Enantioselective enzyme catalysed ammoniolysis of amino acid derivatives. Effect of temperature

Article abstract of DOI:10.1016/S0957-4166(01)00023-4

The lipases from Candida antarctica (B type), Thermomyces lanuginosus and Pseudomonas alcaligenes catalysed the enantioselective ammoniolysis of free amino acid esters. In the ammoniolysis of phenylalanine methyl ester catalysed by T. lanuginosus lipase a

Full text of DOI:10.1016/S0957-4166(01)00023-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 235–240
Enantioselective enzyme catalysed ammoniolysis of amino acid
derivatives. Effect of temperature
Paloma Lo´pez-Serrano, Margreth A. Wegman, Fred van Rantwijk and Roger A. Sheldon*
Laboratory of Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136,
2628 BL Delft, The Netherlands
Received 2 November 2000; accepted 11 January 2001
Abstract—The lipases from Candida antarctica (B type), Thermomyces lanuginosus and Pseudomonas alcaligenes catalysed the
enantioselective ammoniolysis of free amino acid esters. In the ammoniolysis of phenylalanine methyl ester catalysed by T.
lanuginosus lipase a decrease in temperature to −20°C significantly enhanced the enantioselectivity up to an enantiomeric ratio (E)
of 84. Several proteases efficiently catalysed the ammoniolysis of N-BOC protected amino acid esters with nearly absolute
enantioselectivity. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
activation, DS‡ the free entropy of activation and DG‡
the free energy of activation.
Since the first reports by us1,2 and Gotor et al.,3 the
number of publications on the ammoniolysis of car-
boxylic esters catalysed by lipases has substantially
increased.4 We recently showed5 that the enantioselec-
tivity of the Candida antarctica lipase B (CaLB)
catalysed ammoniolysis of phenylglycine methyl ester 1
increased substantially on lowering the temperature of
the reaction. The enantiomeric ratio (E) increased from
17 at 40°C to 52 at −20°C. We now present a more
detailed study of the effect of temperature on the
ammoniolysis of three structurally related amino acid
esters catalysed by three lipases from widely different
sources. We also report our further investigation of the
enantioselective ammoniolysis of two N-tert-butoxycar-
bonyl (N-BOC) protected amino acid esters.
DDG‡=−RT ln E
(1)
and
DDG‡=DDH‡−TDDS‡
(2)
(3)
Hence,
ln E=DDS‡/R−DDH‡/(RT)
Another relevant parameter is the racemic temperature
(Tr in Eq. (4)), at which there is no discrimination of
the enantiomers.
Tr=DDH‡/DDS‡
(4)
Studies on the effect of the temperature on the enan-
tioselectivity of lipases are few7,11 and, with a single
exception,5 concern the enantiodiscrimination of
alcohols.
Variation of the temperature to enhance the enantio-
selectivity has been known for years,6 but the use of
temperatures below 0°C in enzymatic reactions is not
common because most enzymes are not active at these
temperatures. Lipases, in particular Pseudomonas
cepacea lipase, are an exception, and have been
reported to maintain their activity even at temperatures
as low as −60°C.7
2. Results and discussion
2.1. Ammoniolysis of a-amino esters with lipases
We have investigated the ammoniolysis of the methyl
The enantiomeric ratio E8 in enzymatic kinetic resolu-
tions is often temperature dependent.9,10 This can be
explained by Eqs. (1)–(3) where DH‡ is the enthalpy of
esters of phenylglycine 1, phenylalanine
2
and
homophenylalanine 3 (see Fig. 1). They form a series of
three amino acid esters in which the phenyl group is
separated from the stereogenic centre by zero, one and
two methylene groups, respectively.
* Corresponding author. E-mail: r.a.sheldon@tnw.tudelft.nl
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00023-4

236
P. Lo´pez-Serrano et al. / Tetrahedron: Asymmetry 12 (2001) 235–240
The reactions were carried out in sealed 2 mL vials.
