Enzymatic syntheses and selective hydrolysis of O-β-d- galactopyranosides using a marine mollusc β-galactosidase

Article abstract of DOI:10.1016/j.bmcl.2004.10.016

The use of crude extract of the hepatopancreas of Aplysia fasciata, a large mollusc belonging to the order Anaspidea containing a β-galactosidase activity, was reported for the synthesis of different galactosides. Good yields with polar acceptors and the

Full text of DOI:10.1016/j.bmcl.2004.10.016

Bioorganic & Medicinal Chemistry Letters 15 (2005) 139–143
Enzymatic syntheses and selective hydrolysis of
O-b-D-galactopyranosides using a marine mollusc b-galactosidase
Assunta Giordano, Annabella Tramice, Giuseppina Andreotti, Ernesto Mollo and
Antonio Trincone*
Istituto di Chimica Biomolecolare C.N.R., Via Campi Flegrei 34, 80072 Pozzuoli, Napoli, Italy
Received 29 July 2004; revised 7 October 2004; accepted 7 October 2004
Available online 28 October 2004
Abstract—The use of crude extract of the hepatopancreas of Aplysia fasciata, a large mollusc belonging to the order Anaspidea con-
taining a b-galactosidase activity, was reported for the synthesis of different galactosides. Good yields with polar acceptors and the
uncommon b-1-3 selectivity in the transgalactosylation reactions with most of the acceptors were observed. A b-1-2 selectivity in the
hydrolytic conditions was also observed and discussed.
Ó 2004 Elsevier Ltd. All rights reserved.
O-b-D-galactopyranosyl moiety linked to xylopyranose
ring represents an interesting disaccharidic template
found in different oligosaccharides of biological interest.
The b-1-2 interglycosidic linkage is present in the oligo-
saccharins, substances possessing hormone-like effects
on plants1 while the b-1-4 is found in the region between
glycosaminoglycan (GAG) chains and protein parts, in
serine-linked connective tissue proteoglycan. Interest-
ingly this structure contains Gal-(b-1-3)-Gal as terminal
disaccharidic unit.2 In addition, free b-1-3 and b-1-4
Gal-Xyl disaccharides are useful substrates, as noninva-
sive diagnostic tool, for intestinal lactase, which is an en-
zyme involved in adult-type alactasia.3 Chemical and
enzymatic procedures were used for the preparation of
these compounds, the former3 being cumbersome due
to numerous protection–deprotection steps. The enzy-
matic procedures are based on glycosyl hydrolases, able
to catalyze the formation of glycosidic bonds in stereo-
specific manner, and on other enzymes such as glycosyl
transferases.4
forming any desired glycosidic bond, being of current
significance in this field.7
Recently we focused our attention on the sea hare Aply-
sia fasciata Poiret 1789, a large mollusc easily collectable
and very common in Mediterranean habitats belonging
to the order Anaspidea.8
We found a potent b-galactosidase activity in the
hepatopancreas of this organism, an order of magnitude
higher with respect to other mollusc extracts.5 In the
preliminary reactions performed, this catalytic activity
attracted our attention in that it was able to synthesize
b-galactosides in good yield with interesting regioselec-
tivity towards secondary hydroxyl groups.6
In this communication we report on the synthesis of dif-
ferent b-galactopyranosides of xylose-containing mole-
cules obtaining interesting compounds in high yield by
simple procedures avoiding protein purification steps.
The screening of acceptors has also been enlarged to
hexopyranose structures and to a 1,2-unsaturated enol
ether derivative (glucal).
The marine environment furnished different sources of
glycosyl hydrolases5 and we are actively involved in
the search for these enzymes from marine organisms;6
the effort for the identification of different enzymes, each
The crude extracts from hepatopancreas9 of A. fasciata
contained different b-glycosyl hydrolases the most sig-
nificant being the b-galactosidase activity (40% of the
total b-D-fuco-b-D-gluco-b-D-galacto- and b-D-manno-
sidase activities). Lactose, p-nitrophenyl b-D-galacto-
pyranoside and o-nitrophenyl b-D-galactopyranoside
are substrates for the b-galactosidase; the latter
Keywords: b-Galactosidase, Aplysia fasciata; Enzymatic synthesis.
