Penicillin acylase in the industrial production of β-lactam antibiotics

Article abstract of DOI:10.1021/op9700643

Immobilized penicillin acylase is a biocatalyst suitable for the kinetically controlled industrial synthesis of semi-synthetic antibiotics in aqueous environments. Amoxiciliin and ampicillin are obtained by condensing 6-aminopenicillanic acid with the amide or ester of D-( - )-4-hydroxyphenyIglycine and D-( - )phenylglycine, respectively. Similarly, the cephalosporin antibiotics cefadroxil and cephalexin can be obtained from 7-aminodesacetoxycephalosporanic acid.

Full text of DOI:10.1021/op9700643

Organic Process Research & Development 1998, 2, 128−133
Penicillin Acylase in the Industrial Production of â-Lactam Antibiotics
Alle Bruggink,† Eric C. Roos,‡ and Erik de Vroom*,§
Chemferm, P.O. Box 3933, 4800 DX Breda, The Netherlands, DSM Research, P.O. Box 18, 6160 MD Geleen,
The Netherlands, and Gist-brocades B.V., P.O. Box 1, 2600 MA Delft, The Netherlands
Abstract:
cephalosporins, SSCs, e.g., cefadroxil and cephalexin) is
outlined in Scheme 1. In the traditional chemical approach,
the fermentation product penicillin G is deacylated to
6-aminopenicillanic acid (6-APA) according to a procedure
based on the formation of an imide chloride.1 This procedure
adequately discriminates between the secondary amide to be
hydrolyzed and the highly labile tertiary amide of the
â-lactam ring. On the other hand, 7-aminodesacetoxycepha-
losporanic acid (7-ADCA), an intermediate used in the
production of two of the best-selling SSCs, can be obtained
by oxidative ring expansion of penicillin G2 followed by a
similar deacylation. Subsequently, 6-APA and 7-ADCA are
transformed into SSPs and SSCs respectively by condensa-
tion with a D-(-)-phenylglycine or a D-(-)-4-hydroxyphe-
nylglycine derivative. Two of the most widely used ap-
proaches are Schotten-Baumann condensation with the acid
chloride and mixed anhydride activated condensation of the
Dane salt of the side chain.3
In the recent past, many companies have replaced chemi-
cal side chain hydrolysis, requiring hazardous chemicals and
solvents such as phosphorus pentachloride and dichlo-
romethane, by penicillin acylase catalyzed hydrolysis in an
aqueous environment. Although microbial conversion of
penicillin G into 6-APA has been known for almost five
decades,4 industrial application of the enzyme involved,
penicillin acylase (penicillin amidase, penicillin amidohy-
drolase, EC 3.5.1.11), has been introduced successfully only
in the past 8 years. This is primarily a result of the fact that
efficient enzyme production and recovery was unavailable
a decade ago. Generally, two types of enzymes can be
distinguished. The penicillin acylases, of bacterial or fungal
origin, display a preference for phenylacetic acid or phe-
noxyacetic acid and their derivatives bearing small substit-
uents on the R-position (amino, hydroxy, methyl, chloro)
and in the aromatic ring. Also other aromatic acetic acid
derivatives are recognized.5 Penicillin acylase is widely
distributed among bacteria, yeast, and filamentous fungi. The
enzyme is a heterodimer with a 20.5-kDa R-subunit and a
69-kDa â-subunit.6 The crystal structure of Escherichia coli
penicillin acylase at 1.9-Å resolution indicates the catalyti-
cally active centre to be the N-terminal serine residue of the
Immobilized penicillin acylase is a biocatalyst suitable for the
kinetically controlled industrial synthesis of semi-synthetic
antibiotics in aqueous environments. Amoxicillin and ampicillin
are obtained by condensing 6-aminopenicillanic acid with the
amide or ester of
D-(-)-4-hydroxyphenylglycine and
D-(-)-
phenylglycine, respectively. Similarly, the cephalosporin an-
tibiotics cefadroxil and cephalexin can be obtained from
7-aminodesacetoxycephalosporanic acid.
