Enzymatic resolution of methyl N-alkyl-azetidine-2-carboxylates by Candida antarctica lipase-mediated ammoniolysis

Article abstract of DOI:10.1016/S0957-4166(97)00632-0

A facile method for the synthesis of optically active azetidine-2- carboxylic acid derivatives is presented Racemic N-alkylated azetidine esters are resolved by lipase from Candida antarctica in an ammoniolysis reaction, and both the S-amide and the R-ester are obtained with excellent stereoselectivity.

Full text of DOI:10.1016/S0957-4166(97)00632-0

Pergamon
Tetrahedron: Asymmetry 9 (1998) 429–435
Enzymatic resolution of methyl N-alkyl-azetidine-2-carboxylates
by Candida antarctica lipase-mediated ammoniolysis
Wim A. J. Starmans, Roy G. Doppen, Lambertus Thijs and Binne Zwanenburg ∗
Department of Organic Chemistry, NSR-Center for Molecular Structure, Design and Synthesis, University of Nijmegen,
Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Received 10 November 1997; accepted 15 December 1997
Abstract
A facile method for the synthesis of optically active azetidine-2-carboxylicacid derivatives is presented. Racemic
N-alkylated azetidine esters are resolved by lipase from Candida antarctica in an ammoniolysis reaction, and both
the S-amide and the R-ester are obtained with excellent stereoselectivity. © 1998 Elsevier Science Ltd. All rights
reserved.
1. Introduction
Optically active azetidines substituted with one or two carboxyl groups are interesting nonproteino-
genic amino acids. They were applied in the synthesis of peptides1 and natural products.1,2 Moreover,
optically active 2- and 2,4-substituted azetidines have been employed as chiral auxiliaries in asymmetric
synthesis.3–5 Because of our interest in N-alkylated azetidines in chirality transfer reactions, we sought a
method for obtaining both antipodes in enantiopure form. Only azetidine-(2S)-carboxylic acid is commer-
cially available and a satisfactory route to the enantiomer is not yet available. An enzymatic resolution
of racemic azetidine-2-carboxylic acid derivatives would in principle be a suitable method to obtain
both antipodes. For this purpose several enzymes were investigated for the enantioselective hydrolysis
of racemic methyl-N-benzyl-azetidine-2-carboxylate and N-benzyl-azetidine-2-carboxamide. However,
these resolutions were unsuccessful. We therefore turned our attention to biocatalytic transacylation
reactions.
Lipases usually are effective in the transacylation of a wide variety of nucleophiles such as hydrogen
peroxide, amines, hydrazines and oximes.6–10 The enzymatic formation of carboxamides from carboxylic
esters by Candida antarctica, whereby ammonia serves as the nucleophile, was successfully accom-
plished with esters, such as β-ketoesters,11,12 α,β-unsaturated esters,13 dimethyl succinate14 and fatty
∗
Corresponding author. E-mail: zwanenb@sci.kun.nl
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(97)00632-0

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esters.15,16 Amino acid esters have only been reported once as substrates in this reaction.17 However, the
results reported were promising and this prompted us to investigate the lipase catalyzed ammoniolysis of
azetidine-2-carboxylic esters for the purposes indicated above.
2. Results
Starting from γ-butyrolactone, methyl 2,4-dibromobutyrate
1
was obtained by
a
Hell–Volhard–Zelinski bromination. Several primary amines were then used to synthesize racemic
azetidine-2-carboxylic esters 2 following modified literature procedures,18,19 i.e. one equivalent amine
and potassium carbonate as a co-base were used in refluxing acetonitrile/water instead of three
equivalents of amine in refluxing acetonitrile.18 The esters 2 were then subjected to ammoniolysis by
Candida antarctica lipase in tert-butyl alcohol saturated with ammonia (Scheme 1).
Scheme 1. Synthetic route to optically active azetidines by enzymatic ammoniolysis
The enzymatic reactions were monitored in time and the selectivity was determined as a function of
conversion (Fig. 1). The very high substrate specificity of Candida antarctica for 2c is demonstrated by
the almost straight line for the conversion versus the e.e., with only minimal levelling off.
The results of the enzymatic resolution are collected in Table 1. Blank experiments showed that no
appreciable ammoniolysis takes place in the absence of the enzyme. The enantiomeric excess of the
esters and amides were determined by chiral capillary GC and capillary electrophoresis. Furthermore,
the specific rotation of enantiopure (2R)-2a was compared with that reported by Seebach and coworkers1
for the antipode: the enantiopure ester obtained after enzymatic resolution showed [α]D20: +125.2 (c=1,
CH2Cl2), whereas the literature reports [α]D20: −115 (c=1.3, CH2Cl2). This suggests that Seebach’s route
Fig. 1. Selectivities of Candida antarctica mediated ammoniolysis versus time and conversion