*
Corresponding author. Tel.: +39 0818675095; fax: +39 0818041770;
e-mail: atrincone@icmib.na.cnr.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.016

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A. Giordano et al. / Bioorg. Med. Chem. Lett. 15 (2005) 139–143
Table 1. Syntheses conducted with crude extract of the hepatopancreas of the mollusc Aplysia fasciata with different acceptors
Donor
Acceptor
Products
EMa
Ya (%)
2-NP-Gal
2-NP-Gal
2-NP-Gal
2-NP-Gal
2-NP-Gal
2-NP-Gal
2-NP-Gal
2-NP-Gal
4-NP-Gal
4-NP-Gal
2NP-Gal
2NP-Gal
2NP-Gal
2NP-Gal
2NP-Gal
D-Xylose
1, 2
10
0.5
5
60
12
48
75
33
30
18
50
35
20
—
38
13 + 5
75
3
Allyl b-D-xylopyranoside
Allyl b-D-xylopyranoside
Methyl b-D-xylopyranoside
Benzyl a-D-xylopyranoside
Benzyl b-D-xylopyranoside
4-NP a-D-xylopyranoside
4-NP b-D-xylopyranoside
Glucal
3
3, 4
5, 6
7, 8
10
5
9, 10
11, 12
13, 14, 15
16
5
10
10
5
N-Acetyl-D-glucosamine
N-Acetyl-D-galactosamine
Methyl b-D-galactopyranoside
2NP-Gal
17
—
5
10
10
—
10
0.5
18, 19
20, 21
22, 23
24
D-Galactose
5
a EM = acceptor/donor molar ratio; Y = yield. 2-NP-Gal = o-nitrophenyl b-D-galactopyranoside; 4-NP-Gal = p-nitrophenyl b-D-galactopyranoside.
resulted the best one (456U/mg) while p-nitrophenyl
b-D-galactopyranoside (156U/mg) and lactose (3.5U/
mg) have intermediate and poor activity, respectively.
ative 3 was formed in 12% yield (with respect to the
amount of the acceptor used) using the donor in molar
excess (2:1). For the structural assignment of 3 diagnos-
tic signals are the anomeric doublets of the xylose (ppm
98.5/4.52, J = 6.5Hz) and of the galactose (ppm 101.1/
4.60, J = 7.9Hz), and the galactosylated C3/H3 of xylose
found at 75.9/3.88ppm). Two additional disaccharidic
products due to the galactosylation of the donor and
accounting for 14% yield, were also observed in this
reaction. The yield of 3 can be increased to 48% by using
a 5-molar excess of the acceptor obtaining in this case a
mixture of galactosides in which 3 predominated (70%)
with respect to other two regioisomers 4, b-1-4 (29%)
and trace amount (<1%) of the remaining b-1-2
compound.
In Table 1 are reported the syntheses10 conducted by
using nitrophenyl b-D-galactopyranosides as donor
and different xylose based acceptors. D-Galactopyranose
based acceptors were also tested as well as a 1,2-unsatu-
rated enol ether derivative (glucal). All the products
(Fig. 1) were characterized as native or acetylated deriv-
atives after chromatographic purification as single prod-
ucts or in mixtures. 1H NMR spectra, DEPT and COSY
and 1H–13C NMR correlations generally permit the
assignments of chemical shifts of compounds obtained
and the unambiguous structure assignment. In the
COSY spectrum starting from the aglycon-linked or free
sugar anomeric signals and following the correlations
through pyranosidic protons it is easy to detect the posi-
tion of glycosylation for the upfield shift of the signal
due to the absence of acetyl group.
The yield of reaction with alkyl aglycon could be further
increased reaching a value as high as 75% increasing the
molar excess of acceptor (methyl b-D-xylopyranoside) to
10-fold. Also in this case the products obtained were in
the ratio (b-1-3, 5 and b-1-4, 6) 73:26 as for allyl group.
Unfortunately the overlap of diagnostic signals in the
1H NMR spectrum of the mixture of acetylated deriva-
tives hampered the direct unambiguous signal assign-
ment of regioisomers, thus the structures of 5 and 6
were established by COSY spectra of purified acetylated
derivatives. Starting from H1 of xylose at 4.40ppm, H3
was found at 3.82ppm (galactosylated position) for
compound 5 and starting from H1 of xylose at
4.38ppm, H4 was found at 3.81ppm (galactosylated
position) for compound 6. No detectable presence of
b-1-2 isomer was noticed.