Enzyme and zeolite NaA were added to a solution of
the free amino acid ester in tert-butanol saturated with
ammonia. Reactions at temperatures below 20°C were
performed with addition of ammonia saturated tert-
butyl methyl ether (TBME) to avoid solidification of
the reaction medium.
enantioselectivity (Eꢀ1) and no significant improve-
ment at −10°C was detected.
This order of enantioselectivity could be explained
based on the notion that the more inefficient the inter-
action of the (S)-enantiomer with the active site the
higher the (R)-enantioselectivity. The high reactivity
and low enantioselectivity of 2 at all temperatures
suggests that (S)-2 (as well as (R)-2) is a good substrate
with a favourable orientation. In the case of (S)-1, the
phenyl group cannot adopt this orientation (Fig. 2),
resulting in a less effective fit in the active site. Conse-
quently, the enantioselectivity towards (R)-1 would be
enhanced. In the case of (S)-3, the molecule is more
flexible, and the phenyl group could adopt a closer
orientation to that of (S)-2, but not the same. This
species would then interact less efficiently than (S)-2
(higher enantioselectivity towards the (R)-enantiomer
than in the case of 2), but more than (S)-1 (lower
(R)-enantioselectivity than in the case of 1).
Besides C. antarctica lipase B (CaLB, Novo SP611), we
have used the lipases from Thermomyces lanuginosus
(TlL, Novo SP398) and Pseudomonas alcaligenes (PaL).
All were immobilised on Accurel EP100.12 Taking the
ammoniolysis of 2 as a test reaction, a number of other
lipases, as well as related hydrolases† proved to be
inactive. The effect of the temperature on the ammo-
niolysis of 1–3 in the presence of the three lipases
mentioned above was investigated (Tables 1–3).
CaLB showed a preference for the (R)-enantiomer in
all reactions (Table 1). The E value of the reaction of 1
and 3 increased at low temperature, the reaction of 1
being the most selective one. The ammoniolysis of 2 in
the presence of this enzyme proceeded with very low
TlL and PaL, which did not accept 1 as substrate,
preferentially converted the (S)-enantiomers of 2 and 3
(Tables 2 and 3); E was higher in the ammoniolysis of
2 than in that of 3. Enhanced E factors resulted when
the reactions were performed at −20°C, even reaching a
value of 84 in the ammoniolysis of 2 catalysed by TlL.
This lipase acted more enantioselectively than PaL in
the ammoniolysis of both 2 and 3. The ammoniolysis of
2 catalysed by PaL did not show an increase in selectiv-
† C. rugosa lipase, R. arrhizous lipase, cholesterol esterase from C.
cylindracea, porcine pancreas lipase, amino acylase I from A.
melleus, penicillin acylase from E. coli and the proteases SP458,
SP539 (serine type endoprotease), Maxatase (alkaline protease,
subtilisin, from Bacillus licheniformis), Maxacal (high alkaline
protease, subtilisin, from Bacillus alkalophilus), chymotrypsine and
trypsine.
Figure 1. Enzyme catalysed ammoniolysis of 1–3.
Table 1. CaLB catalysed ammoniolysis of 1–3 at different temperaturesa
Substrate
Selectivity
Temp. (°C)
Time (h)
Conv. (%)
EeP (%)
Eb
16
1c
1c
R
R
40
−20
46
33
78
94
52
2
2
2
2
R
R
R
R
40
20
4
0.5
0.5
3
46
37
24
15
7
13
11
13
ꢀ1
ꢀ1
ꢀ1
ꢀ1
−10
3
3
3
3
3
3
3
R
R
R
R
R
R
40
20
4
−10
−20
−30
0.5
0.5
5
5
5
29
18
41
33
16
18
56
62
65
74
80
86
4
5
7
9
10
15
24
a Reaction conditions: substrate 50 mM solution in ammonia saturated tert-butyl alcohol, 50 mg mL−1 zeolite NaA, 50 mg mL−1 enzyme. TBME
(30%) was added in reactions below 20°C.
b Calculated as E=ln[1−c(1+eep)]/ln[1−c(1−eep)].
c Data from Ref. 5.

