Synthesis and evaluation of xylopyranoside derivatives as "decoy acceptors" of human β-1,4-galactosyltransferase 7

Article abstract of DOI:10.1039/c0mb00206b

Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled post-translationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosidation of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as "decoy acceptors" of xylosylprotein 4-β-galactosyltransferase, we have heterologously expressed the catalytic domain of the human β-1,4-GalT 7 and studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0mb00206b

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PAPER
Synthesis and evaluation of xylopyranoside derivatives as
‘‘decoy acceptors’’ of human b-1,4-galactosyltransferase 7w
Juan Francisco Garcıa-Garcı
´ ´
a,a Guillermo Corrales,a Josefina Casas,b
a
a
´
´
Alfonso Fernandez-Mayoralas* and Eduardo Garcıa-Junceda*
Received 24th September 2010, Accepted 24th January 2011
DOI: 10.1039/c0mb00206b
Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor
progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal
template and the sugar chains are assembled post-translationally, one sugar at a time, starting
with the linkage of xylose to a serine residue of the core protein and followed by galactosidation
of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime
heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells.
To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability
to act as ‘‘decoy acceptors’’ of xylosylprotein 4-b-galactosyltransferase, we have heterologously
expressed the catalytic domain of the human b-1,4-GalT 7 and studied the ability of a variety of
synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme.
Introduction
Proteoglycans are highly active and complex biomolecules
involved in a wide range of physiological and pathological
processes through their interactions with several proteins.1
They are composed of a core protein usually attached to the
cell membrane and one or more glycosaminoglycan (GAG)
chains attached at specific Ser residues of the core protein.
Scheme 1 Biosynthesis of the proteoglycan tetrasaccharide primer.
GAGs are linear polysaccharides composed of alternating
N-acetylated or N-sulfated glucosamine units and either
glucoronic or iduronic acid. The GAG chains are linked to
the protein core by a tetrasaccharide linker that is shared by all
proteoglycans. The assembly of this tetrasaccharide is carried
out in the Golgi and began with the transfer of a xylose residue
to a specific Ser of the protein chain, catalysed by a xylosyl-
transferase and is continued by the sequential action of specific
glycosyltransferases (Scheme 1).2
biosynthesis and thereby inhibit the assembly of glycos-
aminoglycan chains on endogenous proteoglycan core proteins.3
Okayama et al.3a named these compounds, that being sub-
strates for a particular enzyme are able to inhibit a metabolic
pathway, ‘‘decoy acceptors’’ to distinguish them from
inhibitors, which are compounds that block the enzyme with-
out being modified by it. This concept has been also applied to
suppress the synthesis of the mucin chain in mucoproteins,4 to
down-regulate the expression of sLex ligands5 or to restore
the streptomycin antibiotic activity against aminoglycoside
resistant Escherichia coli strains.6
It is known for more than 30 years that b-D-xylopyranoside
with hydrophobic aglycones can act as primers for GAG
a
´
Departamento de Quımica Bioorganica, Instituto de Quımica
Organica General, CSIC, Madrid 28006, Spain.
´
´
´
Synthesis of GAG chain can be initiated by b-D-xylosides by
joining the intracellular enzyme machinery at the second
glycosyltransferase step (galactosyltransferase I, Scheme 1)
and their ability to prime the assembly of GAG chains and
their composition depends on the structure of the aglycone
moiety.7 In most cases, xylosides prime chondroitin sulfate/
dermatan sulfate (CS/DS) efficiently and heparan sulfate (HS)
only weakly.7b Xylosides containing polycyclic structures,
such as naphthol-derivatives, will prime a mixture of both
HS and CS/DS chains.7b,8 The xyloside-primed GAG chains
E-mail: eduardo.junceda@iqog.csic.es, mayoralas@iqog.csic.es;
Fax: +34 915 644 853; Tel: +34 915 622 900
b
´
Research Unit on BioActive Molecules, Departamento de Quımica
´
´
´
Organica Biologica, Instituto de Quımica Avanzada de Catalun˜a,
CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
E-mail: jcbqob@iiqab.csic.es; Fax: +34 932 045 904;
Tel: +34 934 006 115
w Electronic supplementary information (ESI) available: Cloning and
purification of enzyme b-1,4-GalT 7, experimental details and Table S1
containing data of antiproliferative activity of xylopyranoside deri-
vatives 4, 7, 8, 9, 13, 14 and peracetates 24 and 25. See DOI: 10.1039/
c0mb00206b
c
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can be retained inside the cells but in general are secreted into
the medium in a nearly quantitative way, since the small
hydrophobic aglycones are not able to tie the chains to cell
membranes.