When the free sugar D-xylose was the acceptor in a reac-
tion conducted using it in a 10-fold molar excess, good
yield (60% with respect to the donor added) of the
galactosides 1 (O-b-D-galactopyranosyl (1-3)-D-xylose,
60%) and 2 (O-b-D-galactopyranosyl (1-4)-D-xylose,
35%) was obtained. The minor b-1-2 isomer was also pre-
sent in ca. 5%, as established by the integral of anomeric
signals in the 1H NMR spectra of acetylated deriva-
tives.11 The structure of the most abundant regioisomers
1 and 2 were secured by the analysis of diagnostic signals
in the 13C NMR spectra of native mixture (D2O).11 The
yield of this reaction was satisfactorily high if compared
to those obtained using other enzymes from different
sources12 in similar conditions. This mixture could be
directly suitable for diagnostic purposes without further
purification of single regioisomers.11 As a comparison
pure compound 2 was obtained by a chemical approach
in 8.8% yield in seven reaction steps.11
Using benzyl a-D-xylopyranoside as acceptor a mixture
of two galactosylated compounds (3:1) was obtained;
the most abundant, 7 was the b-1-4 isomer.6 The minor
product was obtained in enriched form after extensive
purification procedure and was characterized as b-1-3
isomer, 8. The b-anomer of the same acceptor in the
same conditions furnished a mixture of products in
which the b-1-3 was the predominant isomer 9 (60%)
over the b-1-4, 10 (40%).13 The yields (ca. 33%) in both
cases were moderate at 5-molar excess of acceptors.
When the anomeric hydroxyl group of D-xylose was
protected, similar results were obtained. In the case of
allyl b-D-xylopyranoside a single b-1-3 galactosyl deriv-

A. Giordano et al. / Bioorg. Med. Chem. Lett. 15 (2005) 139–143
141
Table 2. 1H NMR signals (disaccharidic moiety) for 11, 12 and 15
O
R2O
R1O
Product
11a
12
15
Galactose
R
OR3
1
2
3
4
5
6
4.07
5.43
4.61
5.14
4.67
5.20
4.99
5.36
3.88
4.02
1 R = OH; R1 = β-Gal; R2 = H; R3 = H
2 R = OH; R1 = H; R2 =β-Gal; R3 = H
5.05
5.44
5.00
5.35
3 R = O-β-allyl; R1 = β-Gal; R2 = H; R3 = H
4 R = O-β-allyl; R1 = H; R2 = β-Gal; R3 = H
5 R = O-β-methyl; R1 = β-Gal; R2 = H; R3 = H
6 R = O-β-methyl; R1 = H; R2 = β-Gal; R3 = H
7 R = O-α-benzyl; R1 = H; R2 = β-Gal; R3 = H
8 R = O-α-benzyl; R1 = β-Gal ; R2 = H; R3 = H
9 R = O-β-benzyl; R1 = β-Gal ; R2 = H; R3 = H
10 R = O-β-benzyl; R1 = H; R2 = β-Gal; R3 = H
11 R = O-α-p-nitrophenyl; R1 = H; R2 = β-Gal; R3 = H
12 R = O-α-p-nitrophenyl; R1 = β-Gal ; R2 = H; R3 = H
13 R = O-β-p-nitrophenyl; R1 = β-Gal; R2 = H; R3 = H
14 R = O-β-p-nitrophenyl; R1 = H; R2 = β-Gal; R3 = H
15 R = O-β-p-nitrophenyl; R1 = H; R2 = H; R3 = β-Gal
3.43
4.14–4.02
3.87
4.06–4.01
Xylose
1
2
3
4
5
5.53
5.12
5.94
3.74
3.60
5.65
5.61
5.52
3.94
5.14
4.90
3.89
5.12
3.85–3.63
4.14–3.64
In bold are the signals of galactosylated positions.
a In C6D6.
