Resolution of (±)-5-substituted-6-(5-chloropyridin-2-yl)-7-oxo-5,6-dihydropyrrolo [3,4b]pyrazine derivatives-precursors of (S)-(+)-Zopiclone, catalyzed by immobilized Candida antarctica B lipase in aqueous media

Article abstract of DOI:10.1016/S0957-4166(02)00867-4

The enzymatic resolution of different cyclopyrrolone compounds has been performed in aqueous systems using different immobilized preparations of lipase from Candida antarctica (fraction B). The relevance of the immobilization protocol in the results has been shown: lipase immobilized on octadecyl-Sepabeads gave the highest stability and enantioselectivity. Commercial Novozym 435 was scarcely enantioselective in the hydrolytic process. On the other hand, the structure of the cyclopyrrolone was also found to be very important to the outcome of the reaction, the best results being achieved with compounds (±)-1 and (±)-2. Thus, using compound (±)-2, a ee_s of >95% can be achieved under conditions in which the enzyme preparation can be utilized in 10 recycles without any significant detriment to the enzymatic properties (activity, enantioselectivity). Moreover, this enzyme catalyzes the hydrolytic resolution of chloromethyl carbonate (±)-5, a useful intermediate for the synthesis of the hypnotic agent (S)-(+)-Zopiclone.

Full text of DOI:10.1016/S0957-4166(02)00867-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 429–438
Resolution of ( )-5-substituted-6-(5-chloropyridin-2-yl)-
7-oxo-5,6-dihydropyrrolo[3,4b]pyrazine derivatives-precursors of
(S)-(+)-Zopiclone, catalyzed by immobilized Candida antarctica
B lipase in aqueous media
Jose M. Palomo,a Cesar Mateo,a Gloria Ferna´ndez-Lorente,a Laura F. Solares,b Monica Diaz,b
V´ıctor M. Sa´nchez,c Miguel Bayod,c Vicente Gotor,b Jose M. Guisana,* and
Roberto Fernandez-Lafuentea,*
aDepartment of Biocatalysis, Institute of Catalysis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain
bDepartment of Organic and Inorganic Chemistry, Faculty of Chemistry, University of Oviedo, 33071 Oviedo, Spain
cAstur-Pharma S.A. Pol´ıgono industrial de Silvota, Parcela 23,33192 Llanera (Asturias), Spain
Received 31 October 2002; revised 11 December 2002; accepted 13 December 2002
Abstract—The enzymatic resolution of different cyclopyrrolone compounds has been performed in aqueous systems using
different immobilized preparations of lipase from Candida antarctica ( fraction B). The relevance of the immobilization protocol
in the results has been shown: lipase immobilized on octadecyl-Sepabeads gave the highest stability and enantioselectivity.
Commercial Novozym 435 was scarcely enantioselective in the hydrolytic process. On the other hand, the structure of the
cyclopyrrolone was also found to be very important to the outcome of the reaction, the best results being achieved with
compounds ( )-1 and ( )-2. Thus, using compound ( )-2, a ees of >95% can be achieved under conditions in which the enzyme
preparation can be utilized in 10 recycles without any significant detriment to the enzymatic properties (activity, enantioselectiv-
ity). Moreover, this enzyme catalyzes the hydrolytic resolution of chloromethyl carbonate ( )-5, a useful intermediate for the
synthesis of the hypnotic agent (S)-(+)-Zopiclone. © 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
possible to use lipases to resolve precursors of this
compound via hydrolysis of suitable intermediates in
aqueous media or in the presence of water as a co-sol-
vent. Lipases (triacyglycerol acylhydrolases, EC 3.1.1.3)
are the most popular enzymes in biocatalysis5–12
because they combine wide substrate specificity with
high regio- and enantioselectivity, and therefore may be
used in many different reactions. When using lipases as
chemical catalysts, their peculiar mechanism of action
(interfacial activation)13–15 and the dramatic conforma-
tional changes involved16–20 must be considered. The
immobilization of the lipase involving different areas of
the enzyme, thus giving different rigidity to the enzyme
structure or even generating a certain special microenvi-
ronment surrounding the enzyme, can alter the shape of
the final open form of the lipase (Scheme 1).
( )-Zopiclone (4-methyl-1-piperazinecarboxylic acid 6-
(5-chloro-2-pyridinyl)-6,7-dihydro-7-oxo-5H-pyrrolo-
[3,4b]pyrazin-5-yl ester)1 is a hypnotic agent of the
cyclopyrrolone class possessing a pharmaceutical profile
similar to that of benzodiazepines, with high efficiency
and low toxicity. The (S)-(+) enantiomer is less toxic
and more active than its enantiomer.2,3
The preparative resolution of ( )-Zopiclone has been
carried out by the formation of the diastereomeric salts
with (+)-O,O%-dibenzoyltartaric acid3 or (+)- or (−)-
malic acid2 and selective crystallization of the racemic
mixture. Moreover, taking into account that some car-
bonates have been resolved by enzymatic methods,4 it is
This method for modulating the properties of the
enzyme has been used successfully to control the cata-
lytic behavior of different enzymes, which suffer drastic
* Corresponding authors. Tel.: 34 91 585 48 09; fax: 34 91 585 47 60;
e-mail: jmguisan@icp.csic.es; rfl@icp.csic.es
0957-4166/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00867-4

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J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
Scheme 1. Conformational engineering of lipases.
conformational changes during catalysis (e.g. penicillin
G acylase16 and lipases17–20). In the work reported
herein, we have used different immobilization protocols
to produce immobilized preparations of lipase from
Candida antarctica (fraction B) (CAL-B) aiming at
finding the optimal immobilized preparation for the
hydrolytic resolution of ( )-6-(5-chloropyridin-2-yl)-7-
how different immobilization protocols may yield
enzymes with very different specificity for different
substrates.