Introduction
The industrial production of â-lactam antibiotics and their
intermediates is undergoing a remarkable transformation.
Traditional chemical conversions based on stoichiometry are
being replaced by enzyme-catalyzed processes. Still, the
starting materials for these transformations are fermentatively
obtained chiral structures such as cephalosporin C, penicillin
G, and penicillin V. The ever-increasing insight into
biochemical pathways and the resulting optimization of
fermentation processes will make this application of the chiral
pool even more profitable in the years to come.
Currently, the majority of N-deacylations in â-lactam
production processes is carried out enzymatically using
enzymes of the group of penicillin acylases. Present
developments indicate that the same enzymes can also be
exploited successfully in a synthetic fashion. Consequently,
processes for the large-scale enzyme-catalyzed production
of valuable antibiotics such as amoxicillin, ampicillin,
cefaclor, cefadroxil, and cephalexin, based on the condensa-
tion of the appropriate D-(-)-amino acid derivative with a
â-lactam nucleus, are in a well-advanced stage.
In this paper, an overview is given of the current status
of the development of environmentally benign production
processes for â-lactam antibiotics. Illustrative examples are
from our own efforts in the area of enzyme-catalyzed
condensation reactions, which is part of a programme of
Chemferm, the joint venture between DSM, producer of
chiral intermediates, and Gist-brocades, manufacturer of
â-lactam products.
Background
(1) Weissenburger, H. W. O.; van der Hoeven, M. G. Recl. TraV. Chim. Pays-
Bas 1970, 89, 1081.
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1975, 40, 1346.
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(5) Van der Mey, M.; de Vroom, E. BioMed. Chem. Lett. 1994, 4, 345.
(6) Bo¨ck, A.; Wirth, R.; Schmid, G.; Schumacher, G.; Lang, G.; Buckel, P.
FEMS Microbiol. Lett. 1983, 20, 135.
A general production chart of penicillin-derived antibiotics
(semi-synthetic penicillins, SSPs, e.g., amoxicillin and ampi-
cillin) and cephalosporin-derived antibiotics (semi-synthetic
† Chemferm.
‡ DSM Research.
§ Gist-brocades B.V.
128
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Vol. 2, No. 2, 1998 / Organic Process Research & Development
S1083-6160(97)00064-9 CCC: $15.00 © 1998 American Chemical Society and Royal Society of Chemistry
Published on Web 02/28/1998

Scheme 1
Kinetically Controlled Enzyme-Catalyzed Synthesis
Use of penicillin acylase as catalyst in the synthetic
direction was first demonstrated in 1960 by Kaufman and
Bauer, who reported the E. coli penicillin acylase catalyzed
formation of penicillin G from 6-APA and phenylacetic
acid.10 Since then, many more examples have been published
in the scientific literature, of which those leading to
therapeutically useful products are summarized in Table 1.
The number of patent applications on the subject is at least
as large.
(9) Alfani, F.; Cantarella, M.; Cutarella, N.; Gallifuoco, A.; Golini, P.; Bianchi,
D. Biotechnol. Lett. 1997, 19, 175.
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Agric. Biol. Chem. 1983, 47, 2503.
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30, S230.
(13) Kato, K.; Kawahara, K.; Takahashi, T.; Igarasi, S. Agric. Biol. Chem. 1980,
44, 821.
(14) Marconi, W.; Bartoli, F.; Cecere, F.; Galli, G.; Morisi, F. Agric. Biol. Chem.
1975, 39, 277.
(15) Okachi, R.; Hashimoto, Y.; Kawamori, M.; Katsumata, R.; Nara, T. Enzyme
Eng. 1982, 6, 81.
(16) Okachi, R.; Kato, F.; Miyamura, Y.; Nara, T. Agric. Biol. Chem. 1973, 37,
1953.
(17) Blinkovsky, A. M.; Markaryan, A. N. Enzyme Microb. Technol. 1993, 15,
965.
(18) Cole, M. Biochem. J. 1969, 115, 757.
