Enantioselective esterase activity of an industrial glutaryl acylase

Article abstract of DOI:10.1016/j.tetasy.2005.06.024

The unexpected esterase activity of an industrial glutaryl acylase was investigated. Glutaryl esters of a series of primary and secondary alcohols as well as of phenols were all efficiently hydrolyzed, the only exception being the sterically hindered glutarate of thymol. The enantioselectivities of the acylase, which were evaluated with three of these substrates, were quite low (E values ranging between 1.9 and 7.2), but were significantly improved by substrate and/or solvent engineering. Enantiomerically enriched hydrolyzed alcohols and unreacted glutarates can be easily separated by selective extraction, thus avoiding chromatographic steps.

Full text of DOI:10.1016/j.tetasy.2005.06.024

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2509–2513
Enantioselective esterase activity of an industrial glutaryl acylase
Sara Adani,a,b Stefano Raimondi,b Luca Forti,b Daniela Montia and Sergio Rivaa,*
aIstituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., Via Mario Bianco 9, 20131 Milano, Italy
b
`
Dipartimento di Chimica, Universita di Modena & Reggio Emilia, Via Campi 183, 41100 Modena, Italy
Received 1 June 2005; accepted 14 June 2005
Abstract—The unexpected esterase activity of an industrial glutaryl acylase was investigated. Glutaryl esters of a series of primary
and secondary alcohols as well as of phenols were all efficiently hydrolyzed, the only exception being the sterically hindered glutarate
of thymol. The enantioselectivities of the acylase, which were evaluated with three of these substrates, were quite low (E values rang-
ing between 1.9 and 7.2), but were significantly improved by substrate and/or solvent engineering. Enantiomerically enriched hydro-
lyzed alcohols and unreacted glutarates can be easily separated by selective extraction, thus avoiding chromatographic steps.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
adipates can also be efficiently hydrolyzed) but are also
quite flexible concerning the amine substituent, which
can be significantly different from a b-lactam skeleton.
As a matter of fact, not only was a series of glutarylated
amino acids and glutarylated amines quite efficiently
hydrolyzed by an industrial glutaryl acylase commercial-
ized by Recordati SpA (GAR), but this enzyme also
showed a significant enantiopreference for the respective
L-enantiomers.3
Glutaryl acylases (GAs) are enzymes industrially
exploited for the two-step biocatalyzed production of
7-aminocephalosporanic acid 1 (7-ACA, Scheme 1)
from cephalosporin C 2 via glutaryl-7-ACA 3 (Glu-7-
ACA).1 These proteins have been optimized in order
to efficiently hydrolyze 3,1,2 but it has been shown that
they also possess a broad substrate tolerance. Our data3
along with a few other reports4 have demonstrated that
GAs do require an amide carrying a carboxylated side
chain (glutarates are the best substrates, succinates and
Preliminary experiments indicated that GAR was also
able to catalyze the enantioselective hydrolysis of gluta-
ryl esters.3b Herein, we report the results of a much more
detailed investigation on the esterase activity of this
enzyme and on the parameters that can influence its
enantioselectivity.
H
N
S
7
R
3
N
R'
COOH
O
2. Results and discussion
A recent paper has described the structure of the active
site of a GA from Pseudomonas diminuta KAC-1 com-
plexed with 3.5 X-ray analysis of the crystallized protein
identified three substrate moieties that were specifically
recognized by this enzyme: the glutaric chain, the b-lac-
tam nucleus and the acetate substituent in C-3. We have
previously shown that only the glutarate side chain (or a
similar substituent derived by the condensation of 1 or 5
with the anhydride of a dicarboxylic acid) is really
needed by GAR, and we were particularly surprised to
measure significant esterase activity on the model glutar-
yl ester 8a.3
1 : R = H ; R' = CH2OAc (7-ACA)
2 : R = HOOC-CHNH2-(CH2)3-CO ; R' = CH2OAc
3 : R = HOOC-(CH2)3-CO ; R' = CH2OAc
4 : R = HOOC-CH2-O-CH2-CO ; R' = CH2OAc
5 : R = H ; R' = CH3 (7-ADCA)
6 : R = HOOC-CH2-O-CH2-CO ; R' = CH3
Scheme 1. Structures of cephalosporinic amides.