The enantioselectivity was very high (E >100), with a
stereochemical preference for hydrolysis of the R iso-
mer, for our lipase-immobilized preparations while
Novozym 435 preparation did not show any selectivity.
oxo-5,6-dihydropyrrolo[3,4b]pyrazine
derivatives
(Scheme 2), intermediates in the production of (S)-(+)-
Zopiclone.
2.2. Use of higher substrate concentrations
From an industrial point of view, it is convenient to
work at higher substrate concentrations. In order to
achieve that, higher concentrations of co-solvent are
needed. Moreover, it is also necessary to have a biocat-
alyst resistant to these drastic conditions. Thus, it is
important to study the stability of these immobilized
preparations under these conditions.
2. Results and discussion
2.1. Characterization of the different enzyme prepara-
tions: use of low substrate concentration
First, the initial activity displayed by different immobi-
lized preparations from CAL-B in the hydrolysis reac-
tion in fully aqueous media was studied. However, even
at low substrate concentrations (only 0.05 mM) some
co-solvent (10%) was necessary to fully dissolve it.
Table 1 summarizes the most significant results.
All immobilized preparations remained fully active at
25°C and different pH values (7 or 5) after 150 h of
incubation, while the activity of the soluble lipase
decreased slightly. However, at 50°C (Table 2) only the
octadecyl-Sepabeads preparation retained 100% of
activity after this time, while the covalent preparations
suffered a decrease in the activity of at least ca. 50%
(the glyoxyl preparation being slightly more stable than
the glutaraldehyde one). The CNBr preparation
retained only 3% of activity and the PEI preparation
was completely deactivated after 35 h.
The reaction was performed at 25°C and pH 7 using
dioxane as co-solvent. In this case, due to the high
spontaneous chemical hydrolysis of the substrate, spe-
cial care was needed to design rapid experiments (e.g. in
2 h less than 1% of the substrate was chemically
hydrolyzed). The glutaraldehyde and CNBr prepara-
tions showed the higher values of activity, being almost
two-fold more active than the octadecyl, the glyoxyl or
the commercial Novozym 435 preparations. This shows
In the presence of 50% (v/v) 1,4-dioxane, only the
octadecyl and glyoxyl preparations retained 100% of

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
431
Scheme 2. Enantioselective hydrolysis of precursors of Zopiclone catalyzed by different CAL-B immobilized preparations.
Table 1. Enzymatic activity of immobilized preparations of CAL-B in the hydrolysis of (9)-1, 0.05 mM and pH 7, 10%
dioxane and 25°C
Immobilized
preparation
Specific activity (mmol mg lipase−1 min−1
)
Enzyme activity (mmol min−1 g biocatalyst)
E (R/S)
CNBr
Octadecyl
PEI
Glyoxyl
Glutaraldehyde
Novozym sp. 435
0.19
0.10
0.15
0.09
0.16
–
2.24
1.16
1.80
1.08
1.87
1.00
\100
\100
\100
\100
\100
1.00
The relative error was estimated to be 5.
Table 2. Stability of CAL-B immobilized preparations under different conditions. (The numbers refer to the residual activity
after 150 h of incubation)
Immobilized preparation
Residual activity (%)
25°C, 50% dioxane
50°C
37°C, 40% dioxane
Octadecyl
Glyoxyl
Glutaraldehyde
CNBr
100
50
40
3
100
100
93
100
100
57
80
40
PEI
0 (35 h)
65
30
the activity. The glutaraldehyde preparation lost 7% of
the initial activity after 150 h. On the other hand, the
activities of the PEI or CNBr preparations decreased by
35 or 20% of the initial activity in the same time period,
respectively (Table 2). Under these conditions, the
octadecyl-Sepabeads preparation no desorption of the
adsorbed enzyme was seen, while with Novozym 435
the enzyme was partially desorbed.

432
J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
Table 4. Activity of the different immobilized preparations
in the hydrolysis of compound (9)-1 at 0.05 mM, pH 7
and 25°C with 50% (v/v) dioxane
At 37°C and in the presence of 40% (v/v) dioxane
(Table 2), again octadecyl-Sepabeads and glyoxyl
preparations retained 100% of the initial activity while
the glutaraldehyde preparation lost 43% of the activity.
In the cases of the CNBr and PEI preparations, these
maintained 40 and 30% of the initial activity,
respectively.
Immobilized
preparation
Specific activity
Enzyme activity
(×10−3
)
(×10−2
)
Octadecyl
Glutaraldehyde
Glyoxyl
0.59
0.70
0.75
0.71
0.85
0.90
These results suggested that glyoxyl, glutaraldehyde
and octadecyl preparations could be suitable biocata-
lysts for this biotransformation.
Specific activity: mmol/(mg lipase min); enzyme activity: mmol/(g of
catalyst min).
The relative error was estimated to be 5.