Therefore, xylopyranoside derivatives may be used to inter-
fere with the proteoglycans biosynthesis and thus modulate
various cellular activities, such as cell growth. In this sense,
Esko and co-workers have shown that GAG-priming
xylosides render tumor cells more sensitive to the cytostatic
effects of a-difluoromethylornithine (DFMO), an inhibitor of
de novo polyamine biosynthesis.9 Mani and co-workers, in a
series of elegant papers,8,10 have shown that 2-(6-hydroxy-
naphthyl) b-D-xylopyranoside (14) selectively inhibits the
proliferation of transformed or tumor-derived cells both
in vitro and in vivo. Also, treatment with this xyloside reduced
the average tumor load by 70–97% in a SCID mice model.
The ability of the xylosides to prime the assembly of GAG
chains—and hence their potential activity as antitumor
agents—must be related to their ability to act as acceptors of
the xylosylprotein 4-b-galactosyltransferase (b-1,4-GalT 7 in
Scheme 1). Herein, we describe the heterologous expression in
E. coli of a truncated form of the human b-1,4-GalT 7 and its
biochemical characterization. Also, we report the synthesis of
a family of xylopyranoside derivatives and the study of their
ability to act as substrates or inhibitors of the recombinant
b-1,4-GalT 7.
Fig. 1 Schematic representation of the human b-1,4-GalT 7 topology.
increasing the cellular levels of molecular chaperones;17 to fuse
the recombinant protein to a soluble fusion tag.18
Using the latter approach, soluble human b-1,4-GalT 7 has
been lately expressed in E. coli. Lattard and co-workers
expressed a truncated form of the enzyme—lacking the first
81 N-terminal amino acids that include the transmembrane
and stem regions—fused with the MBP.19 On the other hand,
Qasba and co-workers expressed the human b-1,4-GalT 7
using as fusion partner the human lectin galectin-1.20 Very
recently, Fournel-Gigleux and co-workers have reported the
production of the wild-type b-1,4-GalT 7 and some mutants as
truncated fusion proteins (lacking the 60 N-terminal amino
acids) linked to GST.30 These authors also have identified two
functional regions critical for the organization of UDP-Gal
binding. They also demonstrated the central role of Trp224 in
governing interactions with both donor and acceptor substrates.
Several examples in the literature reported the soluble
expression of the catalytic domain of glycosyltransferases
(GT’s).21 Since the human b-1,4-GalT 7 presents only one
potential glycosylation site (Asn154), we decided to express the
soluble domain of the protein (Fig. 1) in E. coli fused to a six
His tag to allow its purification by immobilized metal affinity
chromatography (IMAC). We took advantage of the presence
of a unique recognition sequence for the endonuclease PstI
that allowed to cut between the aminoacids 49–50 of the
enzyme and therefore to remove the transmembrane region
and the cytoplasmic tail (for experimental details see ESIw).
Comparison of the graphic plots of Kyte–Doolittle hydro-
pathicity profiles of wild-type and truncated b-1,4-GalT 7
clearly shows that the transmembrane region had been
eliminated (Fig. 2). Peptide mass fingerprinting from the
SDS-PAGE band verified that the purified protein had the
b-1,4-GalT 7 expected features. Nine peptides covering
the major part of the amino acid sequence of the soluble
domain were identified (Fig. 3). Almost all the predicted
tryptic peptides with molecular masses falling in the analyzed
m/z range were found in the peptide mass fingerprint of the
b-1,4-GalT 7 soluble domain. Other unassigned peptides were
identified using the FindMod tool.22 Thus, the peptide with
m/z = 1800.9094 (dark gray in Fig. 3) was assigned to the
Results and discussion
Heterologous expression of a soluble form of human b-1,4-GalT
7 and its biochemical characterization
Galactosyltransferase I or b-1,4-GalT 7 (UDP-galactose:O-b-
D-xylosylprotein 4-b-D-galactosyltransferase, EC 2.4.1.133) is
involved in the synthesis of the common glycosaminoglycan-
protein linkage region. In particular, it catalyzes the first
galactosylation step of the xylose bound to the core protein
of proteoglycans (Scheme 1). The gene codifying for this
enzyme in humans was identified almost simultaneously by
Almeida et al.11 and Okajima et al.12 Human b-1,4-GalT 7 is a
Type II membrane protein. These proteins share a common
structure comprised of a short N-terminal cytoplasmic tail, a
membrane spanning region followed by a luminal stem region
and a large C-terminal catalytic domain (Fig. 1).13
To facilitate general studies on enzymes, large-scale produc-
tion systems are needed.14 Prokaryotic expression systems,
and in particular E. coli, are the most attractive ones for large
scale production of recombinant proteins because of their
ability to grow rapidly and at high density on inexpensive
substrates.15 However, when a heterologous protein is over-
expressed in E. coli misfolding and aggregation happen
frequently, driving the recombinant protein into inactive
aggregates known as inclusion bodies. These limitations are
especially important for eukaryotic proteins that require post-
translational modifications such as glycosylation. A myriad of
strategies have been developed in the last several years to
increase the yield of soluble mammalian recombinant protein
using E. coli as an expression host. Some of these strategies
are: reduction of the recombinant protein production rate;16
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Table 1 Summary of the kinetic constants of the recombinant
b-1,4-GalT 7 soluble domain
Substrate
KM/mM kcat/sꢀ1 kcat/KM sꢀ1 Mꢀ1 kis/mM
p-NO2Ph-b-D-Xyl (1)a 0.59
0.1
49.8
51.4
8.5 ꢁ 104
—
21.1
UDP-Galb
55.6 ꢁ 104
a
Kinetic parameters obtained from fits to Michaelis–Menten eqn (1).