P. Lo´pez-Serrano et al. / Tetrahedron: Asymmetry 12 (2001) 235–240
Table 2. TlL catalysed ammoniolysis of 2 and 3 at different temperaturesa
237
Substrate
Selectivity
Temp. (°C)
Time (h)
Conv. (%)
EeP (%)
Eb
2
2
2
2
2
S
S
S
S
S
40
20
4
−10
−20
1
1
5
24
5
8
11
29
40
8
84
89
91
93
97
13
20
30
52
84
3
3
3
3
3
S
S
S
S
S
40
20
4
−10
−20
5
5
5
5
5
29
18
41
33
16
68
72
81
83
87
6
7
11
13
15
a Reaction conditions: substrate 50 mM solution in ammonia saturated tert-butyl alcohol, 50 mg mL−1 zeolite NaA, 50 mg mL−1 enzyme. TBME
(30%) was added in reactions below 20°C.
b Calculated as E=ln[1−c(1+eep)]/ln[1−c(1−eep)].
Table 3. PaL catalysed ammoniolysis of 2 and 3 at different temperaturesa
Substrate
Selectivity
Temp. (°C)
Time (h)
Conv. (%)
EeP (%)
Eb
2
2
2
2
S
S
S
S
40
20
4
1
1
5
23
21
45
26
65
67
56
76
6
6
6
9
−20
24
3
3
3
3
S
S
S
S
40
20
4
1
1
5
5
7
7
14
4
26
32
50
69
ꢀ1
2
3
−20
6
a Reaction conditions: substrate 50 mM solution in ammonia saturated tert-butyl alcohol, 50 mg mL−1 zeolite NaA, 50 mg mL−1 enzyme. TBME
(30%) was added in reactions below 20°C.
b Calculated as E=ln[1−c(1+eep)]/ln[1−c(1−eep)].
Figure 2. Model of possible conformations of the (S)-enantiomers of 1–3.
ity at moderately low temperatures. However, at
−20°C, the reactivity of the enzyme decreased consider-
ably and an increase in enantioselectivity was then
detected. In the case of 3, the decrease in reactivity and
increase in enantioselectivity at lower temperatures
were smoother.
using Eq. (1), from the data in Figs. 3–5; they are
summarised in Table 4. The differential activation
enthalpy (DDH‡) is the major contributor to DDG‡ in
all cases; hence, the enantioselectivity is enhanced at
low temperatures. This parameter is related to the steric
interactions of the enantiomers with the active site. The
values for DDS‡ give an indication of losses in rota-
tional and translational freedom caused by interaction
with the system.
Finally, ln E was plotted against 1/T (Eq. (1)). In
general, a good linear correlation was obtained (Figs.
3–5). The ammoniolysis of 2 in the presence of PaL was
an exception, and the data points could not be fitted to
a straight line; E remained constant from 40 to 4°C and
improved only at −20°C (Fig. 5).
2.2. Ammoniolysis of a-amino acid esters with
proteases
The free amino acid esters 1–3 were not converted by
any of the proteases tested, as noted above. Pre-
sumably, an acylation of the amino group is required to
The differential activation parameters DDH‡ and DDS‡
as well as the racemic temperature were calculated,

238
P. Lo´pez-Serrano et al. / Tetrahedron: Asymmetry 12 (2001) 235–240
ensure proper binding of the reactant in the S2 sub-
site.13 We previously showed that proteases can catalyse
the ammoniolysis of N-protected a-amino acid esters
(in particular Z-amino acid esters) with high enantio-
selectivity.14 In a continuation of this study, we have
now performed the ammoniolysis of the N-tert-butoxy-
carbonyl (N-BOC) methyl esters of phenylalanine 7 and
homophenylalanine 8 (see Fig. 6). We surmised that the
BOC group could enhance the enantioselectivity due to
its steric bulk. Moreover, the BOC group is commonly
used in organic chemistry as it can be very easily
removed by treatment with mild acid.