We next performed a study to optimize the concentra-
tion of co-solvent in the reaction medium to allow
higher substrate (( )-1) concentration. To this end, we
used several solvents with different grades of hydropho-
bicity, trying to reach a substrate concentration of 10
mM (Table 3).
immobilized preparations in the hydrolysis reaction
using 10 mM of ( )-1 was examined (Table 5). A
comparison of the activities in Tables 2, 4 and 5
suggested that the decrease in enzyme activity in the
presence of organic solvents may be due mainly to
competitive inhibition by the solvent.
Table 3. Solubility of substrate in a variety of co-solvent
in aqueous media
Co-solvent
1,4-Dioxane
Acetonitrile
Acetone
% (v/v)
Maximum soluble
concentration (mM)
The highest activities were obtained with the octadecyl
and covalently immobilized enzyme preparations.
Moreover, it was possible to observe that, although all
preparations presented a very high enantioselectivity,
the enzyme interfacially activated21 on octadecyl Sepa-
beads preparation was the one with the highest E value,
allowing us to achieve the highest enantiomeric excess
ees >99%, even though 0.5% spontaneous chemical
hydrolysis took place (Table 5).
44
48
52
44
48
52
44
48
52
B10
10
10
B10
B10
10
B10
B10
B10
Taking into account all the parameters (activity, stabil-
ity and enantioselectivity), the octadecyl-Sepabeads
preparation seemed the best for this reaction. The
reaction rate is very rapid and 45% conversion was
reached in 50 min. The reaction became slower at near
to 50% conversion and after 48 h the conversion did
not surpass 51% hydrolysis, showing the high enan-
tioselectivity of the enzyme.
The best results were found for 1,4-dioxane with which
it was possible to dissolve the desired amount of com-
pound ( )-1 using 48% of co-solvent.
The activity of the different immobilized preparations
in the presence of dioxane was analyzed (Table 4). All
immobilized preparations suffered a dramatic loss of
activity in the presence of the organic solvent (by a
factor of 100). However, the activities were still
significant.
2.4. Enzymatic resolution of other precursors of
Zopiclone
2.3. Reactions using a concentration of 10 mM of
substrate
In the previous section it was shown that the enzymatic
resolution of substrate ( )-1 was effective, especially
with lipase immobilized on octadecyl-Sepabeads sup-
port. Unfortunately, the high cost of the vinyl chloro-
formate required for the synthesis of compound ( )-1 is
Next, after the conditions with respect to concentration
and co-solvent were optimised, the activity of the
Table 5. Resolution of substrate (9)-1 catalyzed by different immobilized CAL-B preparations. The hydrolysis was per-
formed using 10 mM substrate concentration at 25°C, pH 7 and 50% dioxane
Immobilized preparation
Specific activitya
Enzyme activityb
Enantiomeric preference
Eesc
Ed
Octadecyl
Glutaraldehyde
Glyoxyl
0.10
0.12
0.13
1.20
1.44
1.56
R
R
R
\99
97
94
\500
277
115
a Specific activity: mmol/(mg lipase min).
b Enzyme activity: mmol/(g of catalyst min). The relative error was estimated to be 5.
c Ees=enantiomeric excess of substrate at 50% of conversion.
d E=enantioselectivity.

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
433
Table 6. Enantioselective hydrolysis of the different compounds under different reaction conditions catalyzed by octadecyl-
Sepabeads preparation
Compound
Substrate concentration
(mM)
Dioxane (%)
Temperature
(°C)
Activitya (mmol mg−1
Relation (w/v) Eesb
Ed
min−1
)
(9)-1
(9)-2
(9)-3
(9)-4
(9)-5
(9)-6
(9)-7
(9)-7
10
10
10
0.5
10
0.1
4
50
30
50
40
50
40
50
50
25
37
25
25
25
25
25
37
0.10
0.66
0.04
0
1/30
1/40
1/40
1/30
1/40
1/40
1/40
1/40
\99
\500
\95c \300
60
Nd
\99
Nd
\99
\99
7.1
Nd
\100
Nd
\100
\100
1.25×10−2
0.50×10−5
1.40×10−3
1.67×10−3
6
a Specific activity: mmol/(mg lipase min). The relative error was estimated to be 5.
b Enantiomeric excess calculated by 1H NMR.
c Ees=enantiomeric excess of substrate at 50% of conversion.
d E=enantioselectivity.
a drawback for the scale-up of the process. Hence,
carbonates ( )-5, ( )-6, and ( )-7, that are also precur-
sors of Zopiclone, were tested under similar conditions.
Several ester derivatives [( )-2, ( )-3, and ( )-4] were
also studied in order to evaluate the performance of the
octadecyl-Sepabeads preparation (Table 6). Because of
the high stability of the enzyme preparation, the limit
for the reaction conditions was fixed by the substrate
stability (conditions where less than 0.5% of the sub-
strate was chemically hydrolyzed) (Scheme 3).
around a factor of 3 due to the enhanced enzyme
activity against this substrate). For compound ( )-3 a
three-fold decrease in activity relative to ( )-1 was
observed.
Compounds ( )-4 and ( )-6 were very water-insoluble
and only 0.5 or 0.1 mM concentrations, respectively,
could be achieved with 40% dioxane. The immobilized
preparation seemed to be inactive towards substrate
( )-4, while it was possible to detect some activity with
substrate ( )-6, although it was very low. On the other
hand, compound ( )-5 presented similar solubility to
( )-1 although the activity was almost 10-fold lower.