*
Corresponding author. Tel.: +39 02 2850 0032; fax: +39 02 2890
1239; e-mail: sergio.riva@icrm.cnr.it
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.06.024

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S. Adani et al. / Tetrahedron: Asymmetry 16 (2005) 2509–2513
In order to obtain a better understanding of this new
GAR hydrolytic activity, a series of glutaryl esters 7a–
13a (Scheme 2) was synthesized. The benzylic alcohol
derivative 7a was chosen as a reference compound, its
initial rate of hydrolysis being 15% that of the ÔnaturalÕ
cephalosporinic substrate 3. Table 1 reports the relative
initial reaction rates of compounds 7a–13a: glutaryl
esters of primary 7a, 9a, 11a and secondary alcohols
8a and 10a as well as of phenols 12a were all efficiently
hydrolyzed, the only exception being the sterically hin-
dered glutarate of thymol 13a.
Three of these substrates 8a–10a were racemates and
therefore GAR enantioselectivity was also evaluated,
although the obtained E-values6 were quite low (be-
tween 1.9 and 7.2, Table 1).
OR
OR
Several methods are currently available to improve the
enantioselectivity of a hydrolytic enzyme, most of them
exploiting molecular biology and, specifically, random
mutagenesis techniques.7 Another approach is based
on the optimization of the reaction conditions via the
so-called Ôsubstrate engineeringÕ8 or Ômedium engineer-
ingÕ.8,9 Considering the Ôsubstrate engineeringÕ first, alco-
hol 8 was condensed with three different dicarboxylic
anhydrides (diglycolic,10 succinic and adipic11 anhy-
drides) and the corresponding esters 8b–d submitted to
GAR action. As shown in Table 1, the hydrolysis rates
of these compounds were lower in comparison with
8a, but the E-values did improve, particularly with
succinyl derivative 8c.
7 : R = H
8 : R = H
7a : R = CO-(CH2)3-COOH
7b : R = CO-CH2-O-CH2-COOH
8a : R = CO-(CH2)3-COOH
8b : R = CO-CH2-O-CH2-COOH
8c : R = CO-(CH2)2-COOH
8d : R = CO-(CH2)4-COOH
OR
O
OR
O
9 : R = H
10 : R = H
10a : R = CO-(CH2)3-COOH
9a : R = CO-(CH2)3-COOH
In the next series of experiments, water miscible organic
cosolvents were added to the reaction mixtures (Ôsolvent
engineeringÕ), as in the past it had been demonstrated
that they can significantly influence the enantioselectiv-
ity of hydrolytic enzymes.8,12 Since, to the best of our
knowledge, no data are available on the effect of organic
cosolvents on GAsÕ activity, initially the hydrolysis of
the glutaryl ester of benzylic alcohol 7a was studied in
the presence of various amounts (v/v) of different
cosolvents. As shown in the first two columns of Table
2, with the exception of acetonitrile all the solvents were
well-tolerated when used at 20% v/v, whereas GAR per-
formances were strongly affected by the presence of
higher amounts (40% v/v) of organic modifiers, the
activity being reduced to 25% in the best case. The ki-
netic resolution of 8a was then performed in the
presence of 20% v/v of the best cosolvents, and, as
shown in the last column of Table 2, a significant posi-
tive effect on the enantioselectivity of the enzyme was
observed.
OR
MeO
OR
MeO
OMe
12 : R = H
11 : R = H
12a : R = CO-(CH2)3-COOH
11a : R = CO-(CH2)3-COOH
OR
13 : R = H
13a : R = CO-(CH2)3-COOH
Scheme 2. Structures of compounds 7–13.
Table 1. GAR-catalyzed hydrolysis of glutaryl, diglycoyl, succinoyl, or
adipoyl esters
Compound
Relative ratea
E-valuesb
In the last experiment, the hydrolysis of the best sub-
strate (the succinyl derivative 8c, E = 18.5, Table 1)
was performed in the presence of 20% v/v MeOH (the
best cosolvent for the hydrolysis of 8a). However, it
was found that the positive effect of these two
7a
8a
100
33
112
16
243
262
0
83
33
3
7.2
3.2
1.9
9a
10a
11a
12a
13a
7b
As a diglycolic anhydride has never been used before for these kind of
reactions, we decided to gain some additional information on the
derivative of this dicarboxylic anhydride. Acylation of 7-ACA 1 and
7-ADCA 5 was quite neat with the corresponding amide derivatives 4
and 6 (Scheme 1) hydrolyzed by GAR with similar relative rates,
7.2% and 7.7%, respectively, in comparison with 3. As diglycolic
anhydride is a quite reactive acylating agent, it might become a useful
alternative to glutaric anhydride for the GAR-catalyzed kinetic
resolutions of racemic amides.