Kinetic parameters obtained from fits to eqn (2).
b
Fig. 2 Kyte–Doolittle hydropathicity profile of (A) wild-type human
b-1,4-GalT 7 and (B) the truncated form of the enzyme.
Fig. 4 Michaelis–Menten representation of the b-1,4-GalT 7 soluble
domain activity (V) as a function of the UDP-Gal concentration,
where the inhibition by substrate excess can be observed.
Fig. 3 Peptide mass fingerprint of b-1,4-GalT 7 soluble domain
incorporating a N-t His tag. The sequences of the identified peptides
are shaded and underlined. Molecular mass of each peptide is indi-
cated in Da.
The KM value obtained towards UDP-Gal for the soluble
domain of the b-1,4-GalT 7, was comparable, although some-
what lower, to the data reported by Lattard and co-workers19
for the MBP-b4GalT7 fusion protein (0.23 mM) and by
Fournel-Gigleux and co-workers30 for the GST-b-1,4-GalT7
(0.28 mM). However, the kcat value obtained for the soluble
domain was about 30-fold higher than that reported by Lattard
and co-workers (Fournel-Gigleux et al. used 4-methylumbelliferyl-
b-D-xylopyranoside as acceptor). These differences observed in
the KM and the kcat values could be due to an entropy loss of
the active site in the fused MBP-b4GalT7 enzyme. Besides, it is
necessary to have in mind that the reported data are apparent
constants and it cannot be discarded that the differences
observed may be due to the different conditions in which these
values have been calculated in both works.
peptide ERFEELLVFVPHMR included in the sequence of
the soluble domain.
The activity of the b-1,4-GalT 7 soluble domain was
initially assayed using UDP-Gal as the glycosyl donor and
p-nitrophenyl-b-D-xylopyranoside (p-NO2Ph-b-D-Xyl; 1) as
the acceptor (Scheme 2). The time course of the reaction was
followed by HPLC. The formation of a new peak with a
retention time of 10.2 min was observed. This peak
showed a m/z of 432.1 (M ꢀ H+), matching the mass of
disaccharide 2.
The recombinant b-1,4-GalT 7 soluble domain showed a
Michaelis–Menten kinetic for p-NO2Ph-b-D-xyl. The apparent
KM value (at saturating concentration of UDP-Gal) was
0.59 mM with an apparent kcat of 49.8 sꢀ1 (Table 1).
The behavior observed towards UDP-Gal was more
complex since the soluble domain of the b-1,4-GalT 7 showed
excess-substrate inhibition from a donor concentration of
1.25 mM (Fig. 4). This excess-substrate inhibition has been
also described by Qasba and co-workers for the galectin1-
b4GalT7 fusion protein.20 The calculated kis for UDP-Gal was
21.1 mM.
Synthesis of xylopyranoside derivatives
In Chart 1 the different xylose derivatives 3–14 used for the
enzymatic study are shown.
A
group of compounds containing
a
common
N-(O-xylopyranosyl)-hydoxylpropylamide moiety with
a
variable acyl group (compounds 8–12) was selected to
compare the effect of aliphatic or aromatic groups on the
activity of the xyloside as decoy acceptor. Acyl groups
containing a carboxyl (10 and 12) were also included, since
it is known that acidic amino acid residues are usually present
near the glycosylation site of both HS and CS/DS.23
Compounds 5–7 were obtained as previously described by
one of us24 and compound 14 was obtained following the
procedure described by Mani et al.8
Scheme 2
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Chart 1
The synthesis of amides 8–12 was carried out from a
common amine intermediate 17, which was prepared by
glycosidation of xylose tetracetate 15 with 3-hydroxy-
propionitrile followed by hydrogenation of the nitrile group
(Scheme 3).
Scheme 3
The reaction of 17 with different acylating reagents gave
the corresponding amides 18–22 that were subsequently
O-deacetylated to afford the targets 8–12. For the synthesis
of glycosyl amino acid 13, xylose peracetate 15 was reacted
with protected L-serine derivative 23 in the presence of
TMSOTf, to give stereoselectively the b-glycoside 24 in 75%
yield (Scheme 3). Subsequent O-deacetylation under mild
conditions (KCN/MeOH) furnished 13.
the activity of these compounds as decoy acceptors is to measure
their ability to inhibit the formation of the natural reaction
product. In our case, we evaluated their ability to inhibit (IC50)
the formation of 2 by the recombinant enzyme (Fig. 5).
The 2-(6-hydroxynaphthyl) b-D-xylopyranoside (14) was
included in this study for comparative purposes. The results
are summarized in Table 2.
