Synthesis of lactams using enzyme-catalyzed aminolysis

Article abstract of DOI:10.1016/j.tetlet.2012.10.133

The formation of caprolactam from 6-aminocaproic acid catalyzed by CALB (N435) is reported. Different lactam ring sizes can be prepared starting from 4-aminobutanoic acid, 5-aminovaleric acid, and 8-aminooctanoic acid. Experiments with mixtures of aminocarboxylic acids have shown that CALB prefers homocyclization of the individual aminocarboxylic acids.

Full text of DOI:10.1016/j.tetlet.2012.10.133

Tetrahedron Letters 54 (2013) 370–372
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
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Synthesis of lactams using enzyme-catalyzed aminolysis
⇑
E. Stavila, K. Loos
Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The formation of e-caprolactam from 6-aminocaproic acid catalyzed by CALB (N435) is reported. Different
Received 15 September 2012
Revised 12 October 2012
Accepted 25 October 2012
Available online 21 November 2012
lactam ring sizes can be prepared starting from 4-aminobutanoic acid, 5-aminovaleric acid, and 8-
aminooctanoic acid. Experiments with mixtures of aminocarboxylic acids have shown that CALB prefers
homocyclization of the individual aminocarboxylic acids.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Aminocarboxylic acid
Lactam
Ring-closure
Lipase
Lactams are widely used monomers for the synthesis of polya-
mides/Nylons, which are polymeric materials used extensively in
our daily life. Industrial synthesis of most lactams involves step-
wise reactions using aggressive chemicals and high temperatures.1
However, biocatalytic approaches might be superior alternative
synthetic routes that can be performed under mild reaction condi-
tions. Currently, there is significant interest in the development of
There are no reports on the in vitro cyclization of aminocarbox-
ylic acids of any length catalyzed by CALB. Therefore, in this study,
we present the enzymatic formation of
e-caprolactam from 6-
aminocaproic acid using CALB as the catalyst. Enzymatic syntheses
were carried out in concentrated and dilute systems.8 We opti-
mized the reaction conditions to obtain e-caprolactam in a high
yield. Furthermore, other aminocarboxylic acids such as b-alanine,
4-aminobutanoic acid (GABA), 5-aminovaleric acid, 8-aminooctanoic
acid, and 12-aminododecanoic acid were used to synthesize
lactams of different ring sizes (Scheme 1).
milder and more efficient methods to prepare e-caprolactam and
other lactams, for example, by using enzymatic catalysis. Reactions
using enzymes are more environmentally friendly. Enzymes are
non-toxic natural catalysts and reactions using enzymes can be
carried out under mild conditions.2
In both the concentrated9 and dilute systems,10
e-caprolactam
was successfully synthesized from 6-aminocaproic acid via intra-
The most commonly used biocatalyst in esterification or
transesterification reactions and polymerizations3 is immobilized
Candida antarctica lipase B (CALB), commercially available as
N435. CALB is extremely useful due to its stability at high temper-
atures and its acceptance of various substrates.4,3a CALB has a
broad specificity toward straight chain fatty acids from butanoic
acid to octadecanoic acid. On the other hand, CALB shows high
activity toward carboxylic acids shorter than hexanoic acid.5
Lactams have previously been synthesized via enzymatic pro-
molecular aminolysis catalyzed by N435. In the concentrated sys-
tem, a low conversion (14%) for the formation of e-caprolactam
was observed (calculated from the 1H NMR spectrum in D2O). Fur-
thermore, no oligomers were observed in the 1H NMR spectrum in
TFA-d of the reaction mixture. In the N435-catalyzed reaction
using 8-aminooctanoic acid, in the concentrated system, no lac-
tams or oligomers were formed. Therefore, it can be concluded that
aminocarboxylic acids cannot be used as monomers for
polymerization.
cesses from aliphatic
a
,x
-dinitriles6 and amino esters.7 In the lat-
In the dilute system, enzymatic formation of e-caprolactam oc-
ter case the cyclization of amino esters catalyzed by crude
pancreatic porcine lipase to form five-, six-, and seven-membered
lactams was reported. However, in that study, the formation of se-
ven membered rings proceeded slowly with low conversion,
resulting in only 10% enzymatic conversion after 7 days.
curred with a higher conversion (40%) compared with the concen-
trated system. This is in accordance with the theory of Jacobson
and Stockmayer11 that increased dilution increases ring formation.