The reactions were carried out in sealed 2 mL vials at
40°C. The protease (immobilised on Accurel EP100)
and zeolite NaA were added to a solution of the
N-BOC amino acid ester in tert-butyl alcohol contain-
ing ammonia (see Section 4). The results are sum-
marised in Table 5.
Figure 5. Plot of ln E versus 1/T for PaL catalysed ammoniol-
ysis of 2 (ꢀ) and 3 (ꢁ).
Table 4. Calculated differential thermodynamic parameters
for the ammoniolysis of 1–3a
Lipase
Substrate
DDS‡ (cal
DDH‡ (kcal
Tr (°C)
K−1 mol−1
)
mol−1
)
CaLB
CaLB
CaLB
TlL
TlL
PaL
1
2
3
2
3
3
−4.30
−0.10
−5.89
−10.93
−3.85
−11.42
−3.09
−0.18
−2.73
−5.00
−2.36
−3.80
443
1525
188
182
337
57
a Difference in DS‡ and DH‡ of the fast reacting enantiomer minus
the slow reacting enantiomer.
All reactions showed an extraordinarily high (S)-selec-
tivity. No traces of the (R)-amide were detected except
for the reaction of 7 with SP539. The presence of the
blocking BOC group gave, in general, higher enantio-
selectivities than the Z group.14 In all cases 7 was a
better substrate than 8, the reactions of 8 being very
slow, with a conversion of only 10% in 24 hours in the
best case (Maxatase). Since these reactions were all
highly enantioselective at room temperature, we did not
examine the effect of decreasing the temperature on
enantioselectivity.
Figure 3. Plot of ln E versus 1/T for CaLB catalysed ammo-
niolysis of 1 (ꢂ), 2 (ꢀ) and 3 (ꢁ).
3. Conclusions
Lipases of C. antarctica (Type B), T. lanuginosus and P.
alcaligenes catalyse the enantioselective ammoniolysis
of free a-amino esters. The enantiorecognition is gener-
ally improved at low temperatures, reaching an E of 84
at −20°C in the ammoniolysis of phenylalanine
catalysed by TlL. CaLB was R selective whereas TlL
and PaL were S selective. Proteases catalysed the (S)-
enantioselective ammoniolysis of N-BOC protected a-
amino acid esters with practically total enantio-
selectivity. Free amino esters were not substrates of
proteases.
Figure 4. Plot of ln E versus 1/T for TlL catalysed ammoniol-
ysis of 2 (ꢀ) and 3 (ꢁ).

P. Lo´pez-Serrano et al. / Tetrahedron: Asymmetry 12 (2001) 235–240
239
Figure 6. Protease catalysed ammoniolysis of 7 and 8.
Table 5. Protease catalysed ammoniolysis of 7 and 8a
Substrate
Protease
Selectivity
Time (h)
Conv. (%)
Eb
Ec
7
7
7
7
Maxatase
Maxacal
SP539
S
S
S
S
2
2
2
2
15
5
7
ꢁ100
ꢁ100
65
54
8
89
SP458
1
8
8
8
8
Maxatase
Maxacal
SP539
S
S
S
S
24
24
24
24
10
B1
3
ꢁ100
ꢁ100
SP458
B1
a Reaction conditions: substrate 50 mM solution in tert-butyl alcohol–ammonia saturated tert-butanol (9:1, v/v), 50 mg mL−1 zeolite NaA, 30 mg
mL−1 enzyme, 40°C.
b Calculated as E=ln[1−c(1+eep)]/ln[1−c(1−eep)].
c E values for the ammoniolysis of N-Z-PheOMe (Ref. 14).