Substrate ( )-7 was less soluble than ( )-5, and only 4
mM (at 25°C) and 6 mM (at 37°C) solutions could be
prepared using the maximum concentration of dioxane
studied (50%). Activity of the octadecyl preparation at
both temperatures was lower than that with ( )-1.
Regarding the enantioselectivity of these reactions, the
immobilized preparation presented a very low value of
E in the hydrolysis of compound ( )-3 affording a 60%
ees at 50% of conversion.
By contrast, using the other substrates, high enan-
tiomeric excesses after purification were obtained for
1
ester ( )-2 (ee >95% by H NMR analysis) and carbon-
ates ( )-5 and ( )-7 (ee >99% by chiral HPLC analysis).
In the hydrolysis of compound ( )-2 and octadecyl-Sepa-
beads preparation as biocatalyst, 50% conversion was
obtained in less than 45 min using a ratio of 1:37 grams
of biocatalyst/reaction volume (Fig. 1). The specific
activity and enantioselectivity of the preparation
remained the same over 10 reaction cycles. Fig. 2 shows
that the reaction times required to reach a 50% hydrol-
ysis were identical in each cycle, and the ee achieved
was in all cases higher than 99%.
Scheme 3. ( )-5-Substituted-6-(5-chloropyridin-2-yl)-7-oxo-
5,6-dihydro pyrrolo[3,4b]pyrazine derivatives, precursors of
(S)-(+)-Zopiclone.
The highest activity for the octadecyl-Sepabeads cata-
lyst was observed in the hydrolysis of the acetyl ester
( )-2, a substrate significantly more stable than 1. The
hydrolysis of ( )-2 was six times faster than that of the
reference compound ( )-1 (increasing almost by a fac-
tor of 2 as a result of the higher temperature and
From the results shown in Table 6, it can be concluded
that the size of the side chain (R) of the carbonate has
a remarkable effect on the activity of the catalyst. Good
reaction rates can be obtained only when the R group

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J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
3. Conclusions
The effect of enzyme immobilization on the catalytic
properties of different CAL-B preparations in the reso-
lution of several cyclopyrrolone compounds in aqueous
media has been studied. This work has demonstrated
the strong influence of the enzyme preparation and the
nature of the substrate.
The octadecyl-Sepabeads preparation was the most
efficient among those examined. This preparation pre-
sented the highest enantioselectivity, good activity, and
high stability under the harsh reaction conditions.
Moreover, it presents an additional advantage, which is
the possibility of re-use of the support, thanks to the
reversible adsorption of the enzyme on the support. In
the presence of surfactant (Triton X-100 4%) it is
possible to desorbe all enzyme and to immobilize fresh
enzyme.21
Figure 1. Reaction course of the enzymatic hydrolysis of ( )-3
catalyzed by octadecyl-CAL-B preparation. Experiments were
performed using a concentration of substrate of 10 mM with
30% (v/v) 1,4-dioxane and pH 7, 37°C, as described in Section
4.
With this improved catalyst, it has been possible to
resolve vinyl carbonate ( )-1 and chloromethyl carbon-
ate ( )-5 with high enantioselectivity, thus obtaining the
remaining (S)-(+) isomers of 1 and 5 with high ees.
Besides, the acetyl derivative ( )-2 was the best sub-
strate for the enzyme, giving enantiomeric excess higher
than 95% and E >100.
is sufficiently small; either with ester (2 and 3) or with
carbonate (1 and 5) derivatives. The best results for the
enzymatic resolution were obtained with ester ( )-2,
which possesses the smallest R group (methyl). Unfor-
tunately, this compound cannot be transformed into
enantiopure Zopiclone due to racemization at the
stereogenic center in the subsequent steps.22,23 Never-
theless, the values of activity and enantioselectivity for
the resolution of vinyl carbonate ( )-1 and
chloromethyl carbonate ( )-5 are also quite good, thus
this procedure can be employed for the preparation of
(S)-Zopiclone.
Therefore, the enzymatic resolution of carbonates ( )-1
and ( )-5 with octadecyl-Sepabeads immobilized prepa-
ration in aqueous media is a comparable alternative to
the resolution in organic media with commercial
enzyme preparations.22,23
Figure 2. Different cycles of hydrolysis reaction of compound ( )-3 catalyzed by octadecyl-Sepabeads CAL-B-preparation.
Experiments were performed using a substrate concentration of 10 mM with 30% (v/v) 1,4-dioxane and pH 7, 37°C and 50% of
conversion was obtained after 45 min of reaction and it was maintained during 10 cycles.

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
435
4. Experimental
reduce the reactive groups, a volume of 100 mM
sodium bicarbonate pH 10 containing 2 mg/mL of
sodium borohydride was added. After 30 min the
preparation was washed with an excess of distilled
water.
4.1. Materials
The lipase from Candida antarctica ( fraction B)
(Novozym 525 L) (CAL-B) was from Novo Nordisk
(Denmark). Octyl-agarose 4BCL and cyanogen bro-
mide (CNBr-activated Sepharose 4BCL) were pur-
chased from Pharmacia Biotech (Uppsala, Sweden).
Octadecyl-Sepabeads was generously donated by Resin-
dion Srl (Mitsubishi Chem. Coorp) (Milan, Italy).