8b
8c
5.4
18.5
12.4
8d
10
a Reactions conditions: see Experimental part.
b See Ref. 6. Reactions were monitored by chiral HPLC (compounds
8a–d) or chiral GC (compounds 9a and 10a) in order to evaluate the
respective ee and degrees of conversion (c) values (for details see
Experimental).

S. Adani et al. / Tetrahedron: Asymmetry 16 (2005) 2509–2513
2511
Table 2. GAR-catalyzed hydrolysis of glutaryl ester 7a and 8a in the
presence of organic cosolvents
to significantly improve the performances of a biocata-
lyst in terms of enantioselectivity.
Cosolvent
7a, Relative ratesa 8a, Relative ratesa E-valuesb
20% v/v 40% v/v 20% v/v
4. Experimental
—
100
89
71
76
41
93
106
3
100
17
5
100
84
58
80
61
61
62
n.d.
7.2
15.8
7.6
11.6
5.9
11.6
12.5
n.d.
MeOH
i-PrOH
Acetone
Dioxane
DMSO
DMF
4.1. Materials and methods
7
Glutaryl Acylase (GAR) and a sample of glutaryl-7-
ACA 3 were a gift from Recordati S.p.A. (Opera, MI,
Italy). All other reagents and solvents were from Al-
drich. TLC: precoated silica gel 60 F254 plates (Merck).
Flash chromatography: silica gel 60 (70–230 mesh,
Merck).
n.d.
10
26
n.d.
Acetonitrile
a Reactions conditions: see Experimental.
b See Ref. 6. Reactions were monitored by chiral HPLC.
Hydrolytic reactions were monitored at 25 ꢁC using a
718 STAT Titrino automatic titrator (Metrohm Ltd).
HPLC analyses: Jasco HPLC instrument (model 880-
PU pump, model 870-UV/vis detector, k: 200 nm) and
a Licrospher 100 RP-18 (5 lm, Merck) reverse phase
analytical column or a Chiralcel OD column. GC anal-
yses: Hewlett Packard 5890 series II instrument and a
capillary chiral column (DMePentilBETACDX column,
25 m · 0.25 mm ID · 0.15 lm film thickness, MEGA).
1H spectra at 200 MHz were recorded on a Bruker
DPX-200.
parameters was not additive, as the measured E value
was only 9.8.
Reactions with compounds 8a (in the presence of 20%
v/v MeOH), 8c and 8d were scaled up (Table 3). The
transformations of 8a and 8d were found to be even
more enantioselective than in the small scale experi-
ments, while the data obtained with 8c were worse than
expected. A possible explanation might be related to the
longer reaction times needed with this substrate, which
in the meantime might suffer concomitant aspecific
spontaneous chemical hydrolysis. In all cases, the alco-
hol product and the residual unreacted esters could be
separated by selective extraction from the aqueous reac-
tion solutions, avoiding chromatographic or distillation
steps during work-up.