4. Experimental
4.1. Materials and methods
4.2. Synthesis of substrates and standards
4.2.1. Homophenylalanine methyl ester 3. Hydrogen
chloride was bubbled through
a
dispersion of
1H and 13C NMR spectra were recorded in CDCl3
solution with TMS as internal standard using a 400
MHz Varian-VXR 400S spectrometer. The reactions
were monitored by chiral HPLC on a Daicel Chemical
Industries Ltd 4.6×150 mm 5 m Crownpak CR (+)
column using a Waters 625 pump and a Waters 486 UV
detector operating at 215 nm. The eluent was aqueous
HClO4 at a flow rate of 0.6 mL min−1. Details about
pH and temperature are specified for each case. Sol-
vents were dried over zeolite CaA (Uetikon, activated
at 400°C for 24 hours). All substrates and final prod-
ucts used as standards were obtained from common
commercial sources except homophenylalanine methyl
ester and homophenylalanine amide, which were syn-
thesised (see below). All enzymes were immobilised on
Accurel EP100 according to a published procedure.12
SP539, SP458 and SP398 were kindly donated by Novo
Nordisk; C. antarctica lipase B on Accurel EP100 was
received from Uniqema as a gift. P. alcaligenes lipase,
penicillin acylase from E. coli, Maxacal and Maxatase
were obtained from Gist-brocades (now DSM-Life Sci-
ences), as gifts. R. arrhizus lipase, chymotrypsine,
trypsine and cholesterol esterase were gifts from Roche
Diagnostics. C. rugosa lipase and porcine pancreas
lipase were purchased from Sigma; amino acylase I
from A. melleus was from Fluka.
homophenylalanine (710 mg) in methanol (60 mL) for
30 minutes and stirred at room temperature for 16
hours. The solvent was evaporated in vacuo. Then
NaHCO3 saturated aqueous solution (50 mL) was
added and the mixture extracted with dichloromethane
(3×30 mL). The organic layer was washed with water
(50 mL), dried over anhydrous Na2SO4 and evaporated.
Homophenylalanine (61%) was obtained as a colourless
1
liquid. H NMR: 7.23 (m, 5H, Ar); 3.70 (s, 3H, CH3);
3.48 (m, 1H, CH-a); 2.78 (m, 2H, CH2-g); 2.01 (d, 2H,
NH2); 2.08 (m, 1H, CH2-b1); 1.87 (m, 1H, CH2-b2). 13C
NMR: 176.18 (CO); 141.16, 128.47, 128.45, 126.05
(Ar); 53.85 (CH-a); 52.02 (CH3); 36.25 (CH2-g); 31.90
(CH2-b).
4.2.2. Homophenylalanine amide 6. Homophenylalanine
methyl ester (300 mg) was added to saturated aqueous
ammonium hydroxide solution (20 mL) and stirred
at room temperature for 24 hours. Water (30 mL)
was added and the mixture was extracted with
dichloromethane (3×30 mL). The organic solvent was
evaporated in vacuo to afford homophenylalanine
1
amide (95%) as a white solid. H NMR: 7.26 (m, 5H,
Ar); 7.04 (s, 1H, CONH2), 5.86 (s, 1H, CONH2); 3.37
(m, 1H, CH-a); 2.74 (m, 2H, CH2-g); 2.18 (m, 1H,
CH2-b1); 1.84 (m, 1H, CH2-b2); 1.57 (s, 2H, CH-NH2).

240
P. Lo´pez-Serrano et al. / Tetrahedron: Asymmetry 12 (2001) 235–240
13C NMR: 178.07 (CO); 141.12, 128.52, 128.43, 126.11
(Ar); 54.70 (CH-a); 36.62 (CH2-g); 32.09 (CH2-b). Mp
90–91°C.
was complete. An aliquot was then withdrawn and the
same procedure as in the case of lipases was followed.