As it has been commented above, Mani and co-workers8
have demonstrated that compound 14 is able to prime glycos-
aminoglycan synthesis. As expected, 14 was the substrate of
the recombinant enzyme. However, like in the case of the
UDP-Gal, 14 showed a strong inhibition by substrate excess
(kis = 0.38 mM). Therefore, the kinetic parameters were
obtained from fit data to eqn (2) (see Experimental). The
calculated catalytic efficiency was slightly lower than with
acceptor 1. This difference can be attributed to the lower kcat
showed by 14. According to the proposed hypothesis, since
Study of the activity of the xylopyranoside derivatives as decoy
acceptors of the recombinant soluble domain of the b-1,4-GalT 7
The activity as decoy acceptors of the xylopyranoside
derivatives was evaluated by comparison of the catalytic
efficiency (kcat/KM) showed by the recombinant soluble
domain of the b-1,4-GalT 7 towards the different xylosides.
Since these xylopyranosides mimic the natural acceptor of the
b-1,4-GalT 7, they may compete with it and act also as
inhibitors of the enzyme. Therefore, another way to evaluate
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substrate for the recombinant enzyme with a kcat/KM of
118.1 sꢀ1 mMꢀ1. This result is in agreement with earlier
observations by Sobue et al. on the ability of phenyl
b-D-thioxylopyranoside (7) to prime the synthesis of
chondroitin sulfate chains.25 S-Glycosides are interesting
compounds since they are more stable against enzymatic
cleavage than their O-glycoside counterparts26 and, therefore,
their bioavailability must be also higher.
Fig. 5 If the xylopyranoside derivative is a good substrate for the
enzyme, then it can act as a decoy acceptor sequestering the enzyme
activity and thus preventing the formation of the natural product. In
this study, we have evaluated the ability of the xylosides 3–14 to
prevent the formation of 2.
Study of the antiproliferative activity of the xylopyranoside
derivatives
To test if there is a relationship between the catalytic efficiency
of the recombinant b-1,4-GalT 7 with the different xylo-
pyranosides and their antiproliferative activity, we assayed
selected xylosides against the human lung carcinoma cell line
A 549. Xyloside 7 was chosen for its characteristic S-glycoside
group, aliphatic xylosides 8 and 9 were selected as representa-
tives of amide containing compounds, and, finally, the high
Table 2 Evaluation of xylosides 3–14 as decoy acceptors of the
recombinant human b-1,4-GalT 7a
ꢀ1
KM/mM
kcat/sꢀ1
kcat/KM/sꢀ1 mM
IC50/mM
14b,c
3
4
5
6
7
8
9
10
11
12
13c
0.58
N.D
N.D
0.37
0.28
0.74
0.75
0.75
0.56
0.53
0.40
0.53
30.2
N.D
N.D
60.7
57.6
87.4
41.0
64.3
47.2
78.7
106.3
152.0
52.1
0.30
>20
>20
0.95
1.26
0.52
3.69
2.46
3.96
1.04
0.61
2.39
N.D
N.D
164.0
205.7
118.1
54.7
85.7
84.3
148.5
265.7
286.8
k
cat/KM value calculated for xyloside 13 prompted us to also
test it. Xyloside 14 was included in the study as a positive
control since its antiproliferative activity against this cell line
has been already demonstrated.8a Methyl D-xylopyranoside 4
was used as negative control since this compound was not the
substrate for the recombinant enzyme (Table 2). Finally, the
peracetylated derivatives of
4 (compound 25) and 13
(compound 24) were also included in the study since it has
been described that peracetylated glycosides can penetrate
more efficiently into the cells and once inside they can be
deacetylated by endogenous carboxylesterases leading to the
bioactive compound.5
a
All xylopyranosides have b configuration except for compound 3,
b
which was an a and b anomeric mixture. Kinetic parameters
obtained from fits to eqn (2). Reaction contains 10% of DMSO.
c
As expected, xylosides 4 and its peracetylated derivative 25
were unable to inhibit the cell proliferation (Fig. 6 and Table
S1 (ESIw)). This lack of antiproliferative activity may be
related to the inefficacy of 4 to be a substrate of the human
b-1,4-GalT 7 and therefore to prime the synthesis of GAG.
xylopyranoside 14 is a good substrate for the enzyme, it can
act as a decoy acceptor sequestering the enzyme activity and
thus preventing the formation of 2. In fact, 14 was able to
inhibit the formation of 2 with an IC50 of 0.3 mM (Table 2).
Although the IC50 value does not indicate the type of inhibi-
tion promoted by 14, it seems clear that in this context the
inhibition must be competitive. To substantiate this fact, we
calculated the ki for 14. As expected, the data fit to eqn (3) that
describes the competitive inhibition. The calculated ki value
(0.14 mM) was significantly lower than the IC50 value.