Agarose 10 BCL was kindly donated by Hispanagar
(Burgos, Spain). Glyoxyl-agarose,24 glutaraldehyde-
agarose25 and PEI-agarose26 were prepared as previ-
ously described. Polyethyleneimine (PEI) (Mr 25000),
glutaraldehyde, Triton X-100, p-nitrophenyl propionate
(p-NPP) were from Sigma. Other reagents and solvents
used were of analytical or HPLC grade.
(v) Immobilization on CNBr-activated Sepharose 4B
using the protocol from Amershan Biosciences28 (at pH
7, the immobilization should proceed by the most reac-
tive group: the terminal amino).
Activities of suspensions and supernatants were assayed
by using the p-NPP assay as described below. Protein
concentration was determined by the Bradford
method.29 Enzyme load was 12 mg lipase/mL of sup-
port and in all cases more than 95% of the lipase
activity became immobilized on the different supports.
4.4. Hydrolysis of p-NPP
4.2. Purification of CAL-B
This assay was performed by measuring the increase in
the absorbance at 348 nm produced by the release of
p-nitrophenol in the hydrolysis of p-NPP (0.4 mM) in
25 mM sodium phosphate buffer at pH 7 and 25°C. To
start the reaction, 0.05 mL of lipase solution or suspen-
sion was added to 2.5 mL of substrate solution. One
international unit of p-NPP activity was defined as the
amount of enzyme that is necessary to hydrolyze 1
mmol of p-NPP per minute (IU) under the conditions
described above.
In order to purify the lipase, the enzyme preparation
was incubated in the presence of octyl-agarose at low
ionic strength, following the previously described proce-
dure.27 Periodically, activity of suspensions and super-
natants was assayed by using the p-NPP assay as
described below. Final immobilization yield was over
95%. After that, adsorbed lipase preparation was abun-
dantly washed with distilled water. To desorbe the
enzyme, the adsorbed lipase was washed with Triton
X-100 1% in 5 mM sodium phosphate buffer solution
at pH 7 and 25°C.
4.5. Enzymatic hydrolysis of ( )-1–7
4.3. Immobilization of CAL-B on different supports
The activities of different immobilized preparations of
CAL-B were analyzed in the hydrolysis reaction of
compounds ( )-1–7 at different conditions.
Different preparations were produced following the
procedures previously described.
(i) Interfacial adsorption on hydrophobic support
(octadecyl-Sepabeads21). To immobilize the purified
lipase in octadecyl support, the enzyme solution (con-
taining 1% triton) was diluted 500–fold with 5 mM
sodium phosphate buffer at pH 7.
Procedure A: substrate ( )-1 was dissolved at 25°C in a
mixture of 10 mM sodium phosphate buffer (pH 7, 10
mL) with 10 or 50% (v/v) 1,4-dioxane to a concentra-
tion of 0.05 mM and 300 mg of enzymatic preparation
were added to this solution.
(ii) Ionically adsorbed lipase on solid supports coated
with PEI25 (ionic microenvironment surrounding large
areas of the protein). The immobilization was carried
out in 5 mM sodium phosphate buffer pH 7 at 25°C.
After immobilization the preparations were washed
with distilled water.
Procedure B: substrate ( )-1 was dissolved at 25°C in a
mixture of 10 mM sodium phosphate buffer solution
(pH 7, 3.5 mL) with 50% (v/v) 1,4-dioxane to a concen-
tration of 10 mM and enzymatic preparation (120 mg)
was added to this solution.
The enzymatic activity of the octadecyl-Sepabeads
preparation in the hydrolysis of compounds ( )-2–7
was determined under the following conditions.
(iii) Multipoint covalent immobilization on glyoxyl-
agarose beads (through areas with the highest density
of lysine (Lys) groups).24 The immobilization was car-
ried out in 25 mM sodium bicarbonate pH 10 at 25°C.
To end the multipoint covalent attachment, sodium
borohydride was added to a concentration of 1 mg/mL.
After 30 min the preparation was washed with an
excess of distilled water.
Substrate ( )-2 was dissolved at 37°C in 10 mM sodium
phosphate buffer solution (pH 7, 8 mL) at with 30%
(v/v) dioxane to a concentration of 10 mM and octade-
cyl-Sepabeads (200 mg) were added.
Substrate ( )-3 was dissolved at 25°C in a mixture of 10
mM sodium phosphate buffer solution (pH 7, 8 mL)
with 50% (v/v) dioxane to a concentration of 10 mM
and octadecyl-Sepabeads (200 mg) were added.
(iv) Covalent immobilization on glutaraldehyde-agarose
beads.24 The immobilization was carried out in 25 mM
sodium phosphate buffer pH 7 at 25°C. In order to

436
J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
Substrate ( )-4 was dissolved at 25°C in a solution of
10 mM sodium phosphate buffer (pH 7, 8 mL) with
40% (v/v) dioxane to a concentration of 0.5 mM and
the enzymatic preparation (250 mg) was added.
substrate ( )-7, the mobile phase was a mixture of 60%
hexane, 40% i-propanol, at a flow rate of 0.8 mL/min,
254 nm. (S)-(+)-7, tR 26 min; (R)-(−)-7, tR 32.8 min.
The enantiomeric ratio was expressed as the E value
calculated from the enantiomeric excess (ees) of the
remaining ester and the conversion degrees (c) accord-
ing to method previously reported by Chen et al.30
Substrate ( )-5 was dissolved at 25°C in 10 mM buffer
solution (4 mL) at pH 7 with 50% (v/v) dioxane to a
concentration of 10 mM and biocatalyst (100 mg) was
added.