4.3.3. Ammoniolysis of 1. Following the general proce-
dure for lipases, (R)-phenylalanine amide in the same
concentration as the substrate was used as standard.
HPLC conditions: perchloric acid solution pH 1, 35°C.
4.2.3. N-BOC-phenylalanine methyl ester 7. Phenylala-
nine methyl ester (666 mg, 3.72 mmol) was dissolved in
anhydrous dichloromethane (15 mL). Triethylamine
(1.03 mL, 7.44 mmol) was added, the mixture was cooled
in an ice-water bath and then a solution of di-tert-butyl-
dicarbonate (487 mg, 2.23 mmol) in dichloromethane (10
mL) was added dropwise. The reaction mixture was left
stirring at room temperature for 17 hours and then
washed with 0.1N HCl (2×20 mL), NaHCO3 saturated
solution (20 mL) and finally water (30 mL). The product
7 was obtained as a white solid (520 mg, 50%). 1H NMR:
7.32–7.11 (m, 5H, Ar); 4.98 (d, 1H, NH); 4.58 (m, 1H,
CH-a); 3.71 (s, 3H, OCH3); 3.08 (m, 2H, CH2-b); 1.41
(s, 9H, CH3-BOC). 13C NMR: 172.36 (COOMe); 155.08
(CO-BOC); 136.03, 129.30, 128.55, 127.03 (Ar); 79.91
(C(CH3)3); 54.43 (CH-a); 52.19 (CH3O); 38.37 (CH2-b);
28.29 (CH3-BOC). Mp 46°C.
4.3.4. Ammoniolysis of 2 and 7. Following the general
procedure for lipases and proteases, (R)-phenylglycine
amide in the same concentration as the substrate was
used as standard. HPLC conditions: perchloric acid
solution pH 1, 35°C.
4.3.5. Ammoniolysis of 3 and 8. Following the general
procedure for lipases and proteases, (R)-phenylglycine
amide in the same concentration as the substrate was
used as internal standard. HPLC conditions: perchloric
acid solution pH 2, 49°C.
Acknowledgements
4.2.4. N-BOC-homophenylalanine methyl ester 8. Follow-
ing the same procedure described for 7, from
homophenylalanine (719 mg), the methyl ester 8 was
The authors express their thanks to Novo Nordisk A/S
(Bagsværd, Denmark), Uniqema (Gouda, The Nether-
lands), DSM-Life Sciences (Delft, The Netherlands) and
Roche Diagnostics (Penzberg, germany) for generous
donations of enzymes. This work was partially financed
by a Marie Curie Research Training Grant from the
European Community.
1
obtained as a white solid (625 mg, 57%). H NMR:
7.32–7.14 (m, 5H, Ar); 5.10 (d, 1H, NH); 4.35 (m, 1H,
CH-a); 3.71 (s, 3H, OCH3); 2.67 (m, 2H, CH2-g); 2.14
(m, 1H, CH2-b1); 1.95 (m, 1H, CH2-b2); 1.45 (s, 9H,
CH3-BOC). 13C NMR: 173.17 (COOMe); 155.36 (CO-
BOC); 140.77, 128.49, 128.41, 126.16 (Ar); 79.94
(C(CH3)3); 53.25 (CH-a); 52.02 (CH3O); 34.37 (CH2-g);
31.64 (CH2-b); 28.32 (CH3-BOC). Mp 102°C.
References
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Butyl alcohol and TBME were saturated with ammonia
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.
butanol saturated with ammonia, 50 mg enzyme and 50
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tert-butanol saturated with ammonia (9:1, v/v), protease
immobilised on Accurel EP100 (30 mg) and zeolite NaA
(50 mg) were added. Reactions with free amino esters
were performed following the same procedure described
for lipases. For reactions with N-BOC protected amino
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