With respect to xylopyranosides 3–13, only those lacking
hydrophobic aglycone (compounds 3 and 4) showed no
activity with the soluble domain of the b-1,4-GalT 7. Their
kinetic parameters were not calculated since the transglyco-
silation rates were in both cases lower than the 5% of the
reaction rate of the recombinant enzyme with p-NO2Ph-b-D-
Xyl (1) as acceptor. Moreover, they showed no inhibition even
at concentration as high as 20 mM. All other xylosides were
both substrates and inhibitors of the recombinant enzyme. The
xyloside that showed the highest catalytic efficiency was the
xylopyranosyl-serine derivative 13. This result was not
surprising since 13 was designed to mimic the natural acceptor
of the enzyme, that is, a xylose bound to a serine of the protein
core. Thus, the kcat/KM value for 13 was more than 5-folds
higher than the kcat/KM for xyloside 14, which showed the
lowest value. Interestingly, the S-glycoside 7, in which a sulfur
atom has replaced the glycosidic oxygen atom, was a good
Fig. 6 Effect of xylopyranoside derivatives 7 (K), 13 (’), 14 (J)
and peracetate 24 (&) on the proliferation of human lung carcinoma
cells A549. Values correspond to the mean of 3 different experiments
performed in triplicate. For the sake of clarity only results from 4
compounds are shown. See Table S1 (ESIw) for antiproliferative values
of xylosides 4, 8, 9, and peracetate 25 (mean ꢂ SD).
c
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The only other tested xyloside that did not present anti-
proliferative activity was the S-glycoside 7. This result is in
agreement with Mani’s findings which showed that 2-naphthyl
S-b-D-xylopyranoside was also unable to inhibit the growth
of lung fibroblasts, lung carcinoma cells, or transformed
endothelial cells.8a The rest of xylopyranosides tested showed
IC50 values than 2-(6-hydroxynaphthyl) b-D-xylopyranoside
(14). The xyloside that showed the lowest IC50 (54 mM) was the
peracetate 24. This value was lower than the IC50 calculated
for deacetylated 13 (88 mM) and this difference may be
explained because of a higher uptake of the peracetylated
compound by the cell. Compounds 8 showed an IC50 of
68 mM, substantially lower than the IC50 showed by xyloside
9, in which the only difference is that the alkyl chain is four
carbons longer. Finally, it is interesting to note that all the
xylopyranosides that showed antiproliferative activity were
cytotoxic at high concentrations. Xylosides 8 and 13 and
peracetate 24 were cytotoxic at concentrations higher than
100 mM, xylosides 9 and 14 exhibited cytotoxic effect at
concentrations higher than 150 and 250 mM, respectively,
whereas xylosides 4, 7 and peracetate 25 were not cytotoxic
(Table S1, ESIw).
obtained at 125 or 100 MHz using CDCl3 or CD3OD as a
solvent at room temperature. Chemical shift values are
reported in parts per million (d). Coupling constant values
(J) are reported in hertz (Hz), and spin multiplicities are
indicated by the following symbol: s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet). Mass spectroscopy
spectra were registered on a hp series 1100 MSD spectrometer.
Peptide mass fingerprint analysis
Peptide mass fingerprint analysis from the SDS-PAGE
band corresponding to the recombinant b-1,4-GalT 7 was
performed at the Proteomic Unit of the Spanish National
Center of Biotechnology (CNB-CSIC). Samples were digested
with sequencing grade trypsin O/N at 37 1C. The analysis by
MALDI-TOF mass spectrometry produces peptide mass
fingerprints and the peptides observed can be collated and
represented as a list of monoisotopic molecular weights. Data
were collected in the m/z range of 800–3600.
Synthesis of xylopyranose derivatives
2-Cyanoethyl b-D-2,3,4-tri-O-acetyl-xylopyranoside (16). To
solution of tetra-O-acetyl-xylopyranose (15, 5.0 g,
a
15.7 mmol) in CH2Cl2 (125 mL), 3-hydroxypropionitrile
(1.3 mL, 18.8 mmol, 1.2 eq.) and BF3ꢃOEt2 (1.86 ml,
15.7 mmol, 1 eq.) were added. The mixture was stirred for
1 h at room temperature under an argon atmosphere, then
diluted with CH2Cl2 (50 mL) and washed with NaHCO3 and
H2O. The organic layer was concentrated in vacuo and the
resulting residue was crystallized from methanol to give 16
(4.3 g, 82%) as a solid. Mp: 161–165 1C. [a]D25: ꢀ57.1 (c 1.49,
CH2Cl2). 1H NMR (300 MHz, CDCl3): d 5.17 (t, 1H, J =
8.4 Hz), 4.98–4.88 (m, 2H), 4.56 (d, J = 6, 6 Hz), 4.15 (dd, J =
4.8, 12.0 Hz), 4.00 (dt, 1H, J = 5.7, 9.9 Hz), 3.72 (dt, J = 6.3,
9.9 Hz), 3.40 (dd, 1H, J = 8.4, 12.0 Hz), 2.63 (t, 2H, J =
6.0 Hz), 2.09 (s, 3H), 2.06 (s, 6H). 13C NMR (133 MHz,
CDCl3): d 170.0, 169.8, 169.6, 117.2, 100.75, 70.8, 70.0, 68.5,
63.8, 61.9, 20.7, 19.0. Anal. calc. for C14H19NO8: C 51.06,
H 5.82, N 4.25. Found: C 50.86, H 5.78, N 4.16%.