4.7. Synthesis of compounds
Substrate ( )-6 was dissolved at 25°C in 10 mM sodium
phosphate buffer solution (pH 7, 4 mL) at with 40%
(v/v) dioxane to a concentration of 0.1 mM of substrate
and immobilized preparation (100 mg) was added.
Optical rotations were measured using a Perkin–Elmer
241 polarimeter and are quoted in units of 10−1 deg cm2
1
g−1. H and 13C NMR spectra were obtained with TMS
(tetreamethylsilane) as internal standard using a Bruker
AC-300 (1H-300 MHz and 13C-75.5 MHz) spectrome-
ter. Mass spectra were recorded on a Hewlett–Packard
1100 LC/MSD.
Finally, substrate ( )-7 was dissolved at 25°C in solu-
tion of 10 mM sodium phosphate buffer (8 mL) at pH
7 with 50% (v/v) dioxane to a concentration of 4 mM
of substrate and the biocatalyst (200 mg) was added.
Moreover, it was possible to dissolve up substrate to a
concentration of 6 mM at 37°C using the same condi-
tions in the activity analyses.
4.7.1. ( )-6-(5-Chloropyridin-2-yl)-7-oxo-5-(vinyloxycar-
boyloxy)-5,6-dihydropyrrolo[3,4b]pyrazine, ( )-1. Vinyl
chloroformate (0.40 mL, 4.38 mmol, 1.15 equiv.) was
added to a suspension of ( )-8 (1.0 g, 3.81 mmol) and
anhydrous pyridine (1.2 mL) in anhydrous
dichloromethane (8 mL) under N2 at 0°C. The mixture
was stirred at 25°C for 10 h (it was monitored by TLC
until the initial product was consumed). The mixture
was extracted with water and dichloromethane. The
organic phase was dried over NaSO4, filtered and con-
centrated under reduce pressure. Yield: 75%, white
powder, mp=130–132°C; IR (KBr): 1770, 1736 cm−1,
1H NMR (CDCl3), l (ppm): 8.91 (dd, 2H, 2CH), 8.52
During the course of the reactions, the pH of the
mixture was kept constant by automatic titration using
a pH-stat Mettler Toledo DL50 graphic. The specific
and enzymatic activities were defined as mmol of sub-
strate hydrolyzed per minute per mg of immobilized
protein or per gram of immobilized preparation in each
case.
The degrees of hydrolysis were quantified by reverse-
phase HPLC (Spectra Physic SP 100) coupled with an
UV detector (Spectra Physic SP 8450) on a Kromasil
C18 (25×0.4 cm) column supplied by Analisis Vinicos
(Spain). The elution was isocratic with a mobile phase
of acetonitrile (50%) and 10 mM ammonium phosphate
buffer (50%) at pH 6.9, a flow rate of 1.5 mL/min, the
adsorbance at 270 nm and at 20°C. The retention times
(tr) were: ( )-8 (2.98 min), ( )-1 (11.3 min), ( )-2 (6.33
min), ( )-3 (14.75 min), ( )-4 (18.5 min), ( )-5 (11 min),
( )-6 (27.5 min), ( )-7 (9.9 min).
3
4
(d, 1H, CH, JHH=8.98 Hz), 8.40 (d, 1H, CH, JHH
=
4
2.58 Hz), 8.02 (s, 1H, CH), 7.82 (dd, 1H, CH, JHH
=
2.58 Hz, 3JHH=8.98 Hz), 7.20 (dd, 1H, CH,
3Jtrans=6.16 Hz, Jgem=13.84 Hz), 4.95 (dd, 1H, CH,
2
3Jcis=2.30 Hz, 2Jgem=13.84 Hz), 4.69 (dd, 1H, CH,
3Jcis=2.30 Hz, 3Jtrans=6.14 Hz). 13C NMR (CDCl3):
162.3 (CꢀO), 153.9 (CꢀO), 151.0 (C), 148.3 (CH), 148.2
(CH), 147.2 (C), 146.5 (CH), 143.7 (C), 142.2 (CH),
138.1 (CH), 128.3 (C), 115.6 (CH), 98.8 (CH2), 80.3
(CH). EM (ESI+, m/z): 334 (M+H)+, 356 (M+Na)+.
4.6. Determination of enantiomeric excess and E value
4.7.2. 6-(5-Chloropyridin-2-yl)-5-(O-acetyl)-7-oxo-5,6-
dihydropyrrolo[3,4b]pyrazine, ( )-2. Acetyl chloride
(0.33 mL, 4.57 mmol, 2 equiv.) was added to a suspen-
sion of ( )-8 (600 mg, 2.29 mmol) and anhydrous
pyridine (0.74 mL) in anhydrous dichloromethane (10
mL) under N2 at 0°C. The mixture was stirred at 25°C
for 5 h (it was monitored by TLC until the initial
product was consumed). The mixture was extracted
with water and dichloromethane. The organic phase
was dried over sodium sulfate, filtered and evaporated
under reduced pressure. Yield: 85%; mp 170–173°C; IR
With substrate ( )-1, the enantiomeric excess (ees) of
the remaining ester was analyzed by Chiral HPLC at
different conversion degrees. The column was
Chiracel OD, the mobile phase was an isocratic mixture
of 40% hexane, 60% ethanol and 0.1% triethylamine at
a flow rate 0.6 mL/min; detection at 254 nm. Optical
purity analyses of compound ( )-2 was performed by
1H NMR using europium tris[3-(heptafluoropropyl-
hydroxy methylene)-(+)-camphorate]) as a chiral shift
reagent in CDCl3.