(19) Kasche, V.; Haufler, U.; Riechmann, L. Ann. N.Y. Acad. Sci. 1984, 434,
99.
(20) Kasche, V.; Haufler, U.; Zo¨llner, R. Hoppe-Seyler’s Z. Physiol. Chem. 1984,
365, 1435.
(21) Kasche, V. Biotechnol. Lett. 1985, 7, 877.
(22) Boccu`, E.; Ebert, C.; Gardossi, L.; Gianferrara, T.; Zacchigna, M.; Linda,
P. Farmaco 1991, 46, 565.
(23) Ospina, S.; Barzana, E.; Ram´ırez, O. T.; Lo´pez-Mungu´ı, A. Enzyme Microb.
Technol. 1996, 19, 462.
â-subunit.7 In contrast with other serine proteases, penicillin
acylase does not appear to have a histidine residue in the
vicinity of the active site that may act as base in the catalytic
process. A more narrow substrate specificity is found in a
second class of enzymes, the R-amino acid ester hydrolases,
found in Acetobacter and Xanthomonas species.8 As the
name suggests, substrate specificity requires the presence of
an amino group R to the carboxylic acid function.
In the production of cephalosporin antibiotics, not only
is the above-mentioned penicillin G ring expansion strategy
applied but also the cephalosporin nucleus can be obtained
from the fermentation product cephalosporin C. However,
the application of enzyme catalysis in the hydrolysis of the
R-aminoadipyl side chain in cephalosporin C to give 7-ami-
nocephalosporanic acid (7-ACA) is less well developed since
extensive screening has not provided an enzyme capable of
hydrolyzing the R-aminoadipyl side chain. Presently, a two-
enzyme process can accommodate the removal of this side
(24) Kim, M. G.; Lee, S. B. J. Mol. Catal. B 1996, 1, 181.
(25) Kim, M. G.; Lee, S. B. J. Mol. Catal. B 1996, 1, 201.
(26) Nara, T.; Okachi, R.; Misawa, M. J. Antibiot. 1971, 24, 321.
(27) Okachi, R.; Misawa, M.; Deguchi, T.; Nara, T. Agric. Biol. Chem. 1972,
36, 1193.
(28) Shimizu, M.; Masuike, T.; Fujita, H.; Kimura, K.; Okachi, R.; Nara, T.
Agric. Biol. Chem. 1975, 39, 1225.
(29) Fuganti, C.; Rosell, C. M.; Rigoni, R.; Servi, S.; Tagliani, A.; Terreni, M.
Biotechnol. Lett. 1992, 14, 543.
(30) Baldaro, E. M.; Kostadinov, M.; Nikolov, A.; Tsoneva, N.; Petkov, N. Appl.
Biochem. Biotechnol. 1992, 33, 177.
(31) Fernandez-Lafuente, R.; Guisa´n, J. M.; Pregnolato, M.; Terreni, M.
Tetrahedron Lett. 1997, 38, 4693.
(32) Takahashi, T.; Yamazaki, Y.; Kato, K.; Isono, M. J. Am. Chem. Soc. 1972,
94, 4035.
(33) Takahashi, T.; Yamazaki, Y.; Kato, K. Biochem. J. 1974, 137, 497.
(34) Kim, I. H.; Nam, D. H.; Ryu, D. D. Y. Appl. Biochem. Biotechnol. 1983,
8, 195.
(35) Rhee, D. K.; Lee, S. B.; Rhee, J. S.; Ryu, D. D. Y.; Hospodka, J. Biotechnol.
Bioeng. 1980, 22, 1237.
(36) Choi, W. G.; Lee, S. B.; Ryu, D. D. Y. Biotechnol. Bioeng. 1981, 23, 361.
(37) Nam, D. H.; Kim, C.; Ryu, D. D. Y. Biotechnol. Bioeng. 1985, 27, 953.
(38) Hyun, C. K.; Kim, J. H.; Kim, Y. J. Biotechnol. Lett. 1989, 11, 537.
(39) Hyun, C. K.; Choi, J. H.; Kim, J. H.; Ryu, D. D. Y. Biotechnol. Bioeng.