Experimental
Materials and general procedures
The clone IRAUp969H0626D containing the cDNA sequence
of the human UDP-galactose:O-b-D-xylosylprotein 4-b-D-
galactosyltransferase was purchased from imaGenes. The
E. coli strain BL21(DE3) (Stratagene) was used as expression
hosts and E. coli DH5a (Promega) served as a host for plasmid
maintenance. Restriction enzymes and T4-DNA ligase were
purchased from MBI Fermentas AB and the pET-28b(+)
expression vector was purchased from Novagen. DNase I was
from Roche and lysozyme was from USB. Pyruvate kinase
(PK) and lactate dehydrogenase (LDH) were purchased from
Sigma-Aldrich. Isopropyl-b-D-thiogalactopyranoside (IPTG)
was purchased from Applichem GmBH. A plasmid purifica-
tion kit was from Sigma and DNA purification kit from
agarose gels was from Eppendorf. All other chemicals were
purchased from commercial sources as reagent grade.
3-Acetamido-propyl b-D-xylopyranoside (8). 16 (100 mg,
0.3 mmol) was dissolved in a EtOAc–MeOH solution (2 : 1,
v/v, 9 mL) and Pd/C (50 mg) and trifluoroacetic acid (34 mL,
1.5 eq.) were added. The mixture was stirred for 18 h at room
temperature under an H2 atmosphere. After this time, the
reaction mixture was filtered through Celites and the solids
were washed with a EtOAc–MeOH solution (2 : 1, v/v). The
combined filtrate and washing were concentrated to give
UV/Visible spectra were recorded on a Spectra Max Plus
384 spectrophotometer at 25 1C. HPLC analyses were carried
out on a chromatograph Dionex, Dual Gradient Pump, with a
detector Diodo Array UV/VIS PDA-3000 (Dionex Co.).
SDS-PAGE was performed in a Mini-Protean 3 Cell Electro-
phoresis Unit (BioRad) using 10% and 5% acrylamide in the
separating and stacking gels, respectively. Gels were stained
with Coomassie brilliant blue R-250 (Applichem GmBH).
Electrophoresis was always run under reducing conditions,
in the presence of 5% b-mercaptoethanol. Protein and DNA
gels were quantified by densitometry using a GeneGenius Gel
Documentation and Analysis System (Syngene). Nickel-
iminodiacetic acid (Ni2+-IDA) agarose was supplied by
Agarose Bead Technologies.
1
amine 17. H NMR (400 MHz, CD3OD): d 5.23 (t, 1H, J =
9.2 Hz), 4.98–4.82 (m, 2H), 4.61 (d, 1H, J = 7.3 Hz), 4.08
(dd, 1H, J = 5.5, 11.7 Hz), 3.94 (m, 1H), 3.69 (m, 1H), 3.47
(dd, 1H, J = 9.7, 11.7 Hz), 3.02 (t, 2H, J = 7.1 Hz), 2.02
(s, 3H,), 2.01 (s, 3H,), 2.00 (s, 3H), 1.98–1.88 (m, 2H). 13C
NMR (100 MHz, CD3OD): d 171.5, 102.1, 73.2, 72.7, 70.4,
68.0, 63.3, 38.9, 28.6, 20.5.
17 (137 mg, 0.3 mmol) was dissolved in dry pyridine (2 mL)
and acetic anhydride (2 mL) was added. The mixture was
stirred for 18 h at room temperature, then concentrated and
the residue was purified by column chromatography
(EtOAc–MeOH, 20 : 0 - 20 : 1) to give 18 (130 mg, 100%).
Optical rotations were determined on a Perkin Elmer 241
1
Polarimeter (l = 589 nm, 1 dm cell). H NMR spectra were
registered at 500, 400 or 300 MHz, and 13C NMR were
c
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Mol. BioSyst., 2011, 7, 1312–1321 1317

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1H NMR (300 MHz, CD3OD): d 5.25 (t, 1H, J = 9.0 Hz),
5.05–4.79 (m, 2H), 4.60 (d, J = 7.3 Hz), 4.10 (dd, 1H, J = 5.1,
11.7 Hz), 3.88 (m, 1H), 3.66–3.39 (m, 2H), 3.31–3.19 (m, 2H),
2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.95 (s, 3H), 1.88–1.70
(m, 2H).
d 175.1, 173.4, 103.8, 76.6, 73.7, 70.0, 67.2, 65.7, 36.5, 30.4,
29.1, 28.6. Anal. calc. for C12H20NNaO8: C 43.77, H 6.12,
N 4.25, Na 6.98. Found: C 43.08, H 6.12, N 3.90%.