a
1
(KBr): 1735 cm−1. H NMR (CDCl3) l 8.87 (dd, 2H, 2
3
CH), 8.51 (d, 1H, CH, JHH=8.70 Hz), 8.37 (d, 1H,
4
With chloro carbonates ( )-5 and ( )-7 the enan-
tiomeric excesses (ees) were determined by chiral HPLC
analysis using a CHIRALPAK AS column (4.6×250
mm). Two peaks were obtained for ( )-5 with a mobile
phase of 60% hexane, 35% ethanol and 5% i-propanol,
at a flow rate of 1 mL/min, at 254 nm. (S)-(+)-5, tR
10.34 min; (R)-(−)-5, tR 15.28 min. In the case of
CH, JHH=2.56 Hz), 8.13 (s, 1H, CH), 7.80 (dd, 1H,
CH, 4JHH=2.56 Hz, 3JHH=8.70 Hz), 2.11 (s, 3H, CH3);
13C NMR (CDCl3) l (ppm): 170.0 (CꢀO), 163.5 (C),
155.7 (CꢀO), 149.0 (CH), 148.6 (CH), 148.2 (C), 147.4
(CH), 144.6 (C), 138.8 (CH), 129.0 (C), 116.7 (CH),
77.9 (CH), 21.4 (CH3). MS (ESI+) m/z (%): 327 [(M+
Na)+, 100%].

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
437
4.7.3. 6-(5-Chloropyridin-2-yl)-5-(O-butyryl)-7-oxo-5,6-
dihydropirrolo[3,4b]pyrazine, ( )-3. Butyryl chloride
(0.47 mL, 4.57 mmol, 2 equiv.) was added to a sus-
pension of ( )-8 (600 mg, 2.29 mmol) and anhydrous
pyridine (0.74 mL) in anhydrous dichloromethane (10
mL) under N2 at 0°C. The mixture was stirred at
25°C for 5 h (it was monitored by TLC until the
initial product disappeared). The mixture was
extracted with water and dichloromethane. The
organic phase was dried over sodium sulfate, filtered
and evaporated under reduced pressure. Yield: 87%;
mp 139–142°C; IR (KBr): 1724 cm−1. 1H NMR
(CDCl3) l 8.85 (dd, 2H, 2CH), 8.49 (d, 1H, CH,
3JHH=8.76 Hz), 8.33 (d, 1H, CH, 4JHH=2.55 Hz),
CH2), 13C NMR (CDCl3), l (ppm): 163.0 (CꢀO),
154.5 (CꢀO), 152.8 (C), 149.1 (CH), 148.0 (C), 147.4
(CH), 144.5 (C), 139.0 (CH), 129.2 (C), 116.3 (CH),
81.6 (CH), 73.2 (CH2). EM-ESI+: [M+Na]=376.9,
[M+H]=355.0.
4.7.6. 6-(5-Chloropyridin-2-yl)-7-oxo-5-(2,2,2-trychloro-
ethyloxycarbonyloxy)-5,6-dihydropyrrolo-[3,4b]pyrazine,
( )-6. 2,2,2-Trichloroethyl chloroformate (0.8 mL)
was added to a solution of ( )-8 (1 g) and anhydrous
pyridine (1.2 mL) in anhydrous dichloromethane (30
mL) under N2 at 0°C. The mixture was stirred at
25°C for 5 h (it was monitored by TLC until the
initial product disappeared). After that, it was
extracted with water and dichloromethane. The
organic phase was treated with sodium sulfate,
filtered and evaporated under reduced pressure. Yield:
4
8.13 (s, 1H, CH), 7.78 (dd, 1H, CH, JHH=2.55 Hz,
3JHH=8.76 Hz), 2.30 (t, 2H, CH2), 1.62 (q, 2H, CH2),
0.97 (t, 3H, CH3); 13C NMR (CDCl3) l (ppm): 172.6
(CꢀO), 163.5 (C), 155.8 (CꢀO), 149.0 (CH), 148.5
(CH), 148.2 (C), 147.3 (CH), 144.6 (C), 138.8 (CH),
129.0 (C), 116.6 (CH), 77.7 (CH), 36.5 (CH2), 18.8
(CH2), 14.1 (CH3). MS (ESI+) m/z (%): 411 [(M+Na)+,
100%], 355.
1
98%, mp 201–203°C; IR (cm−1): 1788, 1745, H NMR
(CDCl3), l (ppm): 8.91 (dd, 2H, 2CH), 8.52 (d, 1H,
CH, 2JHH=8.77 Hz), 8.34 (d, 1H, CH, 3JHH=2.31
2
Hz), 8.03 (s, 1H, CH), 7.82 (dd, 1H, CH, JHH=8.72
Hz, 3JHH=2.56 Hz), 4.91 (m, 2H, CH2), 13C NMR
(CDCl3), l (ppm): 162.4 (CꢀO), 153.9 (CꢀO), 152.5
(C), 148.3 (CH), 147.3 (C), 146.6 (CH), 143.8 (C),
138.3 (CH), 128.6 (C), 115.8 (CH), 93.8 (C), 80.8
(CH), 76.4 (CH2). EM-ESI+: [M+Na]=458.9.