To increase the formation of amide bonds (yield) the reaction
conditions were optimized (water content of the solvent and the
enzyme, etc.). The water content of N435 was reduced by drying
under vacuum for 24–96 h at 55 °C, over P2O5. The dried N435
was subsequently used in the enzymatic formation of e-caprolactam
at 55 and 90 °C for 3 days. The highest conversions were obtained
with N435 that had been dried for 24 h and employing a reaction
⇑
Corresponding author. Tel.: +31 50 363 6867.
E-mail address: k.u.loos@rug.nl (K. Loos).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.10.133

E. Stavila, K. Loos / Tetrahedron Letters 54 (2013) 370–372
371
O
O
CALB
toluene
m
H2N
n
OH
m
m
H2O
+
HN
n
aminocarboxylic acids
n = 1 - 4
lactam
Scheme 1. Cyclization of aminocarboxylic acids catalyzed by CALB in toluene.
temperature of 90 °C for the e-caprolactam synthesis. Drying the
enzyme for more than 24 h decreased the synthetic activity of
N435 as shown in Figure 1. This reduction in activity is probably
due to partial denaturation and to excessive removal of water from
the enzyme; it is known that a certain amount of water is neces-
sary to keep enzymes catalytically active.12
The formation of e-caprolactam increased from 3 to 5 day reac-
tion time; after this time, the conversion decreased (see Fig. 2). The
optimal conditions are therefore reactions for 5 days at 90 °C using
dried N435 under a nitrogen atmosphere.
Control reactions13 were carried out to determine whether for-
Figure 2. Influence of the reaction time on the formation of e-caprolactam.
mation of
the absence of CALB. The control reaction, which contained only 6-
aminocaproic acid showed no formation of -caprolactam after
e-caprolactam from 6-aminocaproic acid also occurred in
Table 1
Lactam formation using different aminocarboxylic acids
e
5 days stirring under exactly the same reaction conditions. Reac-
Aminocarboxylic acid
Conversion (%)
tions using deactivated N435 resulted in an 11% conversion into
Control reactiona
Reaction with N435a
e
-caprolactam. From activity tests,13b we know that N435 cannot
be deactivated completely by heat treatment for 4 or 24 h at
150 °C (Table S1 in Supplementary data), as N435 still showed
residual transesterification and synthetic activity after this treat-
ment. Therefore, we also tested macroporous acrylic polymer
beads without immobilized enzymes (identical to the N435 resins)
for control reactions. From these it became obvious that the carrier
4-Aminobutanoic acid
5-Aminovaleric acid
6-Aminocaproic acid
8-Aminooctanoic acid
0
6
0
0
30
70
83
41
a
Reaction was carried out in toluene at 90 °C for 5 days.
for N435 did not catalyze the formation of
e
-caprolactam. Thus we
Unlike the formation of
e-caprolactam, the formation of d-
can conclude that the formation of
to the synthetic activity of CALB.
e-caprolactam occurs only due
valerolactam can occur in the absence of N435, as d-valerolactam
was formed in the control reaction without enzyme. However,
addition of N435 to the reaction increased the conversion of d-
valerolactam up to 11 times compared to the reaction without
enzymes, which makes the enzymatic route a superior alternative
to the chemical synthesis.
For reactions using 8-aminooctanoic acid as the substrate, N435
did not catalyze the formation of caprylolactam. Instead, the corre-
sponding dimer and trimer lactams were formed,15 see Scheme 2.
The formation of macrocyclic bislactams from reactions between
diesters and diamines using porcine pancreatic lipase as the cata-
lyst was previously reported.7 However, in the enzymatic reaction
of 8-aminooctanoic acid using N435, different macrocyclic dimer
and trimer lactams were formed in a ratio of 3:2. The ratio was
calculated from the mass spectrometric peak intensities of the
product.
Besides 6-aminocaproic acid, we found that N435 was able to
catalyze the formation of five- and six-membered lactams with
good conversions from 4-aminobutanoic acid and 5-aminovaleric
acid,14 as shown in Table 1. We found that N435 did not catalyze
the formation of a lactam from b-alanine and aminocarboxylic
acids longer than 8-aminooctanoic acid, such as 12-aminododeca-
noic acid. These results are in agreement with previous reports, in
which N435 showed poor activity in esterification reactions with
straight chain fatty acids shorter than butanoic acid and longer
than octanoic acid.5
n
HN
O
O
NH
dimer
O
CALB
toluene
n
H2N
n
H2O
+
OH
7
8-aminooctanoic acid
O
O
HN
HN
n
NH
O
trimer
Figure 1. Influence of drying N435 at 55 °C under vacuum over P2O5 for 0–96 h on
Scheme 2. Formation of macrocyclic dimer and trimer lactams catalyzed by CALB
the enzymatic formation of
e-caprolactam at 55 and 90 °C.
in toluene.