1993, 41, 654.
(40) Dharmarajan, T. S.; Deshpande, J. V.; Divekar, K. Indian J. Pharm. Sci.
1994, 56, 126.
(41) Varga, M.; Nys, P. S.; Frank, J. Biocatalysis 1994, 11, 315.
(42) Konecny, J.; Sieber, M.; Schneider, A. Biotechnol. Lett. 1981, 3, 507.
(43) Fumian, C.; Lizhao, Z.; Wenzhen, H.; Min, W.; Zhenxiang, W. Acta
Microbiol. Sinica 1984, 24, 376.
chain.
A D-amino acid oxidase (EC 1.4.3.3) catalyzes
oxidative deamination, the resulting R-keto acid spontane-
ously loses carbon dioxide in the presence of hydrogen
peroxide, and finally the resulting glutaryl side chain is
hydrolyzed using a glutaryl acylase.9
(44) Maladkar, N. K. Enzyme Microb. Technol. 1994, 16, 715.
(45) Fujii, T.; Matsumoto, K.; Watanabe, T. Process Biochem. 1976, 21.
(46) Fernandez-Lafuente, R.; Rosell, C. M.; Piatkowska, B.; Guisa´n, J. M.
Enzyme Microb. Technol. 1996, 19, 9.
(7) Duggleby, H. J.; Tolley, S. P.; Hill, C. P.; Dodson, E. J.; Dodson, G.;
Moody, P. C. E. Nature 1995, 373, 264.
(47) Fernandez-Lafuente, R.; Alvaro, G.; Blanco, R. M.; Guisa´n, J. M. Appl.
Biochem. Biotechnol. 1991, 27, 277.
(8) Kato, K.; Kawahara, K.; Takahashi, T.; Kakinuma, A. Agric. Biol. Chem.
1980, 44, 1075.
(48) Fernandez-Lafuente, R.; Rosell, C. M.; Guisa´n, J. M. Biotechnol. Appl.
Biochem. 1996, 24, 139.
Vol. 2, No. 2, 1998 / Organic Process Research & Development
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129

Table 1. Enzymatic synthesis of â-lactam antibiotics
Scheme 3. Kinetically-controlled synthesisa
product
enzymea
source
ref
amoxicillin
amoxicillin
amoxicillin
amoxicillin
ampicillin
ampicillin
ampicillin
ampicillin
cefadroxil
cefadroxil
cefamandole
cefazolin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephalexin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephaloglycin
cephalothin
cephalothin
AH
AH
PA
PA
AH
AH
PA
PA
AH
PA
PA
PA
AH
AH
AH
AH
AH
AH
AH
AH
AH
AH
PA
PA
PA
?
AH
AH
AH
AH
AH
AH
AH
AH
AH
PA
PA
AH
PA
Pseudomonas melanogenum 11
X. citri
E. coli
12, 13
14
15
14, 16
17
13, 18-25
14, 26-28
14
KluyVeromyces citrophila
Ps. melanogenum
Xanthomonas sp.
E. coli
K. citrophila
Ps. melanogenum
E. coli
13
E. coli
E. coli
A. pasteurianum
Acetobacter turbidans
29
30, 31
32
11, 32, 33
Gluconobacter suboxydans 32
Microbacterium dimorpha 32
a The substrate SA is an amino acid amide (RCONH2) or an amino acid ester
(RCO2R′). In contrast with equilibrium-controlled synthesis (Scheme 2), the
enzyme-substrate complex can be formed. Unfortunately, this complex is subject
not only to synthesis (attack of NH to give product SN) but also to hydrolysis
(attack of water to give unreactive amino acid SOH).
Proteus alboflaVus
Ps. melanogenum
X. citri
Xanthomonas compestris
Xanthomonas oryzae
Xanthomonas sp.