4.2. Synthesis of glutaryl and diglycoyl amides 4 and 6
7-ACA or 7-ADCA (5 mmol) was dissolved in 20 mL of
1 M NaHCO3. The anhydride (1 equiv) was dissolved in
5 mL of acetone and the two solutions mixed and left to
react for 3 h (TLC: n-BuOH–AcOH–H2O = 6:2:2). Ace-
tone was evaporated, the water solution acidified to
pH 1.5 with 1 M HCl and extracted three times with
100 mL of EtOAc. The organic layer was evaporated
and the solid residue washed on a Buchner funnel with
20 mL of EtOAc and then dried. Products structures
Table 3. Preparative scale GAR-catalyzed hydrolysis of 8a, 8c, and 8da
Substrate Cosolvent Reaction Conv. eeP
time (h)
eeS
E-valuesb
1
8a
20% v/v
MeOH
—
8
48.7
79.5 75.6 19.8
were confirmed by H NMR. Compound 4 (200 MHz,
DMSO-d6) d: 8.72 (1H, d, NH); 5.71 (1H, dd,
J1 = 8.4 Hz, J2 = 5.0 Hz, NH–CH); 5.11 (1H, d,
J = 5.0 Hz, CH–S); 4.99 and 4.68 (1H each, d each,
J = 18.0 Hz, CH2–O); 4.17 and 4.12 and 4.09 (2H each,
s each, CH2COOH, CH2CONH, CH2O); 2.07 (3H, s,
CH3). Compound 6 (200 MHz, DMSO-d6) d: 8.63 (1H,
d, NH); 5.60 (1H, dd, J1 = 8.6 Hz, J2 = 4.6 Hz,
NH–CH); 5.05 (1H, d, J = 4.6 Hz, CH–S); 4.12 and
4.09 (2H each, s each, CH2COOH, CH2CONH); 3.54
and 3.36 (1H each, d each, J = 18.1 Hz, CH2–S); 2.02
(3H, s, CH3).
8c
8d
71
23
50.9
39.8
65.2 67.7
9.4
—
80.4 53.1 15.5
a Reactions conditions: see Experimental.
b See Ref. 6. Reactions were monitored by chiral HPLC.
3. Conclusion
GAR is an industrial enzyme that has been developed
for the efficient hydrolysis of a specific substrate, namely
Glu-7-ACA 3. Due to practical and scientific interests in
this group of enzymes, an increasing number of GAs
have become available13 and, moreover, wild-type en-
zymes have been modified by protein engineering (site-
directed and/or random mutagenesis) in order to better
understand their catalytic mechanism and to alter their
substrate specificity.14 Herein, we have shown that an
industrial GA possesses a significant esterase activity
and that its enantioselectivity can be modulated by
substrate and solvent engineering. All this information
allowed us to conclude that GAs might be ideal targets
for further studies, combining random mutagenesis
techniques and solvent and/or substrate optimization
4.3. Synthesis of glutaryl, diglycoyl, succinyl, and adipoyl
esters 7a–14a, 8b–d
The respective alcohol 7–14 (5 mmol) was dissolved in
10 mL dioxane. Glutaryl anhydride (1 equiv) was dis-
solved in 10 mL of dioxane and the two solutions then
mixed and allowed to react either at 70 ꢁC or by micro-
wave irradiation (TLC: appropriate mixture of hexane–
EtOAc–MeOH). The solvent was evaporated, the
residue redissolved in EtOAc and the products extracted
with a 5% w/v NaHCO3 solution. The water solution
was acidified to pH 1.5 with 2 M HCl and extracted

2512
S. Adani et al. / Tetrahedron: Asymmetry 16 (2005) 2509–2513
three times with 50 mL EtOAc. The organic layer was
evaporated and the residue either used as it was or, in
case of contamination with unreacted glutaryl anhy-
dride, purified by flash chromatography. Product
structures were confirmed by 1H NMR (200 MHz,
DMSO-d6): 7a, d: 7.45 (5H, m, ArH); 5.09 (2H, s,
