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M. Terreni et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2429–2432
When mandelic acid methyl ester (MAME) was used
instead of TZAM, the acylation of 7-ACA was unsuc-
cessful.12 For this reason, we decided to study and to try
to optimize this enzymatic reaction via biocatalyst and
engineering reaction. We prepared different derivatives
of PGA isolated from E. coli (Recordati; Opera, Italy).
This enzyme was immobilized on agarose according to
the procedure previously reported.5 In order to evaluate
the influence of the rigidity of the enzyme structure on
the catalytic properties, PGA was immobilized on agar-
ose activated with 15 and 75 mmol of glyoxyl groups per
mL of support, affording a limited and a multipoint
covalent derivative, respectively. The enzyme was also
immobilized on Eupergit C (Rhon; Darmstadt, Ger-
many) by interaction at pH 8 in presence of 1 M phos-
phate buffer. The properties of the derivatives prepared
were tested in the enzymatic acylation of 7-ACA with
MAME, to obtain 7-[(1-hydroxy-1-phenyl)-acetamido]-3-
acetoxymethyl-Á3-cephem-4-carboxilic acid 1 (Scheme 1),
an intermediate for the preparation of Cephamandole
and other 30-functionalized cephalosporins. The optimi-
zation of the reaction conditions and the assembly of
this enzymatic step with the DAAO/GA catalyzed clea-
vage of Cephalosporin C were also considered in order
to develop a multi-enzymatic one-pot synthesis of
Cephamandole (see Scheme 1).
The Vs/Vh1 ratio was instead evaluated monitoring
directly the formation of the acylation product 1 and
mandelic acid (MA) at the beginning of the acylation
reaction (less than 20% of ester consumed) between 7-
ACA and MAME. The acylations were carried out
using a high excess of the b-lactam nucleus in order to
consider constant its concentration, ensuring a suitable
saturation degree of the enzyme active sites.
The results obtained with the two agarose derivatives
(limited and multipoint linkage) were very similar con-
cerning both the Ve/Va and the Vs/Vh1 ratios, suggest-
ing that the rigidity of the immobilized enzyme does not
influence the catalytic properties (Table 1).
From the comparison of the PGA derivatives immobi-
lized on agarose and Eupergit C, significant differences
of the Ve/Va ratio were not observed. In fact, all deri-
vatives showed a low value due to a high hydrolytic
activity against the amide (Cephamandole). The effects
of the immobilization were more significant for the Vs/
Vh1 ratio and the most interesting values were obtained
with the agarose derivatives (Table 1).
The derivatives were also studied in extreme experi-
mental conditions (high ionic strength or presence of
water mixable solvents). Comparing the enzyme immo-
bilized on Eupergit C and agarose by multipoint cova-
lent interaction, the effects of the different tested
conditions were very different; agarose–multipoint deri-
vative always showed the best catalytic properties. For
this derivative, high concentrations of phosphate buffer
increased the Ve/Va ratio, whereas methanol decreased
the Vs/Vh1 ratio. High ionic strength decreased the
amidase activity against Cephamandole. In fact,
although the esterasic activity was unaffected, when
phosphate buffer concentration was 1 M, the Ve/Va
ratio was 7-fold higher than the value obtained with low
ionic strength media. The Vs/Vh1 ratio was unaffected
by high ionic strength, but increased (from 8 to up to
20) in the presence of methanol (Table 1). For the
enzyme immobilized on Eupergit C, high concentrations
of phosphate buffer allowed a very high activity to be
obtained (Ve around 50 mmol/minꢁg of enzyme
derivative), but the Ve/Va ratio slightly decreased. In
the presence of methanol, the value of Vs/Vh1 ratio was
only 7 (Table 1).
In the kinetically controlled acylation catalyzed by
PGA, the yields depend on the balance of three different
reactions catalyzed by the same enzyme (Scheme 1):12
the synthesis of the b-lactamic compound (S), the
undesired hydrolyses of the activated acyl donor (h1)
and of the product (h2). The yield depends on the ratio
between the rate of synthesis (Vs) and the rate of the
two hydrolyses (Vh1 and Vh2): to obtain good yields the
ratios Vs/Vh1 and Vs/Vh2 must be as high as possible.
For the study of the catalytic properties, the ratio
between the esterase and amidase activities (Ve/Va) was
considered as a parameter strictly related to Vs/Vh2.
The enzyme showing the highest Ve/Va ratio may be
expected to present the highest synthetase/amidase
activity ratio.12 This ratio was evaluated measuring,
separately, the hydrolytic activity against MAME (Ve)
and against Cephamandole (Va). Cephamandole was
selected as reference substrate in order to evaluate the
capacity of the catalyst to hydrolyze the amidic bond of
cephalosporin compounds bearing R-mandelic acid as side
chain. The esterasic activity is, instead, a measure of the
capacity of the catalyst to accept the acylating agents in the
active center, the first step of the synthetic process.12
Table 1. Influence of the enzyme derivative and of the reaction con-
ditions on the catalytic properties of PGA (4 ꢂC; pH 6.5)
Support
Phosphate
buffer
Solvent
(20%)
Vea Ve/Va Vs/Vh1
Eupergit C
10 mM
10 mM
—
—
15
12
0.67
1.09
2.4
7.5
Agarose–limited
linkage
Agarose–multipoint
10 mM
—
—
—
25
31
29
4.7
44
3.3
0.94
4.70
8.43
0.61
0.51
0.75
8.2
8.0
9.4
Agarose–multipoint 500 mM
Agarose–multipoint
Agarose–multipoint
Eupergit C
1 M
10 mM
1 M
Methanol
—
Methanol
>20
1.8
7.1
Eupergit C
10 mM
aActivity expressed in U/g (mmol of substrate hydrolyzed/minꢁg of
enzyme derivative).
Scheme 1.