4.7.4. 6-(5-Chloropyridin-2-yl)-5-(O-benzoyl)-7-oxo-5,6-
dihydropyrrolo[3,4b]pyrazine, ( )-4. Benzoyl chloride
(0.44 mL, 3.81 mmol, 2 equiv.) was added to a sus-
pension of ( )-8 (500 mg, 1.90 mmol) and anhydrous
pyridine (0.62 mL) in anhydrous dichloromethane (10
mL) under N2 at 0°C. The mixture was stirred at
25°C for 4 h (it was monitored by TLC until the
initial product disappeared). The mixture was
extracted with water and dichloromethane. The
organic phase was treated with sodium sulfate,
filtered and concentrated until completely dry. Yield:
4.7.7. 5-(2-Chloroethyloxycarbonyloxy)-6-(5-chloropy-
ridin-2-yl)-7-oxo-5,6-dihydropyrrolo-[3,4b]pyrazine, ( )-
7. 2-Chloroethyl chloroformate (0.8 mL) was added
to a suspension of ( )-8 (1 g) and anhydrous pyridine
(1.2 mL), in anhydrous dichloromethane (20 mL)
under N2 at 0°C. The mixture was stirred at 25°C for
7 h (it was monitored by TLC until the initial
product disappeared). After that, it was extracted
with water and dichloromethane. The organic phase
was dried over sodium sulfate, filtered and evaporated
under reduced pressure. Yield: 98%, mp 177–178°C;
IR (cm−1): 1766, 1741, 1H NMR (CDCl3), l (ppm):
8.86 (dd, 2H, 2CH), 8.52 (d, 1H, CH, 2JHH=8.98
Hz), 8.39 (d, 1H, CH, 3JHH=2.58 Hz), 7.99 (s, 1H,
1
89%; mp 212–216°C; IR (KBr): 1732 cm−1. H NMR
(CDCl3) l 8.86 (dd, 2H, 2CH), 8.56 (d, 1H, CH,
3JHH=8.72 Hz), 8.41 (s, 1H, CH), 8.28 (d, 1H, CH,
4JHH=2.52 Hz), 7.96 (dd, 1H, CH, 4JHH=2.52 Hz,
3JHH=8.72 Hz), 7.79 (dd, 2H, 2CH), 7.55 (d, 1H,
CH), 7.42 (t, 2H, 2CH); 13C NMR (CDCl3) l (ppm):
166.1 (CꢀO), 163.6 (C), 155.9 (CꢀO), 149.1 (CH),
148.6 (CH), 148.1 (C), 147.5 (CH), 144.7 (C), 138.8
(CH), 134.4 (CH), 130.6 (CH), 129.3 (C), 129.1 (CH),
129.0 (C), 116.5 (CH). MS (ESI+) m/z (%): 389 [(M+
Na)+, 60%], 405 [(M+K)+, 100%].
2
3
CH), 7.82 (dd, 1H, CH, JHH=8.98 Hz, JHH=2.58),
4.53 (m, 2H, CH2), 3.74 (t, 2H, CH2), 13C NMR
(CDCl3), l (ppm): 162.8 (CꢀO), 154.6 (CꢀO), 153.6
(C), 148.6 (CH), 148.6 (CH), 147.8 (C), 147.1 (CH),
144.3 (C), 138.6 (CH), 128.9 (C), 116.3 (CH), 80.8
(CH), 68.5 (CH2), 41.3 (CH2). EM-ESI+: [M+Na]=
391.0.
4.7.5.
5-(Chloromethyloxycarbonyloxy)-6-(5-chloropy-
ridin-2-yl)-7-oxo-5,6-dihydropyrrolo[3,4b]pyrazine, ( )-
5. Chloromethyl chloroformate (0.8 mL, 7.61 mmol)
was added to a solution of the ( )-8 (1 g) and anhy-
drous
pyridine
(1.2
mL)
in
anhydrous
dichloromethane (10 mL) under N2 at 0°C.
Acknowledgements
The mixture was stirred at 25°C for 17 h (it was
monitored by TLC until the initial product disap-
peared). After that, it was extracted with water and
dichloromethane. The organic phase was treated with
sodium sulfate, filtered and evaporated under reduced
pressure. Yield: 86%, mp 135–137°C; IR (cm−1): 1748,
1804, 1H NMR (CDCl3), l (ppm): 8.89 (dd, 2H,
The authors gratefully recognize the support from the
Spanish CICYT with the project BIO2000-0747-C05-
02 FEDER 2FD97-1255-C02-02. The authors thank
CAM for a PhD. fellowship for Mr. Palomo. We
thank Resindion srl for the gift of Sepabeads resin
and we gratefully recognize the help from Mr. Dami-
nati (Resindion). We acknowledge support from Dr.
Martinez (Novo) and the interesting correction of
English from Angel Berenguer.
2
2CH), 8.50 (d, 1H, CH, JHH=8.85 Hz), 8.37 (d, 1H,
3
CH, JHH=8.85 Hz), 7.97 (s, 1H, CH), 7.80 (dd, 1H,
CH, 2JHH=2.52 Hz, 3JHH=8.88 Hz), 5.81 (dd, 2H,

438
J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 429–438
R.; Mateo, C.; Guisa´n, J. M. Bioorg. Med. Chem. Lett.
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