Enantioselective aminolysis of an alpha-chloroester catalyzed by Candida cylindracea lipase encapsulated in sol-gel silica glass.

Article abstract of DOI:10.1021/ol015989o

[reaction: see text] Lipase from Candida cylindracea (CcL) encapsulated in porous silica glass by a sol-gel method catalyzes enantioselective aminolysis of ethyl 2-chloropropionate. A silica matrix enhances the enzyme activity, i.e., improves the yield. T

Full text of DOI:10.1021/ol015989o

ORGANIC
LETTERS
2001
Vol. 3, No. 13
2025-2028
Enantioselective Aminolysis of an
r-Chloroester Catalyzed by Candida
cylindracea Lipase Encapsulated in
Sol−Gel Silica Glass
Jovica D. Badjic´, Ekaterina N. Kadnikova, and Nenad M. Kostic´*
Department of Chemistry, Iowa State UniVersity, Ames Iowa 50011-3111
nenad@iastate.edu
Received April 16, 2001
ABSTRACT
Lipase from Candida cylindracea (CcL) encapsulated in porous silica glass by a sol−gel method catalyzes enantioselective aminolysis of ethyl
2-chloropropionate. A silica matrix enhances the enzyme activity, i.e., improves the yield. The scope and limitations of the aminolysis reaction
were investigated, and dynamic kinetic resolution of the ester was achieved. Encapsulated lipase remains active when used repeatedly.
Encapsulation much improves the chiral discrimination by lipase and makes this enzyme even more useful in organic chemistry.
Glasses made of silica and its various organic derivatives
may be prepared at room temperature by the sol-gel method,
that is, by condensation-polymerization of suitable alkox-
ides.1 Mild reaction conditions allow encapsulation in these
glasses of enzymes, catalytic antibodies, and other proteins.2
The resulting biocomposite materials hold great promise as
sensors and heterogeneous catalysts, but the fulfillment of
this promise requires study of the interactions between the
protein and the surrounding glass matrix. This study has only
begun,3 and much remains to be learned about the various
effects of the matrix on activity and stability of encapsulated
biomolecules.
this great utility of lipases, their encapsulation in sol-gel
glasses has been studied more than that of any other
enzymes.5 Research by Reetz and co-workers showed that
the enzyme activity and stability can be adjusted, even
improved, by varying the hydrophobicity of the matrix.5a
Making an enzyme into a heterogeneous catalyst is very
advantageous in synthetic practice, and encapsulated lipases
can be purchased.
The main advantage of lipases as catalysts in organic
chemistrystheir great enantioselectivityshas barely been
studied in sol-gel glasses.6 This is the subject of our Letter.
Lipase from Candida cylindracea catalyzes enantioselective
aminolysis of esters.7 We compare the effectiveness of this
Lipases, obtained from various organisms, are the most
useful enzymes available to organic chemists today.4 They
catalyze esterification, transesterification, amidation, trans-
amidation, and hydrolysis of many substrates. Because of
(4) (a) Lopez-Serrano, P.; Wegman, M. A.; van Rantwijk, F.; Sheldon,
R. A. Tetrahedron: Asymmetry 2001, 12, 235-240. (b) Lau, R. M.; van
Rantwijk, F.; Seddon, K. R.; Sheldon, R. A. Org. Lett. 2000, 2, 4189-
4191. (c) Theil, F. Chem. ReV. 1995, 95, 2203-27.
(5) (a) Reetz, M. T.; Wenkel, R.; Avnir, D. Synthesis 2000, 6, 781-
783. (b) Reetz, M. T. AdV. Mater. 1997, 9, 943-954. (c) Reetz, M. T.;
Zonta, A.; Simpelkamp, J. Biotech. Bioeng. 1996, 49, 527-534. (d) Reetz,
M.; Zonta, A.; Simpelkamp, J. Angew. Chem., Int. Ed. Engl. 1995, 34, 301-
303. (e) Furukawa, S.; Ono, T.; Ijima, H.; Kawakami, K. Kagaku Kogaku
Ronbunshu 2000, 26, 882-886.
(6) Jaeger, K.-E.; Schneidinger, B.; Rosenau, F.; Werner, M.; Lang, D.;
Dijkstra, B. W.; Schimossek, K.; Zonta, A.; Reetz, M. T. J. Mol. Catal. B:
Enz 1997, 3, 3-12.
(1) (a) Husing, N.; Schubert, U. Angew. Chem., Int. Ed. Engl. 1998, 37,
22-45. (b) Dunn, B.; Zink, J. I. Chem. Mater. 1997, 9, 2280-2291. (c)
Avnir, D. Acc. Chem. Res. 1995, 28, 328-334.
(2) (a) Bhatia, R. B.; Brinker, C. J.; Gupta, A. K.; Singh, A. K. Chem.
Mater. 2000, 12, 2434-2441. (b) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi,
M. Chem. Mater. 1994, 6, 1605-1614.
(3) (a) Badjic´, J. D.; Kostic´, N. M. Chem. Mater. 1999, 11, 3671-3679.
(b) Gottfried, D. S.; Kagan, A.; Hoffman, B. M.; Friedman, J. M. J. Phys.
Chem. B 1999, 103, 2803-2807. (c) Chen, Q.; Kenausis, G. L.; Heller, A.
J. Am. Chem. Soc. 1998, 120, 4582-4585.
10.1021/ol015989o CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/07/2001