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431
Table 1
Selectivities of C. antarctica mediated ammoniolysis of N-alkylated azetidine esters
toward optically active (2S)-2a, using base induced alkylation of the nitrogen atom, is accompanied by
partial racemization.
3. Conclusions
The azetidine ester 2d, with the bulky benzhydryl group, was not accepted by Candida antarctica,
probably due to steric factors. The other three substrates were converted with a remarkably high
selectivity to the primary amides. This enzymatic resolution leads to the formation of N-alkylated
azetidine-(2S) and (2R)-carboxylic acid derivatives of high enantiopurity.
4. Experimental
4.1. General remarks
Melting points were determined using a Reichert thermopan microscope and are uncorrected. Optical
rotations were measured with a Perkin–Elmer automatic polarimeter, model 241 MC, using concen-
1
trations c in g/100 ml at 20°C in the solvents indicated. H and 13C NMR spectra were recorded
with a Bruker AC 100 (100 MHz, FT) or a Bruker AM-400 (400 MHz, FT) spectrometer. IR spectra
were recorded on a Perkin–Elmer 298 spectrophotometer. For (high resolution) mass spectra a double
focussing VG7070E mass spectrometer was used. GC–MS were measured using a Varian Saturn II
GC–MS by on-column injection (DB-1 column, length 30 m, internal diameter 0.25 mm, film thickness
0.25 µm). Elemental analyses were performed using a Carlo Erba Instruments CHNS-O EA 1108
element analyzer. Capillary electrophoresis was performed on a Hewlett–Packard CE apparatus, using
β-cyclodextrine saturated Tris.HCl/phosphate buffer (pH 2.5) atꢀ30 kV and 10°C, while detection was at
254 nm. Chiral capillary GC was performed using a Beta-DEX™ 120 fused silica column (length 50 m,
internal diameter 0.25 mm, film thickness 0.25 µm) by Supelco.