3-Benzamido-propyl b-D-xylopyranoside (11). Amine 17
(274 mg, 0.6 mmol), obtained as described above, was
dissolved in dry pyridine (2 mL) and benzoyl chloride
(215 mL, 3 eq.) was added. The mixture was stirred for 18 h
at room temperature, then washed with AcOEt and concen-
trated. The residue was purified by column chromatography
18 (100 mg, 0.27 mmol) was dissolved in methanol (15 mL)
and treated with 0.5 M NaOMe (5 mL) for 1 h at room
temperature, then neutralized with Amberlite IR-120 (H+)
resin, and filtered. The solvent was evaporated to give 8
(67.3 mg, 98%). [a]D25: ꢀ41.6 (c 1.25, H2O). 1H NMR
(400 Mz, CD3OD): d 4.20 (d, 1H, J = 7.4 Hz), 3.92–3.82
(m, 2H), 3.57 (m, 1H), 3.47 (m, 1H), 3.36–3.12 (m, 5H), 1.92
(s, 3H), 1.78 (m, 2H). 13C NMR (100 MHz, CD3OD): d 172.1,
103.9, 76.3, 73.7, 70.0, 67.2, 65.8, 36.6, 29.1, 21.4. MS (ES) m/z
(calc. 249.1): 250.1 (M + 1), 272.0 (M + 23). Anal. calc. for
C10H19NO6: C 48.19, H 7.68, N 5.62. Found: C 47.99, H 7.88,
N 5.57%.
1
(hexane–AcOEt 3 : 1 - 1 : 1) to give 21 (54 mg, 20%). H
NMR (300 MHz, CDCl3): d 7.77 (m, 2H), 7.53–7.38 (m, 3H),
6.69 (s, 1H), 5.18 (t, 1H, J = 9.0 Hz), 5.01–4.84 (m, 2H), 4.48
(d, 1H, J = 6.9 Hz), 4.08 (dd, 1H, J = 6.6, 5.1 Hz), 3.93
(m, 1H), 3.71–3.39 (m, 3H), 3.34 (dd, 1H, J 9.3, 2.4 Hz), 2.03
(s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.89 (m, 2H).
21 (54 mg, 0.12 mmol) was deacetylated under similar
conditions as described for 18 to give 11 (37 mg, 99%).
[a]D25: ꢀ34,4 (c 1.42, H2O); H NMR (400 MHz, CD3OD):
1
3-Hexanamido-propyl b-D-xylopyranoside (9). Amine 17
(274 mg, 0.6 mmol), obtained as described above, was
dissolved in dry pyridine (2 mL) and hexanoic anhydride
(278 mL, 2 eq.) was added. The reaction was stirred for 3 h
at room temperature, then concentrated and the residue was
purified by column chromatography (hexane–EtOAc 1 : 1) to
d 7.82–7.78 (m, 2H), 7.52 (m, 1H), 7.45 (m, 2H), 4.22 (d, 1H,
J = 7.4 Hz), 3.92 (m, 1H), 3.85 (dd, 1H, J = 5.5, 12.3 Hz),
3.64 (m, 1H), 3.57–3.42 (m, 3H), 3.31 (m, 1H), 3.24–3.15
(m, 2H), 1.91 (m, 2H). 13C NMR (100 MHz, CD3OD):
d 169.0, 134.6, 131.4, 128.4, 127.0, 104.0, 76.7, 73.8, 70.0,
67.4, 65.8, 37.1, 29.2. Anal. calc. for C15H21NO6: C 57.87,
H 6.80, N 4.50. Found: C 57.58, H 6.78, N 4.76%.
1
give 19 (129 mg, 50%). H NMR (300 MHz, CDCl3): d 5.95
(s, 1H), 2.15 (t, J = 9.0 Hz), 5.02–4.82 (m, 2H), 4.42 (d, 1H,
J = 7.2 Hz), 4.10 (dd, 1H, J = 5.4, 11.7 Hz), 3.89 (m, 1H),
3.53 (m, 1H), 3.45–3.18 (m, 3H), 2.15 (t, 2H, J = 7.5 Hz), 2.05
(s, 3H), 2.03 (s, 6H), 1.82–1.68 (m, 2H), 1.59 (m, 2H),
1.37–1.21 (m, 4H), 0.88 (t, J = 6.6 Hz).
Sodium N-[O-(b-D-xylopyranosyl)-3-hydroxypropyl]-phthalamate
(12). Amine 17 (274 mg, 0.6 mmol), obtained as described
above, was dissolved in dry pyridine (2 mL) and phthalic
anhydride (221 mg, 2 eq.) was added. The mixture was
stirred for 2 h at room temperature, then concentrated
and the residue was purified by column chromatography
(AcOEt–MeOH 10 : 0 - 10 : 1) to give 22 (360 mg, 80%).
