via di Corticella 133,
I-40129 Bologna, Italy
bInstitut de Chimie Organique et Analytique, Université d’Orléans, B.P. 6759, F-45067 Orléans, France
Received 4 November 1999; accepted 9 December 1999
Abstract
Chiral 1,3-oxazolidine-2-thiones were prepared in enantiopure form from renewable resources through an
enzymatic process involving immobilized myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.1). © 2000 Elsevier
Science Ltd. All rights reserved.
1. Introduction
Chiral auxiliary methodologies are proving to be increasingly useful for the asymmetric synthesis of
many classes of compounds. Chiral 1,3-oxazolidin-2-one1 and 1,3-oxazolidine-2-thione2 heterocyclic
templates have proven to be remarkably versatile and efficient auxiliaries (Fig. 1).
Fig. 1.
The development of five membered 2-thioxo-O,N-heterocycles as chiral auxiliaries has motivated the
search for enantiomerically pure representatives. As chiral auxiliaries, oxazolidine-2-thiones5 show some
advantages when compared to their 2-oxo-analogs: they exhibit a strong UV absorption and they have
∗
Corresponding author. E-mail: patrick.rollin@univ-orleans.fr
0957-4166/99/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00574-1
tetasy 3208

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O. Leoni et al. / Tetrahedron: Asymmetry 10 (1999) 4775–4780
been claimed to be more efficient in aldol-type reactions involving complex molecules.6 Moreover, they
can be readily transformed into their oxo counterparts.7
In contrast with 1,3-oxazolidinones which require N-metallation prior to acylation, 2-thioxo-O,N-
heterocyclic systems can be acylated using mild procedures.8 An extra advantage of oxazolidinethiones
consists in their easy removability under smooth conditions.9
Enantiomerically pure oxazolidine-2-thiones are generally prepared in basic medium from chiral α-
aminoalcohols and carbon disulfide.10 We herein report a chemo-enzymatic process to furnish miscella-
neous enantiopure 1,3-oxazolidine-2-thiones from vegetal sources.
2. Results and discussion
Glucosinolates (GSL) are naturally occurring thio-sugars mainly found in Brassicaceae11-mustard,
cabbage, broccoli, turnip, radish, rape, etc. More than 100 GSL have been identified to date, differing
only in the nature of the aglyconic side chain A (Fig. 2).
Fig. 2.
In the Brassicaceae family (and also in Capparidaceae), every plant species displays a characteristic
individual ‘GSL profile’ which can be relatively simple in some cases. Enzyme catalysed hydrolysis
of GSL by myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) affords D-glucose, sulfate anion and
a series of sulfur and nitrogen-containing compounds principally isothiocyanates, whose bioactivity
is well documented.12 In cases when the side-chain A carries a hydroxyl group in β-position to the
isothiocyanate function, spontaneous cyclization occurs to yield enantiopure 1,3-oxazolidine-2-thiones
(Fig. 3).
Fig. 3.
Using a bioreactor containing myrosinase immobilized on Nylon 6.6,13 we have developed a simple
and effective process to prepare different kinds of bioactive compounds starting from pure native GSL
isolated from different cruciferous seeds. This process was shown to be very efficient for the production
on a gram scale of diverse 1,3-oxazolidine-2-thiones in enantiopure form (see Table 1). Those molecules
are being currently tested in our laboratory to perform stereoselective aldol reactions6 and Michael
additions.14
3. Experimental
Melting points were determined with a Tottoli apparatus (Büchi SMP 20) and are uncorrected. Specific
1
rotations were measured at 20°C using a Perkin–Elmer polarimeter 141. H and 13C NMR spectra

O. Leoni et al. / Tetrahedron: Asymmetry 10 (1999) 4775–4780
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Table 1
were recorded in CDCl3 solution on a Bruker Avance DPX250 instrument operating at 250 MHz and
62.89 MHz, respectively. The coupling constants (J) are reported in Hz and the chemical shifts (δ)
in ppm downfield from tetramethylsilane as the internal standard. IR spectra were measured using a
Perkin–Elmer FT Paragon 1000 PC spectrometer. Mass spectra (MS) were obtained on a Perkin–Elmer
SCIEX API 300 spectrometer (Ionspray® mode). HPLC profiles were recorded on a Hewlett Packard
1090 L with diode array detector, and GC–MS spectra were measured on a Hewlett Packard GCD 1800
apparatus.