372
E. Stavila, K. Loos / Tetrahedron Letters 54 (2013) 370–372
2008, 29, 794–797; (d) Schwab, L. W.; Baum, I.; Gregor, F.; Loos, K. Green
Reactions between two different aminocarboxylic acids were
Polymer Chemistry: Biocatalysis and Biomaterials In Vol. 1043; wiley, 2010. pp
265–278; (e) Biocatalysis in Polymer Chemistry; Loos, K. Ed.; Wiley-VCH, 2010;
(f) Baum, I.; Elsässer, B.; Schwab, L. W.; Loos, K.; Fels, G. ACS Catal 2011, 1, 323–
336; (g) Schwab, L. W.; Kloosterman, W. M. J.; Konieczny, J.; Loos, K. Polymers
2012, 4, 710–740; (h) Schwab, L.W.; Kloosterman, W. M. J.; Konieczny, J.; Loos,
K.; Journal of Renewable Materials 2012. http://dx.doi.org/10.7569/
JRM.2012.634102.
performed to determine whether mixed macrocyclic lactams could
be formed as well.16 However, instead of forming macrocycles
comprised of the two aminocarboxylic acids, in all cases, mixtures
of two or three different lactams were formed. In fact, the products
resemble the lactams formed in the reactions with individual ami-
nocarboxylic acids as outlined above. The products were formed in
a ratio of 1:1 (calculated from the 1H NMR spectra of the crude
products) which perfectly matched the feed ratio. Thus it is obvi-
ous that CALB has a preference for homocyclization of the individ-
ual aminocarboxylic acids.
Other enzymes such as CALA (immobilized on immobead 150
and in form of CLEA) and cutinase in the form of CLEA were tested
for the synthesis of homocyclic and macrocyclic lactams as well.
However, these enzymes did not catalyze the cyclization reactions
and no lactams were formed.
4. (a) Gotor-Fernández, V.; Busto, E.; Gotor, V. Adv. Synth. Catal. 2006, 348, 797–
812; (b) Rejasse, B.; Lamare, S.; Legoy, M.-D.; Besson, T. Org. Biomol. Chem.
2004, 2, 1086–1089.
5. Kirk, O.; Björkling, F.; Godtfredsen, S. E.; Larsen, T. O. Biocatal. Biotransform.
1992, 6, 127–134.
6. (a) Di Cosimo, R.; Fallon, R. D.; Gavagan, J. E.; Herkes, F. E. U.S. Patent 5,814,508,
1998; Chem. Abstr. 1998, 128, 48589.
7. Gutman, A. L.; Meyer, E.; Yue, X.; Abell, C. Tetrahedron Lett. 1992, 33, 3943–
3946.
8. Enzymatic syntheses of lactams were carried out in concentrated (ꢀ1 M) and
dilute systems.
9. Synthesis of lactams in concentrated systems (ꢀ1 M):
A mixture of
aminocarboxylic acid (2.5 mmol) (6-aminocaproic acid or 8-aminooctanoic
acid), N435 (100 mg), and dry toluene (2.5 mL) was stirred for 120 h at 90 °C
under an N2 atmosphere. Subsequently, the toluene was removed by rotary
evaporation. An aliquot from the crude reaction mixture was analyzed by 1H
NMR in D2O or in TFA-d.
In summary, this study has shown that dried CALB is able to cat-
alyze the cyclization of 6-aminocaproic acid to
moderate yields of up to 60% after 5 days. The anhydrous reaction
conditions play a crucial role in increasing the yield of -caprolactam
formation. Other lactams such as butyrolactam, d-valerolactam,
and dimer and trimer lactams can be formed from enzymatic
reactions of 4-aminobutanoic acid, 5-aminovaleric acid, and
8-aminooctanoic acid using N435 as the catalyst. Reactions with
mixtures of the individual aminocarboxylic acids showed that
CALB prefers homocyclization of the aminocarboxylic acids thus
preventing the formation of mixed macrocyclic lactams.
e-caprolactam in
10. (a) Synthesis of lactams in dilute systems: A mixture of 6-aminocaproic acid
(100 mg), N435 (100 mg), and dry toluene (5 mL) was stirred for 72–120 h, at
90 °C under an N2 atmosphere. After cooling to room temperature, the toluene
was removed by rotary evaporation. CHCl3 (20 mL) was added to the residue to
extract the lactam product. After filtration and evaporation of the CHCl3, the
e
remaining product (e-caprolactam) was purified using short path distillation
(Kugelrohr); yielding a white crystalline product (60%); mp 68.68 °C (Lit. 68–
71 °C).