enzyme when suspended in the reaction mixture and when
encapsulated in sol-gel silica powder that is suspended in
the reaction mixture. We are interested in the stability and
catalytic activity of the encapsulated enzyme, enantioselec-
tivity of the aminolysis reaction, and recyclability of this
catalyst. Our results show a considerable advantage of the
encapsulated over the free enzyme.
Objective of this Study. We examine the effect of silica
matrix on the stability of encapsulated C. cylindracea lipase
and its enantioselectivity. Our very recent study of catalysts
other than lipase showed that the silica matrix may lower
the enantioselectivity of sulfoxidation by indirectly enhancing
the “background” reaction, which is not enantioselective.8
Clearly, enantioselectivity of reactions in the silica matrix
cannot be taken for granted.
Table 1. Enantioselective Aminolysis of Ethyl
2-Chloropropionate by p-Anisidine Catalyzed by C. cylindracea
Lipase at 60 °C
entry
catalyst
ester:amine time (h) yield (%) ee (%)
1
2
3
4
5
6
7
8
wsCcLa
2:1
24
48
217
24
30
24
1.3
6.0
21
64
54
62
85
6.0
2.0
94
96
86
71c
74
CcL@glassb
2:1
5:1
1:1
1:2
2:1
25
26
116c
4
CcL@glassd
a Water-soluble fraction of C. cylindracea lipase. b C. cylindracea lipase
encapsulated into sol-gel silica glass. c Slower-reacting enantiomer of ester
also reacts under these conditions (see text). d After 24 h at 60 °C.
Choice of Enantioselective Reaction. Crude C. cylin-
dracea lipase (CcL) suspended in an organic solvent
catalyzes the reaction of racemic esters with various amines
as shown in eq 1. Yield and enantioselectivity generally are
excellent enantioselectivity. Entries 4 and 1 show that
encapsulated lipase under the given conditions is more
reactive than free lipase but that the reaction ceases after
ca. 24 h.
Entries 4-7 show the effects of the reactant ratio.
Lowering the ester-to-amine ratio (entries 6 and 7) raises
the yield but lowers the enantioselectivity. A yield greater
than 100%, accompanied by a decline in enantioselectivity
(entry 7), shows that the slower-reacting enantiomer of the
ester also reacts when the amine is present in large excess.
Since amine was never in excess in our other experiments,
this anomaly was avoided.
good.7 We use this known reaction to study the possible
effects of organically modified sol-gel silica glass on
enzyme stability, reaction yield, and enantioselectivity. For
kinetic resolution of the racemic ester, a 2:1 ratio of ester to
amine should be used. Ideally, the faster-reacting enantiomer
of the ester would be converted to a single enantiomer of
the amide, and the slower-reacting enantiomer of the ester
would remain. When the ester-to-amine ratio is greater than
2:1, the amine is the limiting reagent; when this ratio is lower
than 2:1, the faster-reacting enantiomer of the ester is the
limiting reagent.
To test the recyclability, the catalyst was repeatedly
recovered from the reaction mixture, washed with CCl4 and
acetone, and reused. As Figure 1 shows, after just three 24
Encapsulation of the Lipase. The enzyme was encapsu-
lated into hydrophobic sol-gel glass using a modification
of the published method.5c The water-soluble fraction of the
enzyme (designated wsCcL) was extracted from the pur-
chased crude enzyme.9 A 5:1 molar ratio of trimethoxy-
propylsilane to tetramethoxysilane was used for the sol-
gel method. Experimental procedure can be found in the
Supporting Information.
Enantioselective Aminolysis of Ethyl 2-Chloropropi-
onate by p-Anisidine at 60 °C. An aminolysis reaction in
the presence of the water-soluble fraction of CcL (see Table
1, entry 1) after 24 h gave a minute yield, but good
enantioselectivity. After several days, the yield increased
somewhat, but the enantioselectivity almost vanished (entries
2 and 3). Encapsulated CcL (designated CcL@glass) yielded
much more amide than the free enzyme did, and with
(7) (a) Gotor, V. Bioorg. Med. Chem. 1999, 7, 2189-2197. (b) Gotor,
V.; Brieva, R.; Gonzalez, C.; Rebolledo, F. Tetrahedron 1991, 47, 9207-
14.
(8) Kadnikova, E. N.; Kostic´, N. M. J. Non-Cryst. Solids 2001, 283,
63-68.
(9) Lewis, G.; Bevan, J.; Rawas, A.; McMichael, P.; Wisdom, R.;
McCague, R.; Watson, H.; Littlechild, J. Acta Crystallogr., D: Biol.
Crystallogr. 1997, D53, 348-351.
Figure 1. Effect of recycling of C. cylindracea lipase encapsulated
into sol-gel silica glass on the yield of amide (top) and enantio-
meric excess of its (S)-enantiomer (bottom). All the experiments
lasted for 24 h, and the ratio of racemic ester to amine was 2:1.
Temperature was 60 (b) and 25 (2) °C.
2026
Org. Lett., Vol. 3, No. 13, 2001