Bacillus megaterium
K. citrophila
32
32
11, 32, 34-39
40
32
16, 41
42, 43
14, 28
11, 13, 44
45
32
32
this is done, a reactive substrate is obtained that will have
its amino group partly uncharged at pH values that are
optimal for the enzyme (i.e., 6-8). In biological systems,
the energy required for this activation is delivered by ATP;
in industrial processes, chemical activation is needed for the
conversion. Once an amide or ester (SA) is available,
kinetically controlled formation of product SN can take place,
accompanied by undesired processes such as hydrolysis of
SA and SN to inactive SOH. The molar ratio of product
SN to SOH is referred to as synthesis/hydrolysis ratio (S/H
ratio) and is a function of reaction conditions and biocatalyst
properties and formulation. The mechanism of kinetically
controlled synthesis (Scheme 3) was established by Nam et
al.38 for the Xanthomonas citri catalyzed formation of
cephalexin from D-(-)-phenylglycine methyl ester (D-(-)-
PGM) and 7-ADCA and holds equally well for other
products such as amoxicillin, ampicillin, cefaclor, and
cefadroxil.
E. coli
Achromobacter sp.
A. pasteurianum
A. turbidans
G. suboxydans
M. dimorpha
Pr. alboflaVus
Ps. melanogenum
X. citri
32
32
32
32
32
32
X. oryzae
Xanthomonas sp.
E. coli
K. citrophila
16
46
28
K. citrophila
E. coli
47
46, 48
a AH: R-amino acid ester hydrolase. PA: penicillin acylase.
Scheme 2a
The early work on enzymatic synthesis at Gist-brocades
focused on the use of R-amino acid ester hydrolases from
Acetobacter pasteurianum and X. citri in the synthesis of
ampicillin (2a in Scheme 4) from 6-APA and PGM. First
of all, it was found that ampicillin synthesis proceeds
particularly smoothly at low temperatures (i.e., 5 °C), giving
a conversion of 88% based on 6-APA when optically pure
D-(-)-PGM was used. Secondly, ampicillin could also be
obtained from the racemic form of PGM (77% conversion,
ee 69%).51 Although the ee values reached are remarkable
compared to those for the penicillin acylases, where ste-
reospecificity for the R-position of the side chain is only
moderate, the idea of introducing optical activity in the side
chain during the condensation reaction was eventually
abandoned. For economic reasons, asymmetry should be
introduced in a production pathway as early as possible so
as to avoid the transport of isomeric ballast throughout the
a Both the substrate SOH and the nucleophile NH are amino acids. In SOH
the carboxylic acid takes part in the reaction with the amino group of NH.
However, the favoured uncharged form of SOH (RCO2H) can only be achieved
with concurrent protonation of its amino group. The molecule thus obtained
will not be recognized by the enzyme.
Enzyme-catalyzed condensation processes can, in prin-
ciple, be carried out under equilibrium control or under
kinetic control. In the first case (Scheme 2), the enzyme
catalyzes the establishment of the thermodynamic equilibrium
between, on the one hand, substrate (SOH, i.e., an amino
acid) and nucleophile (NH, i.e., a â-lactam nucleus) and, on
the other hand, the product (SN, i.e., a semisynthetic â-lactam
antibiotic) and water. The known penicillin acylases do not
accept charged amino functions. If phenylglycine is to serve
as substrate, the carboxyl function should be uncharged.
However, when this situation is achieved, the amino group
will be charged and enzymatic catalysis is not possible.49,50
In kinetically controlled synthesis, non-equilibrium con-
centrations of the product SN can be reached by activation
of the substrate SOH to an amide, ester, or anhydride. When
(49) Schroe¨n, C. G. P. H.; Nierstrasz, V. A.; Kroon, P. J.; Bosma, R.; Janssen,
A. E. M.; Beeftink, H. H.; Tramper, J. Submitted to Enzyme Microb.
Technol.
(50) Diender, M. B.; Straathof, A. J. J.; van der Wielen, L. A. M.; Ras, C.;
Heijnen, J. J. Submitted to J. Mol. Catal. B: Enzym.
(51) Van der Laken, C. J. Eur. Patent Appl. EP 339,751, 1989.