OH
OH
The anomeric control of regioselectivity observed with
the latter two aryl acceptors is a well-known phenome-
non reported for these enzymes.14
O
O
HO
β-Gal-O
HO
β-Gal-O
OH
17 NHAc
16
The predominant b-1-4 product obtained with the a-
anomers was also previously detected6 in the galactosyl-
ation of N-acetyl glucosamine 17 with a moderate 20%
yield while the N-acetyl-D-galactosamine was not sub-
strate at all even using it in 10-fold molar excess (Table
1). These results, compared with the clear capability of
the enzyme for the transfer of galactose to methyl b-
galactopyranoside and to o-nitrophenyl b-D-galactopyr-
anoside itself, allow us to conclude that the N-acetyl
group, more than the 4 axial hydroxyl group of N-ace-
tyl-D-galactosamine, is responsible for the nonproduc-
tive orientation of this acceptor into the enzyme
active site and for a different orientation of N-acetyl-
D-glucosamine, which produce in fact the b-1-4
regioisomer.
OH
O-β-Gal
HO
HO
HO
O
O
CH3
O
O
CH3
β-Gal-O
OH
OH
18
19
O2N
O
O2N
OH
O-β-Gal
HO
HO
HO
O
O
O
β-Gal-O
OH
OH
20
21
O-β-Gal
O
OH
HO
β-Gal-O
HO
HO
O
OH
OH
OH
OH
Methyl b-D-galactopyranoside as acceptor furnished
moderate–high yield of two products 18 and 19 (b-1-
3:b-1-6, 81:19, respectively); the reaction with the aryl
version of this galactose acceptor, o-nitrophenyl b-D-
galactopyranoside, was less efficient both in terms of
yield of 20 and 21 (13%) and selectivity: 20 b-1-3
(62%), 21 b-1-6 (38%). In the 1H NMR and COSY spec-
tra of acetylated derivatives of compounds 18 and 19 the
signals of protons at galactosylated positions correlated
with proper anomeric signals: 18 (3.83ppm/4.29ppm
22
23
Figure 1. Galactosyl derivatives synthesized by b-galactosidase of
Aplysia fasciata.
Using as acceptor p-nitrophenyl b-D-xylopyranoside
(molar excess 10) the yield of reaction was 50% and all
the three possible regioisomers were formed with the
b-1-3 being predominant over the other two b-1-4 and
b-1-2 (2:1:1, respectively). These three purified disacchar-
ides were characterized as acetylated derivatives by
COSY experiments. The spectra of 13 (b-1-3) and 14
(b-1-4) regioisomers are in accord to those reported.13
The spectrum of b-1-2 regioisomer, 15 was characterized
by the diagnostic xylose H2 signal at 3.94ppm (Table 2).
Compounds 13, 14 and 15 after deprotection were used
for the analysis of hydrolytic activity of the enzyme (see
below). The a-anomer of the acceptor, p-nitrophenyl a-
D-xylopyranoside, in the same conditions furnished 18%
yield of a mixture of products containing the b-1-4 iso-
mer 11 predominating (62%) over the b-1-3 12 (38%)
(Table 2).
J1,2 = 7.99Hz), 19 (3.85–3.75ppm/4.37ppm, J1,2
7.95Hz). NMR data for compounds 20 and 21 are in
=
accord to those reported.15
Reaction with free D-galactose furnished higher yield
(75%). Three regioisomers were present in the purified
disaccharidic fraction as established by 13C and DEPT
experiments; the b-1-3 and the b-1-6 regioisomers are
in 1:1 ratio; a minor unidentified disaccharide, comigrat-
ing with the former, is also detectable.
Finally glucal can also be used as acceptor furnishing
easy access to the b-1-3 isomer 16 (90% selectivity,
35% yield), which is synthetically interesting for the

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A. Giordano et al. / Bioorg. Med. Chem. Lett. 15 (2005) 139–143
great potential carried out by glucal moiety in chemical
transformation reactions.16
respectively, in the same conditions. These results could
be of a certain interest from a physiological point of
view since galactose was found to be b-1-2 linked to
xylose (xyloglucan) and the enzymatic breakdown of
ingested carbohydrate polymers has been shown to be
very important in this context, in molluscs.20 The b-1-
2 specificity of this b-galactosidase in the hydrolysis
reaction is also of interest in the field of characterization
of large carbohydrate based polymer structures; a num-
ber of commercially available b-galactosidases tested for
b-1-2 hydrolysis was in fact shown to be unsuccessful on
this interglycosidic linkage.21 The b-1-3 selectivity in the
transgalactosylation reactions makes this enzyme very
attractive as alternative catalyst in the synthesis of novel
food components as also found for bovine testes b-
galactosidase.22
Unfortunately modest yields of a mixture of trisaccha-
ridic derivatives was obtained in the two cases reported
in Table 1, namely the autocondensation of o-nitrophen-
yl b-D-galactopyranoside (5%) and the galactosylation
of 5 (3%). The ESI-MS spectra (Q-Tof mass spectro-
meter) of these compounds after purification and
acetylation account for their trisaccharidic nature; no
further efforts were made for their structural
characterization.