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4.2. Methyl 2,4-dibromobutyrate 1
To a cooled suspension of γ-butyrolactone (40.00 g, 0.47 mol) and red phosphorus (4.00 g, 0.13
mol) approximately 10 ml of bromine was carefully added dropwise. The resulting mixture was then
heated to about 100°C and the remaining part of the bromine (total amount 52.50 ml, 1.03 mol) was
gradually added. After an additional 3 h of heating, the reaction mixture was cooled to room temperature
and flushed with a nitrogen flow for 30 min. The suspension was then cooled to 0°C and an excess of
methanol (50 ml) was carefully added. The resulting solution was set aside for 19 h, after which it was
concentrated in vacuo. The residue was taken up in diethyl ether and Na2S2O3 solution was added, the
layers separated and the aqueous solution extracted once with ether. The combined organic layers were
successively washed with Na2S2O3 solution and saturated NaCl solution, dried over MgSO4, filtered and
concentrated in vacuo. The resulting yellow oil was purified by vacuum distillation.
1
Yield: 102.70 g, 85%. Bp.: 53°C (0.03 mmHg). H NMR (CDCl3): δ 4.54 (t, J=7.1 Hz, 1H, CHBr),
3.81 (s, 3H, COOCH3), 3.55 (t, J=6.2 Hz, 2H, CH2Br), 2.53 (dt, J=6.3 Hz and J=9.4 Hz, 2H, CH2).
IR (CCl4): σ 1742 (s, COOCH3). GC–MS: m/z 227 (M+−OCH3, isotope pattern of 2 bromines), 199
79
(M+−COOCH3, isotope pattern of 2 bromines), 179 (M+− Br, isotope pattern of 1 bromine).
4.3. General procedure for the synthesis of racemic azetidine esters
To a refluxing solution of 1 in acetonitrile:distilled water (25:1, v/v) containing one equivalent of
potassium carbonate, a solution of 1.1 equivalent primary amine in acetonitrile was added dropwise.
When no more 1 was present, as was determined by GC, the reaction was stopped by pouring the mixture
into chloroform/water. The layers were separated and the organic layer acidified with 6 N HCl solution
until pH>4. The layers were separated, the aqueous layer neutralized with 6 N NaOH solution until pH<8
and extracted three times with chloroform. The latter organic extracts were then dried over MgSO4,
filtered and concentrated in vacuo. The resulting yellow oil was purified by distillation in vacuo or flash
chromatography.
4.4. (ꢁ)-Methyl 1-benzylazetidine-2-carboxylate 2a
Yield: 45%. Bp.: 107–109°C (1.0 mmHg). 1H NMR (CDCl3): δ 7.27 (m, 5H, Ph), 3.88–3.51 (m, 3H,
NCH and CH2Ph), 3.63 (s, 3H, COOCH3), 3.32 and 2.95 (dt, J=2.9 Hz and J=7.2 Hz, and dt, J=8.4 Hz
and J=6.7 Hz, 2H, NCH2), 2.46–2.15 (m, 2H, CH2). 13C NMR (CDCl3): δ 173.0 (s, C(O)O), 139.0 (s,
aromatic C), 129.1, 128.3, 127.2 (d, aromatic C), 64.3 (d, NCH), 62.4 (t, CH2Ph), 51.7 (q, OCH3), 50.8
(t, NCH2), 21.6 (t, CH2). IR (CCl4): σ 1740 (s, COOCH3), 700 (s, monosubstituted phenyl). Mass (EI):
m/z 205 (M+), 91 (tropilium). HR-MS: calculated for C12H15NO2 205.11028, found 205.11025.
4.5. (ꢁ)-Methyl 1-(4-methoxybenzyl)azetidine-2-carboxylate 2b
Yield: 39%. Bp.: 85°C (0.07 bar). 1H NMR (400 MHz, CDCl3): δ 7.20 (d, Jortho=9.9 Hz, 2H, aromatic),
6.84 (d, Jortho=9.6 Hz, 2H, aromatic), 3.79 (s, 3H, OCH3), 3.73 and 3.70 (AB quartet, J=8.7 Hz and J=12.3
Hz, 2H, benzylic CH2), 3.64 (s, 3H, COOCH3), 3.54 (d, J=12.4 Hz, 1H, NCH), 3.29 (dt, J=2.3 Hz and
J=7.4 Hz, 1H, NCH2), 2.93 (q, J=8.0 Hz, 1H, NCH2), 2.36 (m, 1H, CH2), 2.21 (m, 1H, CH2). 13C NMR
(CDCl3): δ 173.0 (s, C(O)O), 158.9 (s, aromatic C), 130.3 (d, aromatic C), 129.1 (s, aromatic C), 113.7
(d, aromatic C), 64.2 (d, NCH), 61.7 (t, CH2Ph), 55.2 (q, OCH3), 51.7 (q, COOCH3), 50.5 (t, NCH2),