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Scheme 4
Scheme 5
Enzyme-Catalyzed Synthesis of D-(-)-Phenylglycine-Der-
ived Antibiotics
Table 2. Influence of carrier composition on the S/H ratio in
the enzymatic synthesis of ampicillin by immobilized E. coli
penicillin acylasea
Cephalexin. The first successful application of the
catalyst mentioned above was achieved in the production of
cephalexin (4b in Scheme 5) from 7-ADCA and D-(-)-PGA
or D-(-)-PGM.53 As outlined above, kinetically controlled
synthesis requires delicate optimization of reaction conditions
towards the optimal conversion. The main problem resides
in the formation of unwanted D-(-)-phenylglycine (D-(-)-
PG). In principle, a very high yield based on the â-lactam
nucleus can be obtained when a high molar excess of D-
(-)-PG derivative is used, as was the general strategy in
most of the references mentioned in Table 1. Major
drawbacks of this approach are the necessity for recycling
excess D-(-)-PGA or D-(-)-PGM, and the recovery of D-
(-)-PG. An elegant solution for the recovery of D-(-)-PGA
was found by treating the reaction mixture, following
removal of immobilized enzyme and solid products, with
benzaldehyde.54 The Schiff base of D-(-)-PGA thus ob-
tained crystallizes from the reaction mixture and can be
recovered by filtration.
carrier composition
S/H ratiob
gelatin/alginate amine (1%)
gelatin/alginate amine (3%)
gelatin/chitosan (1%)
gelatin/chitosan (2%)
gelatin/chitosan (3%)
gelatin/pectin (2%)
gelatin/pectin (3%)
3.5
2.9
2.6
2.1
3.4
1.9
2.7
2.4
2.8
2.5
2.8
2.4
gelatin/polyethylene imine (1%)
gelatin/polyethylene imine (2%)
gelatin/polyethylene imine (3%)
polyacrylamidec
polyacrylamided
a Condensations were carried out in water at 20 °C and pH 7.5 (maintained
with HCl) using 6-APA (300 mM) and D-(-)-PGA (500 mM). The amount of
enzyme used was 1 unit mL-1 b The S/H ratio was determined at 10% conversion
.
using reversed-phase HPLC. c Enzygel; commercially available from Boehringer
Mannheim GmbH, Germany. d Commercially available from Recordati, Milan,
Italy.
Yet another complicating factor is the relatively low
solubility of D-(-)-PG under the conditions applied for
synthesis. Since also the required product has a low
solubility, complex downstream processing is unavoidable.
Three solids (i.e., the desired antibiotic, D-(-)-PG, and
immobilized enzyme) need to be separated from the reaction
mixture. Enzyme separation was achieved by workers from
Novo Nordisk who developed an immobilized enzyme with
a density below unity.55 When stirring is stopped, the
immobilized enzyme will float whereas solid products can
be removed from the bottom of the reactor. Alternatively,
a sieving technique as outlined in Figure 1 may be applied.
When an enzyme immobilized on sufficiently large particles
(i.e., diameter > 150 µm) is used, the catalyst can be retained
during drainage of the reactor. By designing an appropriately
spaced sieve, crystalline material can be transported together
with the effluent.56
whole process. Furthermore, in order to reach sufficient
optical purity, conversions must be kept below 50%. This
implies a very complicated downstream processing since,
besides product isolation and enzyme recovery, unreacted
L-(+)-PGM must be racemized and reused, and the sensitive
â-lactam nucleus must be recovered with high efficiency.
With the development of immobilization techniques, the
economical feasibility of enzymatic condensation reactions
became within reach. Covalent attachment of E. coli
penicillin acylase on a gelatin-based carrier yielded a water-
insoluble catalyst that could be recycled manyfold. By
tuning of the composition of the carrier material, the S/H
ratio in synthesis reactions could be optimized as outlined
in Table 2 in the case of ampicillin synthesis.52 Initial S/H
ratios at 10% conversion indicate that gelatin-based carriers
display high S/H ratios as compared to commercially
available immobilized penicillin acylases.
(53) Bruggink, A. Chimia 1996, 50, 431.