Although all these reactions were performed with crude
extract of the hepatopancreas of A. fasciata the
overwhelming presence of b-galactosidase activity, the
regularity of the overall results and the observation of
the known anomeric control of regioselectivity let us
to consider some conclusions about yields and
selectivity.
Acknowledgements
The authors wish to thank E. Pagnotta for skillful
technical assistance and A. Maiello, V. Mirra and S.
Zambardino of the NMR service of ICB-Naples,
for running NMR spectra. The present research was
partially supported by Regione Campania, L.R. N.5
28.03.2002 Research project.
The results reported in Table 1 indicate a clear prefer-
ence of the Aplysia enzyme for the galactosylation of
polar acceptors. Owing to the specificity of acceptor site
of most galactosidases for compounds with phenyl
groups,13 the yields obtained in the reactions using free
or methyl derivative of xylose and methyl b-galactopyr-
anoside and D-galactose, are interestingly high. In fact,
for example, the enzyme from A. oryzae was reported
to have a very low affinity for these polar acceptors thus
resulting in low yield using the same acceptor excesses17
and the E. coli b-galactosidase catalyzed the synthesis of
6 in 33% yield using, as in our case, 10-fold molar excess
of methyl b-D-xylopyranoside.13 Moreover no product
formation was observed using b-galactosidase from
bovine testes and a polar acceptor such as 2-deoxy-D-
galactopyranose.18
References and notes
1. Fry, S. C. Trends Plant Sci. 1996, 1, 326.
2. Fukase, K.; Yasukochi, T.; Suda, Y.; Yoshida, M.;
Kusumoto, S. Tetrahedron Lett. 1996, 37, 6763.
3. Rivera-Sagredo, A.; Fernandez-Mayoralas, A.; Jimenez-
Barbero, J.; Martin-Lomas, M. Carbohydr. Res. 1992, 228,
129.
4. Garcia-Junceda, E.; Garcia-Garcia, J. F.; Bastida, A.;
Fernandez-Mayoralas, A. Bioorg. Med. Chem. 2004, 12,
1817.
Another interesting characteristic of this enzyme is the
uncommon b-1-3 selectivity in the transgalactosylation
reactions with most of the acceptors. Using free xylose
or its b-allyl and methyl derivative the b-1-3 isomer
was always selectively formed as in the case of methyl
b-D-galactopyranoside and glucal. With b-aryl linked
aglycons for both xylose and galactose this b-1-3
selectivity is again expressed although it is lost with
a-anomers. However the influence of aryl groups as
aglycones is not limited to the yield of reaction but also
to the regioselectivity as shown comparing the results of
the reactions using the p-nitrophenyl and benzyl
xylopyranosides.
5. Kusaiykin, M. I.; Burtseva, Y. V.; Svetasheva, T. G.;
Sova, V. V.; Zvyagintseva, T. N. Biochemistry (Moscow)
2003, 68, 384.
6. Giordano, A.; Andreotti, G.; Mollo, E.; Trincone, A. J.
Mol. Catal. B: Enzym. 2004, 30, 51.
7. Scigelova, M.; Singh, S.; Crout, D. H. G. J. Mol. Catal. B:
Enzym. 1999, 6, 483.
8. Carefoot, T. Oceanogr. Mar. Biol. Ann. Rev. 1987, 25, 167.
9. Twenty animals were carefully dissected to obtain 52g of
hepatopancreas, which were homogenized in ca. 2.5–3 vol/
w of acetate buffer 50mM pH5; clear protein solution
(170mL 1.19mg/mL) was obtained after centrifugation.
After dialysis and ultrafiltration the crude extract con-
tained a final concentration of 6mg/mL of total protein.
Activities were measured using nitrophenyl glycosides
substrates as described in Ref. 6 or lactose. One unit
corresponds to the amount of enzyme hydrolyzing one
nanomol of substrate in 1min/mg of total proteins.