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433
21.6 (t, CH2). IR (CHCl3): σ 1720 (s, COOCH3). GC–MS: m/z 236 (M++1), 176 (M+−COOCH3), 121
(methoxytropilium). HR–MS: calculated for C13H17NO3 235.12084, found 235.12027.
4.6. (ꢁ)-Methyl 1-allylazetidine-2-carboxylate 2c
1
Yield: 64%. Bp.: 47–48°C (1.0 mmHg). H NMR (400 MHz, CDCl3): δ 5.86–5.75 (m, 1H,
CH2CH=CH2), 5.20 and 5.11 (d, 17.1 Hz, and d, J=10.1 Hz, 2H, CH2CH=CH2), 3.73 (s, 3H, COOCH3),
3.66 (t, J=8.5 Hz, 1H, NCH), 3.38 and 2.90 (dt, J=2.5 Hz and J=7.5 Hz, and q, J=8.0 Hz, 2H, NCH2),
3.26 and 3.07 (dd, J=6.4 Hz and 12.9 Hz, and dd, J=6.7 Hz and J=12.9 Hz, 2H, CH2CH=CH2), 2.40–2.31
and 2.27–2.20 (m and m, 2H, CH2). 13C NMR (400 MHz, CDCl3): δ 172.8 (s, C(O)O), 133.4 (d,
CH2CH=CH2), 117.5 (t, CH2CH=CH2), 64.0 (d, NCH), 61.0 (t, CH2CH=CH2), 51.4 (q, COOCH3),
50.2 (t, NCH2), 21.3 (t, CH2). IR (CHCl3): σ 1718 (s, COOCH3). GC–MS: m/z 156 (M++1), 96
(M+−COOCH3). HR–MS: calculated for C8H13NO2 155.09463, found 155.09416.
4.7. (ꢁ)-Methyl 1-benzhydrylazetidine-2-carboxylate 2d
Yield: 50%. Bp.: 130°C (0.1 mmHg). 1H NMR (CDCl3): δ 7.52–7.09 (m, 10H, aromatic benzhydryl),
4.46 (s, 1H, benzylic CH), 3.72 (t, J=8.1 Hz, 1H, NCH), 3.50–3.34 (m, 1H, NCH2), 3.34 (s, 3H,
COOCH3), 2.90 (dt, J=7.7 Hz and J=8.4 Hz, 1H, NCH2), 2.49–2.0 (m, 2H, CH2). 13C NMR (CDCl3): δ
172.9 (s, C(O)O), 141.4, 141.2 (s, aromatic C), 128.5, 128.3, 128.2, 127.5, 127.2 (d, aromatic C), 77.5
and 64.6 (d, CHPh2 and NCH), 51.4 (q, COOCH3), 51.4 (t, NCH2), 20.9 (t, CH2). IR (CHCl3): σ 1715
(s, COOCH3). GC–MS: m/z 282 (M++1), 222 (M+−COOCH3), 167 (Ph–CH+–Ph). HR–MS: calculated
for C18H19NO2 281.14158, found 281.14091.
4.8. General procedure for the synthesis of racemic azetidine carboxamides
A solution of azetidine ester 2 (4.87 mmol) in concentrated aqueous ammonia (20 ml) was stirred for
3 h and then extracted three times with chloroform to provide the corresponding carboxamide as a white
solid.
4.9. (ꢁ)-1-Benzylazetidine-2-carboxamide 3a
1
Yield: 81%. Mp.: 104–105°C (ethanol (96%)). H NMR (CD3OD): δ 7.24 (s, 5H, Ph), 4.92 (t, J=9.2
Hz, 1H, NCH), 4.20 (s, 2H, benzylic CH2), 3.97 and 3.73 (t, J=9.4 Hz, and dt, J=4.3 Hz and J=9.5 Hz, 2H,
NCH2), 2.8–2.15 (m, 2H, CH2). IR (CCl4): σ 3500 and 3430 (s, NH2 amide), 1685 (s, CO amide), 700
(s, monosubstituted phenyl). GC–MS: m/z 191 (M++1), 146 (M+−CO(NH2)), 91 (tropilium). Elemental
analysis: calculated for C11H14N2O % C 69.45, % H 7.42, % N 14.72. Found % C 69.43, % H 7.10, %
N 14.45.
4.10. (ꢁ)-1-(4-Methoxybenzyl)azetidine-2-carboxamide 3b
1
Mp.: 109.5–111°C. H NMR (CDCl3): δ 7.18 (d, Jortho=8.6 Hz, 2H, aromatic), 6.84 (d, Jortho=8.6
Hz, 2H, aromatic), 5.71 (broad s, CONH2), 3.79 (s, 3H, OCH3), 3.78 (t, J=9 Hz, 1H, NCH), 3.57 (d,
J=4.5 Hz, 2H, benzylic CH2), 3.33 and 3.49 (dt, J=2.5 Hz and J=7.8 Hz, and dt, J=7.6 Hz and J=7.9 Hz,
2H, NCH2), 2.55–2.04 (m, 2H, CH2). 13C NMR (CDCl3): δ 176.5 (s, CONH2), 129.6 (d, aromatic C),
129.4 (s, aromatic C), 114.0 (d, aromatic C), 66.0 (d, NCH), 61.7 (t, benzylic CH2), 55.3 (q, OCH3),