CH2O); 2.39 (2H, t, J = 6.9 Hz, ROOC-CH2); 2.25
(2H, t, J = 6.9 Hz, CH2COOH); 1.76 (2H, m, J =
6.9 Hz, CH2CH2CH2). Compound 8a, d: 7.35 (5H, m,
ArH); 5.86 (1H, q, J = 6.7 Hz, CH); 2.36 (2H, t, J =
7.2 Hz, ROOC-CH2); 2.23 (2H, t, J = 7.2 Hz, CH2-
COOH); 1.73 (2H, quintet, J = 7.2 Hz, CH2CH2CH2);
1.45 (3H, d, J = 6.7 Hz, CH3). Compound 9a, d: 4.22
(1H, dq, J1 = 6.0 Hz, J2 = 4.2 Hz, H-4); 4.10 (1H, dd,
J1 = 11.4 Hz, J2 = 4.1 Hz, CHaO); 4.00 (1H, dd, J1 =
8.4 Hz, J2 = 6.5 Hz, H-5a); 3.99 (1H, dd, J1 = 11.4 Hz,
J2 = 6.0 Hz, CHbO); 3.65 (1H, dd, J1 = 8.4 Hz, J2 =
6.1 Hz, H-5b); 2.35 (2H, t, J = 7.5, ROOC-CH2); 2.25
(2H, t, J = 7.4, CH2COOH); 1.74 (2H, m, CH2CH2-
CH2); 1.26 e 1.31 (3H each, s each, CH3). Compound
10a, d: 5.07 (1H, m, H-5); 4.77 (1H, m, H-2); 2.27
(2H, t, J = 7.2 Hz, ROOC-CH2); 2.24 (2H, t, J =
7.2 Hz, CH2COOH); 1.95 and 1.48 (2H each, m each,
CH2-3 and CH2-4); 1.72 (2H, m, CH2CH2CH2); 1.64
and 1.55 (3H each, br s each, CH3-7 and CH3-8); 1.14
(3H, d, J = 6.3 Hz, CH3-1). Compound 11a, d: 6.67
(2H, s, ArH); 5.00 (2H, s, CH2O); 3.76 (6H, s, m-
(CH3O)); 3.64 (3H, s, p-CH3O); 2.40 (2H, t, J =
7.4 Hz, ROOCCH2); 2.26 (2H, t, J = 7.4 Hz,
CH2COOH); 1.76 (2H, m, J = 7.4 Hz, CH2CH2CH2).
Compound 12a, d: 7.13 (1H, d, J = 8.2 Hz, H-5); 6.89
(1H, d, J = 2.3 Hz, H-2); 6.82 (1H, dd, J1 = 8.2 Hz,
J2 = 2.3 Hz, H-6); 2.58 (2H, t, J = 7.3 Hz, ROOCCH2);
2.33 (2H, t, J = 7.3 Hz, CH2COOH); 2.20 (6H, br s,
ArCH3); 1.84 (2H, m, J = 7.3 Hz, CH2CH2CH2). Com-
pound 13a, d: 7.22 (1H, d, J = 8.6 Hz, H-3); 7.12 (1H, br
d, J = 8.6 Hz, H-4); 6.83 (1H, br s, H-6); 2.89 (1H, m,
CH(CH3)2); 2.64 (2H, t, J = 7.4 Hz, ROOC-CH2); 2.34
(2H, t, J = 7.4 Hz, CH2COOH); 2.25 (3H, s, CH3-5);
1.86 (2H, m, J = 7.4 Hz, CH2CH2CH2); 1.11 (6H, d,
J = 7.4 Hz, CH(CH3)2).
at least. The initial rates of hydrolysis were calculated
from the amount of NaOH solution added in the time
unit. The initial rate of hydrolysis of compound 3
(10.0 lmol/min) was taken as 100.
Table 1: Total volume, 10 mL: 50 mM substrate in H2O,
1 U/mL GAR. Reaction solutions were stirred at 25 ꢁC
in an automatic titrator maintaining a constant pH va-
lue (7.0) by adding 0.1 M NaOH. The initial rates of
hydrolysis were calculated from the amount of NaOH
solution added in the time unit. Experiments were re-
peated in duplicate at least. The initial rate of hydrolysis
of compound 7a (1.5 lmol/min) was taken as 100.
Table 2: Total volume, 10 mL; organic cosolvent 20% or
40% v/v; 50 mM substrate, 1 U/mL GAR. Reaction
solutions were stirred at 25 ꢁC in an automatic titrator
maintaining a constant pH value (7.0) by adding
0.1 M NaOH. The initial rates of hydrolysis were calcu-
lated from the amount of NaOH solution added in the
time unit. Experiments were repeated in duplicate at
least. The initial rates of hydrolysis of compounds 7a
(1.5 lmol/min) and 8a (0.6 lmol/min) were taken as
100%, respectively.
4.5. Enantioselectivity of GA towards racemic esters
(Tables 2 and 3)
Conversion degrees and ee of the hydrolysis of com-
pounds 8a–d were evaluated by chiral column HPLC
(k 254 nm) using a Chiralcel OD column, eluent: hex-
ane–iPrOH–CF3COOH 98:2:0.1, 0.75 mL/min (8b–d).