3.1. General procedure for the preparation of enantiopure 1,3-oxazolidine-2-thiones
The bioreactor is constituted by a thermostated column (œ 2.6 cm, L 30 cm, 37°C), loaded with 10 g of
Nylon 6.6-supported myrosinase. This enzyme was purified from white mustard seeds13 and immobilized
on Nylon 6.6 granules (18–36 mesh particles).15 The specific activity of the enzyme was ca. 65 units/mg
protein before immobilization, whereas the final immobilized activity reached 325 opponent units/g solid.
GSL were extracted from the ripe seeds of each plant species (see Table 1) homogenizing the seeds
in boiling water. The centrifuged crude extract was loaded into a DEAE-Sephadex A25 ion exchange
chromatographic column. After suitable washing of the column with water, the GSL were eluted by a
0.5 M K2SO4 aqueous solution. The GSL solution was concentrated, dried and solubilized with boiling
methanol. Finally, the GSL were precipitated with ethanol at −20°C.16 The isolation yields ranged from
40 up to 60%, depending on the starting seed species. The substrate was dissolved in phosphate buffer
(pH=6.5) at the concentration of 10 mg/mL and passed through the column with a 2 mL/min flow. The
reaction was followed polarographically and/or by increasing of UV absorption at 240 nm due to the
formation of the 1,3-oxazolidine-2-thione and in all cases the transformation of the starting material was
complete. The final products were purified on a RPC18 column using water as eluent; after freeze drying,
the purity of the oxazolidine-2-thiones were assessed by HPLC, GC–MS and NMR (1H and 13C).

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3.2. (5S)-5-Vinyl-1,3-oxazolidine-2-thione 1
1
[α]D=−70 (c 1, CHCl3);17 mp=48°C; IR (KBr): 3202 (NH), 1532 (CS); H NMR: 3.53 (dd, 1H,
J4b-5=8.3, H-4b), 3.92 (dd, 1H, J4a-4b=9.5, H-4a), 5.33 (ddd, 1H, J5-4a=9.4, H-5), 5.40 (d, 1H, J7Z-6=10.5,
H-7Z), 5.48 (d, 1H, J7E-6=17.0, H-7E), 5.96 (ddd, 1H, J6-5=6.7, H-6), 7.44 (s, 1H, NH); 13C NMR: 49.5
(C-4), 83.8 (C-5), 121.0 (C-7), 133.4 (C-6), 189.7 (CS); MS: MH+=130.
3.3. (5R)-5-Vinyl-1,3-oxazolidine-2-thione 2
1
[α]D=+72 (c 1, CHCl3);18 mp=48°C; IR (KBr): 3215 (NH), 1531 (CS); H NMR: 3.53 (dd, 1H,
J4b-5=8.3, H-4b), 3.92 (dd, 1H, J4a-4b=9.5, H-4a), 5.33 (ddd, 1H, J5-4a=9.4, H-5), 5.40 (d, 1H, J7Z-6=10.5,
H-7Z), 5.48 (d, 1H, J7E-6=17.0, H-7E), 5.96 (ddd, 1H, J6-5=6.7, H-6), 7.40 (s, 1H, NH); 13C NMR: 49.5
(C-4), 83.8 (C-5), 121.0 (C-7), 133.4 (C-6), 189.7 (CS); MS: MH+=130.
3.4. (5S)-5-(Prop-2-enyl)-1,3-oxazolidine-2-thione 3
[α]D=−6 (c 1, CHCl3); mp=60°C;19 IR (KBr): 3188 (NH), 1537 (CS); 1H NMR: 2.50–2.70 (m, 2H,
H-6a and H-6b), 3.48 (dd, 1H, J4b-4a=9.0, H-4b), 3.81 (dd, 1H, J4a-5=8.9, H-4a), 5.00 (m, 1H, J5-4b=7.7,
H-5), 5.17–5.30 (m, 2H, H-8a and H-8b), 5.75 (m, 1H, H-7), 7.42 (s, 1H, NH); 13C NMR: 38.4 (C-6),
48.1 (C-4), 82.1 (C-5), 120.1 (C-8), 130.6 (C-7), 189.8 (CS); MS: MH+=144.