h cycles at 60 °C the yield drops to single digits and the
enantioselectivity declines.
Table 3. Enantioselective Aminolysis of Ethyl
2-Chloropropionate by Amines RNH2 Catalyzed by C.
cylindracea Lipase
After the encapsulated enzyme was heated in CCl4 at 70
°C for 14 days, it was filtered off, and the organic phase
was thoroughly extracted with water. The Bradford test
showed no protein in the extract. Given the sensitivity of
this assay, at most 0.10-0.20% of the encapsulated enzyme
might have leaked from the silica. When the encapsulated
enzyme was kept at 60 °C for 24 h prior to the addition of
the reactants (entry 8), both the yield and the enantioselec-
tivity declined from the values obtained with the fresh
catalyst (entry 4). These experiments show that the decline
in the performance of encapsulated lipase evident in Figure
1 is due to thermal inactivation of the enzyme at 60 °C, not
to the leakage of the enzyme from the glass matrix.
CcL@glassa
wsCcLb
temp
(°C)
time
(h)
yield
(%)
ee
yield
ee
entry
R
(%)
(%)
(%)
1
2
3
4
5
6
7
8
9
p-tolyl
60
25
60
25
25
5
25
5
25
5
24
127
24
170
4
12
6
12
3
28
92
20
43
25
23
40
38
58
22
51
50
76
86
95
97
3c
0.6
2.4
1.4
4.5
2.1
1.5
1.5
1.3
2.0
1.5
1.7
1.5
37
5
PhCH2
allyl
n-Bu
Cy
6
62
0c
0c
39
96
62
37
0
3c
88
95
84
77
13
10
Enantioselective Aminolysis of Ethyl 2-Chloropropi-
onate by p-Anisidine at 25 °C. Both water-soluble (Table
10
11
12
3
4
12
t-Bu
25
5
0
a The enzyme encapsulated into sol-gel silica glass. b Water-soluble
fraction of the enzyme. c Estimated from the enantiomeric excess of starting
material after the reaction (see Supporting Information).
Table 2. Enantioselective Aminolysis of Ethyl
2-Chloropropionate by p-Anisidine Catalyzed by C. cylindracea
Lipase at 25 °C
entry
catalyst
time (h)
yield (%)
ee (%)
with the thermal inactivation of enzyme at a higher temper-
ature, as we also found for p-anisidine (see above). Results
with more volatile aliphatic amines (entries 5-12) were
virtually the same at 25 °C and 5 °C.
Good-to-excellent yields of the corresponding amide, with
excellent enantioselectivities, were achieved for p-toluidine,
benzylamine, n-butylamine, and cyclohexylamine. The com-
parison of the experiments in the presence of CcL@glass
and wsCcL clearly demonstrates the superiority of encap-
sulated over free enzyme.
1
2
3
4
5
6
7
8
9
wsCcLa
24
48
310
24
48
197
25
7
16
31
81
89
100
58
67
94
87
83
94
94
88
98
94
93
CcL@glassb
CcL@glassc
44
235
79
a Water-soluble fraction of the enzyme. a The enzyme encapsulated into
sol-gel silica glass. c After 48 h at 25 °C.
Dynamic Kinetic Resolution of Ethyl 2-Chloropropi-
onate into N-p-Methoxyphenyl-2-chloropropionamide via
Aminolysis in the Presence of the Chloride Ion. Most of
2, entries 1-3) and encapsulated (Table 2, entries 4-6) forms
of lipase afford higher yields and higher enantioselectivities
at 25 °C than at 60 °C (Table 1, entries 1-3 and 4,
respectively). Moreover, at 25 °C encapsulated enzyme is a
much more efficient catalyst than the free enzyme (Table 2,
entries 4-6 vs 1-3). When encapsulated lipase was kept at
25 °C for 48 h before addition of the reactants (Table 2,
entries 7-9), the yield declined somewhat, but the enantio-
selectivity remained high.
Scheme 1. Dynamic Kinetic Resolution of Ethyl
2-Chloropropionate via Aminolysis Catalyzed by Encapsulated
C. cylindracea Lipase in the Presence of Triphenylphosphonium
Chloride Immobilized on Merrifield Resin
As Figure 1 shows, the encapsulated enzyme remains quite
efficient after many cycles at 25 °C. In fact, the relative
decline in yield after 10 cycles at 25 °C is still less than that
after just two cycles at 60 °C. Moreover, enantioselectivity
remains very high after 10 cycles at 25 °C. This good
behavior at room temperature makes encapsulated lipase a
convenient catalyst for synthesis.
the experiments described above were carried out under
kinetic resolution conditions, namely, with a 2:1 ratio of
racemic ester to amine. The two main disadvantages of
kinetic resolution are that one-half of the substrate remains
unspent and that enantioselectivity decreases as the reaction
progresses because the slower-reacting enantiomer becomes
dominant in the substrate. The racemic substrate can be
completely converted to a single enantiomer of the product
Enantioselective Aminolysis of Ethyl 2-Chloropropi-
onate by Other Amines. We also studied several other
amines in the aminolysis reaction. The results are shown in
Table 3. Both p-toluidine (entries 1-2) and benzylamine
(entries 3-4) afforded more amide, with better enantio-
selectivity, at 25 °C than at 60 °C. These results are consistent
Org. Lett., Vol. 3, No. 13, 2001
2027