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50.6 (t, NCH2), 22.9 (t, CH2). IR (KBr): σ 3370 (s, NH), 1620 (s, CONH2). GC–MS: m/z 221 (M++1),
176 (M+−CO(NH2)), 121 (methoxytropilium). HR–MS: calculated for C12H16N2O2 220.12118, found
220.12129.
4.11. (ꢁ)-1-Allylazetidine-2-carboxamide 3c
Mp.: 49–52°C. 1H NMR (300 MHz, CDCl3): δ 7.17 and 5.9 (broad s, 2H, CONH2), 5.81–5.67 (m, 1H,
CH2CH=CH2), 5.19 and 5.11 (d, J=17.1 Hz and d, J=10.2 Hz, 2H, CH2CH=CH2), 3.54 (t, J=8.6 Hz, 1H,
NCH), 3.39 and 2.94 (dt, J=2.7 Hz and J=7.7 Hz, and dt, J=7.9 Hz and J=8.5 Hz, 2H, NCH2), 3.16 and
3.06 (dd, J=6.1 Hz and J=13.3 Hz, and dd, J=6.4 Hz and J=13.2 Hz, 2H, CH2CH=CH2), 2.46–2.34 and
2.22–2.10 (m and m, 2H, CH2). 13C NMR (CDCl3): δ 176.8 (s, CONH2), 133.7 (d, CH2CH=CH2), 117.9
(t, CH2CH=CH2), 65.8 (d, NCH), 60.9 (t, CH2CH=CH2), 50.6 (t, NCH2), 22.7 (t, CH2). IR (CHCl3): σ
1670 (s, CONH2). GC–MS: m/z 141 (M++1), 96 (M+−CO(NH2)). HR–MS: calculated for C7H12N2O
140.09496, found 140.09492.
4.12. General procedure for the ammoniolysis of racemic azetidine-2-carboxylic esters
A stock solution of NH3 saturated tert-butyl alcohol was prepared by bubbling NH3 gas through 20 ml
of tert-butyl alcohol for approximately 60 min. while stirring. A reference line was made by GC-analysis
of solutions of the racemic azetidine ester and 2-bromonaphthalene (internal standard) with different
weight ratios.
A vial was charged with azetidine ester (approximately 1.0 mmol), 2-bromonaphthalene (100 mg,
0.48 mmol), immobilized lipase from Candida antarctica (50 mg, Novozym 435) and NH3 saturated
tert-butyl alcohol (5.0 ml, 12 mmol NH3), capped with a septum and shaken in a thermostatized box at
35°C. At regular time intervals 0.1 ml samples were taken with a syringe, diluted with dichloromethane
(0.4 ml) after which the conversion was measured by GC-analysis (apolar column, 130°C isothermic).
At approximately 50% conversion the reaction was stopped by filtration. The immobilized enzyme was
washed with methanol and the combined organic filtrates were concentrated in vacuo. The residue
was dissolved in water/hexane and the layers were separated. The aqueous layer was extracted twice
with hexane, the combined organic layers dried with MgSO4, filtered and concentrated in vacuo. The
optical purity of the remaining azetidine ester was determined by chiral capillary GC, or when purified
by bulb-to-bulb distillation, by optical rotation. The aqueous layer containing the corresponding amide
was concentrated in vacuo after which the enantiomeric excess of the amide was determined by chiral
capillary GC or chiral capillary electrophoresis.
Acknowledgements
We wish to thank Novo Nordisk A/S for the generous gift of immobilized Candida antarctica
(Novozym 435).
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