3.5. (5R)-5-Phenyl-1,3-oxazolidine-2-thione 4
1
[α]D=−51 (c 1, CHCl3);20 mp=116°C;19 IR (KBr): 3176 (NH), 1530 (CS); H NMR: 3.75 (dd, 1H,
J4a-4b=9.6, H-4b), 4.17 (dd, 1H, J4a-5=9.4, H-4a), 5.90 (t, 1H, J5-4b=8.2, H-5), 7.37–7.46 (m, 5H, H-ar),
7.75 (s, 1H, NH); 13C NMR: 51.2 (C-4), 83.9 (C-5), 125.8, 129.0, 129.4, 136.9 (C-ar), 189.7 (CS); MS:
MH+=180.
3.6. (4S)-4-Methyl-1,3-oxazolidine-2-thione 5
[α]D=+23 (c 1, CHCl3);21 mp=60°C; IR (KBr): 3263 (NH), 1509 (CS); 1H NMR: 1.36 (d, 1H, Jvic=6.0,
CH3), 4.17–4.29 (m, 2H, H-4 and H-5b), 4.77 (m, 1H, J5a-4=6.8, J5a-5b=11.5, H-5a), 7.90 (s, 1H, NH);
13C NMR: 20.4 (CH3), 52.7 (C-4), 76.9 (C-5), 189.9 (CS); MS: MH+=118.
3.7. (4R)-4-Ethyl-1,3-oxazolidine-2-thione 6
[α]D=+47 (c 1, CHCl3);21 mp=32°C; IR (KBr): 3199 (NH), 1526 (CS); 1H NMR: 0.98 (t, 3H, Jvic=7.4,
CH3CH2), 1.68 (m, 2H, CH2CH3), 4.02 (m, 1H, H-4), 4.32 (dd, 1H, J5b-4=6.6, J5b-5a=8.9, H-5b), 4.74
(dd, 1H, J5a-4=8.7, H-5a), 7.73 (s, 1H, NH); 13C NMR: 9.7 (CH3CH2), 27.7 (CH2CH3), 58.1 (C-4), 75.3
(C-5), 190.1 (CS); MS: MH+=132.
3.8. (5S)-5-Ethyl-5-methyl-1,3-oxazolidine-2-thione 7
[α]D=−23 (c 1, CHCl3);22 mp=43°C; IR (KBr): 3184 (NH), 1556 (CS); 1H NMR: 0.99 (t, 3H, Jvic=7.5,
CH3CH2), 1.50 (s, 3H, CH3), 1.79 (q, 2H, CH2CH3), 3.43 (d, 1H, J4b-4a=9.6, H-4b), 3.56 (d, 1H, H-4a),

O. Leoni et al. / Tetrahedron: Asymmetry 10 (1999) 4775–4780
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7.93 (s, 1H, NH); 13C NMR: 8.5 (CH3CH2), 25.5 (CH3), 33.4 (CH2CH3), 54.3 (C-4), 92.0 (C-5), 189.6
(CS); MS: MH+=146.
3.9. 5,5-Dimethyl-1,3-oxazolidine-2-thione 8
Mp=106°C;23 IR (KBr): 3191 (NH), 1531 (CS); 1H NMR: 1.57 (s, 6H, CH3), 3.53 (s, 2H, H-4), 8.06
(s, 1H, NH); 13C NMR: 26.7 (CH3), 55.3 (C-4), 88.4 (C-5), 188.6 (CS); MS: MH+=132.
Acknowledgements
The authors thank the E.U. for financial support through the B.O.P. Project (FAIR CT 95-0260). We
also thank the French M.E.N.R.T. for a grant (D.G.).
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