in the presence of a suitable agent, which racemizes the
substrate much more rapidly than the product. Under
dynamic kinetic resolution, shown in Scheme 1, enantio-
selectivity remains constant throughout the reaction.10
The chloride salt of the triphenylphosphonium cation
bound to the Merrifield resin effectively racemizes R-chlo-
roester.11 We prepared this immobilized catalyst by a
published procedure.12
enantiomeric excess is 71%. Comparison of this result to
the results obtained under kinetic resolution conditions (Table
2, entries 4-6) again shows the importance of the reactant
ratio for efficient resolution.
The reaction in the presence of the racemizing agent
proceeds to very high conversion. As Figure 2 shows, the
enantiomeric excess of amide stays constant throughout. The
final yield of amide now is 92%, and its enantiomeric excess
is 86%. The small difference between the initial enantio-
selectivity (94%, in Table 2, entry 1) in the absence and the
constant enantioselectivity (86%) in the presence of the
phosphonium chloride can be attributed to partial racemiza-
tion of the product. Fortunately, racemization of the product
is much slower than that of the substrate. To our knowledge,
this is the first example of dynamic kinetic resolution of a
racemic ester into an amide via aminolysis.
As Figure 2 shows, the reaction in the absence of this
catalyst slows down after reaching ca. 50% conversion; the
C. cylindracea lipase encapsulated into sol-gel silica glass
catalyzes enantioselective aminolysis of an ester. Silica
matrix stabilizes the enzyme and enhances its activity under
the conditions used in our study. Yield and enantioselective
excess of the product, an amide, depend on temperature,
stoichiometry, and the substrate. The reaction yield is further
improved under dynamic kinetic resolution. The method used
in this study can be applied to other enantioselective reactions
catalyzed by enzymes. Encapsulation in sol-gel glasses
preserves chiral discrimination by lipase and extends the
usefulness of this enzyme in organic chemistry.
Figure 2. Enantioselective aminolysis of ethyl 2-chloropropionate
by an equimolar amount of p-anisidine catalyzed by encapsulated
C. cylindracea lipase in the presence (2) and in the absence (b)
of the racemizing agent, triphenylphosphonium chloride im-
mobilized on Merrifield resin. Reaction mixtures were sampled after
3, 6, 12, 18, 24, 36, 48, 72, 126, 192, 240, and 312 h. The 12
points in each set correspond to the incremental increase in
conversion from left to right.
Acknowledgment. We thank Prof. Jonathan M. J. Wil-
liams for a helpful suggestion and Dr. Kamel Harrata for
help with optimization of chiral GC-MS conditions. This
research was supported by the National Science Foundation.
enantiomeric excess of the product also declines sharply
around this point. The final yield of amide is 70%, and its
Supporting Information Available: Experimental pro-
cedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
(10) (a) Strauss, U. T.; Felfer, U.; Faber, K. Tetrahedron: Asymmetry
1999, 10, 107-117. (b) Stecher, H.; Faber, K. Synthesis 1997, 1-16.
(11) Haughton, L.; Williams, J. M. J. Abstracts of Papers, 220th National
Meeting of the American Chemical Society, Washington, DC, August 2000;
American Chemical Society: Washington, DC, 2000; ORGN-112.
OL015989O
(12) Zheng, A.; Shan, D.; Shi, X.; Wang, B. J. Org. Chem. 1999, 64,
7459-7466.
2028
Org. Lett., Vol. 3, No. 13, 2001