10. The enzymatic syntheses were performed using 0.080–
0.30mmol of the donor dissolved with the molar excesses
of acceptors as indicated (Table 1) in 1–10mL of acetate
buffer 50mM pH5.6. 3000U/mmol of donor of b-galac-
tosidase activity of the crude hepatopancreas extract were
used as biocatalyst; the reactions were started in sealed
vials incubating the mixtures under agitation at 30°C up
to total donor consumption (1–5h; TLC monitoring
CHCl3/MeOH/H2O 65:25:4 by vol or EtOAc/MeOH/
The easy enzymatic synthesis and purification of all gal-
actosides of p-nitrophenyl b-D-xylopyranoside above
reported (compounds 13, 14 and 15) prompted us to
perform kinetic analysis in the hydrolytic conditions
for these products. The hydrolysis reactions19 formed
p-nitrophenyl b-D-xylopyranoside and the rate of
hydrolysis was compared for each isomer. The ratio of
the hydrolysis rates k1-2/k1-4 resulted 4.1 while k1-2/k1-3
resulted 1.9; as a matter of fact after 3h the b-1-2 isomer
was hydrolyzed at an extent of ca. 60% while only 30%
and 12% of b-1-3 and b-1-4 isomers were consumed,

A. Giordano et al. / Bioorg. Med. Chem. Lett. 15 (2005) 139–143
143
H2O 70:20:10 by vol). The reactions were stopped by
heating the mixtures at 90–100°C for 5min and the
products purified by different procedures (reverse phase
(RP18), water/methanol gradients; silica gel chromatogra-
phy, CHCl3/MeOH or EtOAc/MeOH gradients; Biogel
P2, water; preparative TLC, EtOAc/MeOH/H2O 70:20:10
by vol). The products were detected in TLC by a-naphthol
reagent. Acetylation was performed overnight in Ac2O/
Pyr 1:2 at room temperature. NMR studies of acetylated
derivatives were carried out using Bruker instruments 300
or 400MHz in CDCl3 or C6D6; D2O was used for
underivatized compounds.
16. Look, G. C.; Wong, C.-H. Tetrahedron Lett. 1992, 30,
4253.
17. Giacomini, C.; Irazoqui, G.; Gonzalez, P.; Batista-Viera,
F.; Brena, B. M. J. Mol. Catal. B: Enzym. 2002, 19-20,
159.
18. Gambert, U.; Gonzalez Rio, R.; Farkas, E.; Thiem, J.;
Bencomo Verez, V.; Liptak, A. Bioorg. Med. Chem. 1997,
5, 1285.
19. The hydrolysis reaction was conducted solubilizing the
disaccharides (ca. 2.5mM) in acetate buffer (1mL) 50mM
pH5.6 and adding equal amount of the enzyme (9000U/
mmol) for each compound. The p-nitrophenyl b-D-xylo-
pyranoside liberated by the enzymatic hydrolysis was
detected and quantitatively analyzed by HPLC (lBond-
apak-NH2, acetonitrile/water 8:2, 1mL/min, UV detec-
tion). Preliminary experiments with the extract secured
that p-nitrophenyl b-D-xylopyranoside was not substrate
for any enzymatic activity.
11. Aragon, J. J.; Canada, F. J.; Fernandez-Mayoralas, A.;
Lopez, R.; Martin-Lomas, M.; Villanueva, D. Carbohydr.
Res. 1996, 290, 209.
12. Montero, E.; Alonso, J.; Canada, F. J.; Fernandez-
Mayoralas, A.; Martin-Lomas, M. Carbohydr. Res. 1998,
305, 383.
13. Lopez, R.; Fernandez-Mayoralas, A. J. Org. Chem. 1994,
59, 737.
20. Foster, G. G.; Hodgson, A. N.; Boyd, C. S. Comp.
Biochem. Phys. Part B 1999, 122, 47.
14. Crout, D. H. G.; Vic, G. Curr. Opin. Chem. Biol. 1998, 2,
98.
21. Edwards, M.; Bowman, Y. J.; Dea, I. C.; Reid, J. S.
J. Biol. Chem. 1988, 263, 4333.
15. Giraud-Chiffoleau, V.; Spangenberg, P.; Dion, M.; Rab-
iller, C. Eur. J. Org. Chem. 1999, 4, 757.
22. Schroder, S.; Schmidt, U.; Thiem, J.; Kowalczyk, J.;
Kunz, M.; Vogel, M. Tetrahedron 2004, 60, 2601.