Retention times (min) at 0.75 mL/min flow rate: (R)-8:
25.08, (S)-8: 33.42; (S)-8a: 37.17, (R)-8a: 33.42. Reten-
tion times (min) at 0.75 mL/min; flow rate: (R)-8:
17.08, (S)-8: 22.08; (S)-8b: 16.58, (R)-8b: 21.46; (R)-8c:
26.42, (S)-8c: 33.08; (R)-8d: 25.96, (S)-8d: 34.21.
The eeProd of compounds 9 and 10 was evaluated by chi-
ral GC (DMePentil-BETACDX column). Compound 9
(previously acetylated): init. T: 60 ꢁC; init. time: 30 min;
rate: 0.5 ꢁC/min; final T: 120 ꢁC; ret. times: 25.3 and
28.6 min. Compound 10: init. T: 50 ꢁC; init. time:
20 min; rate: 1 ꢁC/min; final T: 200 ꢁC; ret. times: 20.6
and 21.9 min.
The esters 8b–d were prepared with similar protocols.
Compound 8b, d: 7.37 (5H, m, ArH); 5.87 (1H, q,
J = 5.7 Hz, CH); 4.23 (2H, s, ROOC-CH2O); 4.08
(2H, s, OCH2COOH); 1.49 (3H, d, J = 5.7 Hz, CH3).
Compound 8c, d: 7.32 (5 H, m, ArH); 5.79 (1H, q,
J = 7.0, CH); 2.51 (4H, m, CH2–COOR); 1.44 (3H, d,
J = 7.0, CH3); Compound 8d, d: 7.30 (5H, m, ArH);
5.89 (1H, q, J = 6.5, CH); 2.34 (4H, m, CH2COOR);
1.68 (4H, m, CH2CH2), 1.52 (3H, d, J = 6.5 Hz, CH3).
4.6. Preparative-scale kinetic resolution of racemic
glutarates (Table 3)
In a typical experiment 800 mg (3.4 mmol) of 8a was dis-
solved in 40 mL of a 4:1 mixture of H2O–MeOH. The
pH was adjusted to 7.0 and the reaction was started
by adding 100 U of GA and monitored by chiral column
HPLC (see above), keeping the pH constant by adding
0.1 M NaOH via the automatic titrator. The reaction
was stopped at 48.7% conversion (approximately 8 h).
The solvent was partially evaporated to eliminate most
of the MeOH and the remaining solution was extracted
with 50 mL EtOAc (two times) to remove the product 8
(79.5 ee, 25% yields). The water phase was adjusted to
pH 3.0 with 2 M HCl and extracted with 50 mL EtOAc
(three times) to recover unreacted 8a (75.6 ee, 43%
yields).
4.4. Relative rates of hydrolysis of 3, 4, 6 and of the
compounds in Table 1
Compounds 3, 4, and 6: Total volume, 10 mL: 50 mM
substrate in H2O, 1 U/mL GAR (1 Unit is defined as
the amount of GAR that hydrolyzes 1 lmol of 3 per
minute at pH 8.0 and at 25 ꢁC. The specific activity of
the GAR sample used herein was 2.3 U/mg). Reaction
solutions were stirred at 25 ꢁC in an automatic titrator
maintaining a constant pH value (8.0) by adding
0.1 M NaOH. Experiments were repeated in duplicate

S. Adani et al. / Tetrahedron: Asymmetry 16 (2005) 2509–2513
2513
Petrounia, I. P.; Arnold, F. H. Curr. Opin. Biotechnol.
2000, 11, 325–330.
Acknowledgements
8. Faber, K.; Ottolina, G.; Riva, S. Biocatal. Biotransform.
1993, 8, 91–132.
We thank Dr. Marco Sanchini, Recordati S.p.A. (Op-
era, MI, Italy), for a generous gift of their GA (both as
an enzyme solution and as an immobilized preparation)
and for authentic samples of compounds 1 and 5.
9. (a) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39,
2226–2254; (b) Carrea, G.; Ottolina, G.; Riva, S. Trends
Biotechnol. 1995, 13, 63–70; (c) Wescott, C. R.; Klibanov,
A. M. Biochem. Biophys. Acta 1994, 1206, 1–9.
10. De Gonzalo, G.; Brieva, R.; Sanchez, V. M.; Bayod, M.;
Gotor, V. J. Org. Chem. 2003, 68, 3333–3336.
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