(54) Boesten, W. H. J.; Moody, H. M. Int. Patent Appl. WO 95/03420, 1995.
(55) Kaasgaard, S. G.; Karlsen, L. G.; Schneider, I. Int. Patent Appl. WO 92/
12782, 1992.
(52) De Vroom, E. Int. Patent Appl. WO 97/04086, 1997.
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131

Figure 1.
One way to overcome the problem of excessive produc-
tion of D-(-)-PG is to use near-equimolar amounts of
substrates and design a process in which the degree of
conversion is kept relatively low. As a result, an efficient
recycling of â-lactam substrate must be realized. This may
be done in situ, as suggested by Kasche,57 in the case of
D-(-)-PGM, where a constant recycling of D-(-)-PG to D-
(-)-PGM is thought to take place as a result of the formation
of methanol, which can act as a nucleophile. Should this
be the case, the effect may be further enhanced by supplying
additional methanol. Indeed, Kim et al.25 have reported a
2-fold increase in yield in the case of ampicillin synthesis at
a methanol concentration of 40% (v/v). A drawback of this
method is a 3-fold drop in the initial rate. In our hands, this
approach gave only moderate improvements. Alternatively,
the unreacted â-lactam nucleus may be recovered by crystal-
lization after isolation of the product. For the cephalexin
process, this may be a fruitful strategy since the solubility
of 7-ADCA at its isoelectric point is very low and, thus,
losses in mother liquor may be neglected. However, the
fragile â-lactam nucleus requires careful handling when
losses resulting from degradation are to be kept minimal.
This is even more so the case with penicillin derivatives.
Ampicillin. Ampicillin synthesis is hampered by the fact
that the product solubility is high (compared to that of, e.g.,
amoxicillin). Thus, degradation percentages can amount to
unacceptable levels unless special care is taken. Since
penicillanic acid derivatives are more sensitive towards
degradation than cephalosporanic acid derivatives at almost
all pH values, recycling techniques as advocated in the case
of cephalexin are not attractive. A solution must be found
in a design in which all 6-APA present is converted to
product, which, in turn, is rapidly recovered by means of
crystallization. Thus, in a typical procedure, ampicillin is
synthesized from 6-APA and excess D-(-)-PGA or D-(-)-
PGM during which process both crystalline ampicillin and
Figure 2.
crystalline D-(-)-PG are formed. The biocatalyst is removed
by sieving, and all crystals are dissolved at acidic pH. After
concentration, pure ampicillin can be obtained by crystal-
lization at its isoelectric point. In a second concentration
and crystallization stage, D-(-)-PG can be obtained (Figure
2).
Cefaclor. As outlined above, it has become clear that
enzyme-catalyzed synthesis of antibiotics requires the de-
velopment of separate condensation and downstream pro-
cessing strategies for each individual product of interest.
Recent preliminary research into the synthesis of other
antibiotics has emphasized this theory.
Cefaclor (4a in Scheme 5) is a D-(-)-PG-derived anti-
biotic based on the nucleus 7-aminodesacetoxymethyl-3-
chlorocephalosporanic acid (7-ACCA) which can be obtained
by ozonolysis and chlorination of 3-methylene cephams.58
Cefaclor is unstable at pH values above 6.5 whilst the
solubility of 7-ACCA is very low at pH levels below 6.5.
Consequently, it is impossible to design an economically
feasible high-yielding enzymatic conversion at any pH value.
In developing enzymatic condensation processes based on
the labile and relatively expensive nucleus 7-ACCA, com-
plexation of the final product is a promising strategy, as
already shown for cephalexin.59 By coadministration of a
complexing agent (i.e., â-naphthol) to the reaction mixture,
the 2/1 cefaclor-â-naphthol complex crystallizes and very
(56) Kaasgaard, S. G.; Ulrich, C. H.; Clausen, K.; Jensen, T. Int. Patent Appl.
WO 93/23164, 1993.
(57) Kasche, V. AdV. Biosci. 1987, 65, 151.