(b) e
-Caprolactam (yield 60%); 1H NMR (400 MHz, CDCl3): d = 5.89 (s, 1H, NH);
3.21 (dd, J = 9.67, 5.69 Hz, 2H, CH2); 2.47 (m, 2H, CH2); 1.70 (m, 6H, CH2).
(C6H11NO) (113.16): Calcd C 63.68, H 9.80, N 12.38. Found C 63.60, H 9.97, N
12.33.
Acknowledgements
11. (a) Jacobson, H.; Stockmayer, W. H. J. Chem. Phys. 1950, 18, 1600–1606; (b)
Jacobson, H.; Beckmann, C. O.; Stockmayer, W. H. J. Chem. Phys. 1950, 18, 1607–
1612.
The authors wish to thank the Ubbo Emmius Programme of the
University of Groningen for financial support, CLEA Technology
(Delft, The Netherlands) for supplying CALA in the form of CLEA,
Novozymes (Denmark) for supplying Fusarium solani pisi cutinase,
and ChiralVison (Leiden, The Netherlands) for supplying the mac-
roporous acrylic polymer beads.
12. Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141–144.
13. (a) Control reactions were conducted without enzyme, by stirring the
aminocarboxylic acid in toluene for 72–120 h at 90 °C under an N2
atmosphere. Using the same procedure, other control reactions were
performed using deactivated N435 and macroporous acrylic polymer beads.
(b) The determination of the activity from deactivated N435 was accomplished
by the transesterification of 4-nitrophenyl acetate (transesterification assay)
and by enzymatic ring-opening polymerization of e-caprolactone (synthetic
activity assay).3c These assays are required to determine whether there is any
residual activity of N435 left after the thermal deactivation process. N435 was
Supplementary data
deactivated by heating at 150 °C under vacuum for
4 and 24 h, and
subsequently used in the control reactions.
Supplementary data (1H NMR spectra, ESI-MS chromatogram,
and synthetic and transesterification procedures) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.tetlet.2012.10.133.
14. (a) Using similar procedures, b-alanine, 4-aminobutanoic acid, 5-aminovaleric
acid, 8-aminooctanoic acid, and 12-aminododecanoic acid were used to
synthesize the corresponding lactams.
(b) Butyrolactam (conversion 30%); 1H NMR (400 MHz, CDCl3): d = 6.32 (s, 1H,
NH); 3.39 (t, J = 6.94 Hz, 2H, CH2); 2.29 (t, J = 8.07 Hz, 2H, CH2); 2.13 (m, 2H,
CH2).
(c) d-Valerolactam (conversion 70%); 1H NMR (400 MHz, CDCl3): d = 6.54 (s, 1H,
NH); 3.29 (t, J = 5.95 Hz, 2H, CH2); 2.34 (t, J = 6.46 Hz, 2H, CH2); 1.76 (m, 4H,
CH2).
References and notes
15. Dimer and trimer lactams (conversion 41%); 1H NMR (400 MHz, CDCl3):
d = 5.43 (s, 2H, NH); 3.30 (dd, J = 12.36, 6.09 Hz, 4H, CH2); 2.16 (m, 4H, CH2);
1.63 (m, 4H, CH2); 1.47 (m, 4H, CH2); 1.30 (m, 12H, CH2). MS (ESI) dimer: m/
z = 283.24 [M+H]+ and 305.22 [M+Na]+; trimer: m/z = 424.35 [M+H]+ and
446.33 [M+Na]+.
1. (a) Weissermel, K.; Arpe, H.-J. Industrial Organic Chemistry; Wiley VCH GmbH,
2007. pp 237–264; (b) Bellussi, G.; Perego, C. CATTECH 2000, 4, 4–16.
2. (a) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B.
Nature 2001, 409, 258–268; (b) Cheng, H. N. Biocatalysis in Polymer Chemistry;
Wiley VCH GmbH & Co. KGaA, 2010. pp 131–141.
3. (a) Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal. Biotransform. 1998, 16,
181–204; (b) Gross, R. A.; Kumar, A.; Kalra, B. Chem. Rev. 2001, 101, 2097–2124;
(c) Schwab, L. W.; Kroon, R.; Schouten, A. J.; Loos, K. Macromol. Rapid Commun.
16. The syntheses of macrocyclic lactams were performed using two different
aminocarboxylic acids in equimolar amounts (0.485 mmol in 5 mL of toluene).