(58) Chauvette, R. R.; Pennington, P. A. J. Med. Chem. 1975, 18, 403.
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both the side chain derivative and the nucleus are sufficiently
soluble. At pH values above 7, 7-ADCA readily dissolves
a high concentration but the side chain derivative has only
moderate solubility. At pH values below 7, the situation is
completely reversed. Thus, careful investigation is required
to establish a pH at which both reactants are present in
acceptable concentrations, i.e., both the reactivity and the
S/H ratio are acceptable. Secondly, the solubility of ce-
fadroxil under enzymatic conversion conditions is relatively
high in comparison with amoxicillin solubility. For this
reason, chemical degradation and enzymatic hydrolysis of
cefadroxil are unwanted events that may not be neglected.
Thirdly, despite the above-mentioned difficulties, a high
conversion must be achieved in the condensation reaction
in order to obtain a mixture from which cefadroxil can be
recovered by crystallization. If the conversion is too low,
the product will be contaminated with unacceptably high
levels of â-lactam nucleus and/or side chain derivative.
As already mentioned in the case of cefaclor, the situation
can be drastically improved by employing â-naphthol as a
complexing agent during the enzymatic reaction. By crystal-
lization of the cefadroxil formed as its â-naphthol complex,
the product is protected against chemical as well as enzymatic
degradation during the enzymatic conversion and recovery
procedure. In this way, the above-mentioned drawbacks may
be overcome, as this process results in a higher conversion
and S/H ratio.59
Figure 3.
high conversions (>90%) are easily reached. As a result of
the low solubility of the complex, losses in mother liquors
may be neglected. Decomplexation is normally effected at
low pH in a two-phase system. This implies the introduction
of an organic solvent into the process, which may be
considered a drawback of complexation technology.
Enzyme-Catalyzed Synthesis of D-(-)-4-Hydroxyphenylgly-
cine-Derived Antibiotics
Conclusion
Amoxicillin. In the enzyme-catalyzed synthesis of amox-
icillin (2b in Scheme 4) from 6-APA and D-(-)-4-hydroxy-
phenylglycine amide (D-(-)-HPGA) or D-(-)-4-hydroxyphe-
nylglycine methyl ester (D-(-)-HPGM), advantage can be
taken of the very low solubility of the product at conversion
conditions. Due to this phenomenon, the S/H ratio is high
since, in solution, almost no amoxicillin is available for
hydrolysis. Furthermore, for the same reason, degradation
of amoxicillin is minimal. Since amoxicillin is the first
compound to precipitate, a semi-continuous reactor system
for the production of amoxicillin at high substrate concentra-
tions could be developed successfully (Figure 3).60
Cefadroxil. Cefadroxil (4c in Scheme 5) is a semi-
synthetic antibiotic combining the side chain of amoxicillin
(D-(-)-HPG) and the nucleus of cephalexin (7-ADCA).
There are several drawbacks in enzymatic cefadroxil
synthesis when compared to enzymatic amoxicillin synthesis.
First of all, it is difficult to pinpoint a pH value at which
The aqueous enzymatic synthesis of â-lactam antibiotics
is a rapidly evolving technique which has proven its
feasibility. The keystones for this technique are the develop-
ment of a biocatalyst that can be recycled manyfold and the
product-dedicated design of downstream processes. Yields
as high as or better than those obtained in traditional chemical
condensations are achievable, and thus, economically attrac-
tive processes are within reach.
Acknowledgment
This report is based on R & D efforts by many colleagues
at DSM Research and Gist-brocades. In particular, Mr. W.
H. J. Boesten, Mr. H. M. Moody, and Dr. E. Raemakers-
Franken at DSM Research and Dr. T. van der Does, Dr. J.
M. van der Laan, and Dr. H. Slijkhuis at Gist-brocades are
gratefully acknowledged for their valuable contributions.
Received for review December 18, 1997.
OP9700643
(59) Clausen, K. U.S. Patent 5,470,717, 1995.
(60) Clausen, K.; Dekkers, R. M. Int. Patent Appl. WO 96/02663, 1996.
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