1H NMR (300 MHz, CDCl3): d 5.14 (t, 1H, J = 8.8 Hz),
4.95–5.75 (m, 2H), 4.43 (d, 1H, J = 7.1 Hz), 4.02 (dd, 1H, J =
5.4 Hz, J = 11.7 Hz), 3.95–3.83 (m, 3H), 3.67–3.38 (m, 3H),
3.30 (dd, 1H, J = 9.5, 11.7 Hz), 2.01 (s, 3H), 1.99 (s, 3H), 1.98
(s, 3H), 1.87 (m, 2H).
19 (100 mg, 0.23 mmol) was deacetylated under similar
conditions as described for 18 to give 9 (64.2 mg, 91%).
1
[a]D25: ꢀ40.4 (c 1.53, H2O). H NMR (400 MHz, CD3OD):
d 4.19 (d, J = 7.6 Hz), 3.91–3.81 (m, 2H), 3.56 (m, 1H),
3.36–3.12 (m, 5H), 2.16 (t, J = 7.2 Hz), 1.78 (m, 2H), 1.60
(m, 2H), 1.40–1.24 (m, 4H), 0.91 (t, 3H, J = 7.2 Hz). 13C
NMR (100 MHz, CD3OD): d 175.5, 103.9, 76.7, 73.7, 70.0,
67.2, 65.8, 36.4, 36.0, 31.3, 29.1, 25.6, 22.3, 13.1. Anal. calc. for
C14H27NO6: C 55.06, H 8.91, N 4.59. Found: C 54.78, H 9.06,
N 4.52%.
22 (280 mg, 0.64 mmol) was deacetylated under similar
conditions as described for 18 to give 12 (229 mg, 95%).
[a]D25: ꢀ24.3 (c 1.50, H2O) 1H NMR (300 MHz, CD3OD):
d 7.96 (m, 1H), 7.68–7.48 (m, 2H), 7.43 (m, 1H), 4.23 (d, 1H,
J = 7.6 Hz), 3.94 (m, 1H), 3.83 (dd, 1H, J = 5.4, 11.5 Hz),
3.67 (m, 1H), 3.52–3.12 (m, 11H), 1.98–1.84 (m, 2H). Anal.
calc. for C16H20NNaO8: C 50.93, H 5.34, N 3.71, Na 6.09.
Found: C 51.03, H 5.61, N 3.54%.
Sodium N-[O-(b-D-xylopyranosyl)-3-hydroxypropyl]-succinamate
(10). Amine 17 (274 mg, 0.61 mmol), obtained as described
above, was dissolved in dry pyridine (2 mL) and succinic
anhydride (60 mg, 2 eq.) was added. The mixture was stirred
for 2 h at room temperature, then concentrated and the
residue purified by column chromatography (AcOEt–MeOH,
10 : 0 - 10 : 1) to give 20 (387 mg, 75%). 1H NMR
(300 MHz, CDCl3): d 6.49 (s, 1H), 5.21 (t, 1H, J = 9.0 Hz),
5.02–4.86 (m, 2H), 4.42 (d, 1H, J = 7.3 Hz), 4.11 (dd, 1H, J =
5.1, 12.0 Hz), 3.91 (m, 1H), 3.56 (m, 1H), 3.42–3.26 (m, 3H),
2.66 (m, 2H), 2.56 (m, 2H), 2.06 (s, 3H), 2.03 (s, 6H), 1.78
(m, 2H).
Oc-(2,3,4-tri-O-acetyl-b-D-xylopyranosyl)-N-Cbz-L-serine-
methyl-ester (24). N-Cbz-L-serine methyl-ester (23, 200 mg,
0.79 mmol) and tetra-O-acetyl-b-D-xylopyranose (15, 500 mg,
2 eq.) were dissolved in dry CH2Cl2 (15 mL), then molecular
sieve (4 A, 4.5 g) and TMSOTf (78 mL, 0.5 eq.) were added.
The mixture was stirred for 2 h at room temperature under an
argon atmosphere. The reaction mixture was further diluted
with CH2Cl2 (20 mL) and neutralized with triethylamine.
Evaporation of the solvent and purification by column
chromatography (hexane–AcOEt 3 : 1 - 1 : 1) gave a residue
fraction (340 mg) which was further purified by column
chromatography (CH2Cl2–AcOEt 9 : 1) to give 24 as a solid
20 (160 mg, 0.19 mmol) was deacetylated under similar
conditions as described for 18 to give 10 (62 mg, 98%).
[a]D25: ꢀ31.9 (c 1.65, H2O). H NMR (400 MHz, CD3OD):
1
d 4.20 (d, J = 7.8 Hz), 3.92–3.82 (m, 2H), 3.57 (m, 1H), 3.48
(m, 1H), 3.37–3.14 (m, 5H), 2.63–2.55 (m, 3H), 2.51–2.43
(m, 2H), 1.78 (m, 2H). 13C NMR (100 MHz, CD3OD):
c
1318 Mol. BioSyst., 2011, 7, 1312–1321
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(303 mg, 75%). Mp: 102–104 1C. [a]D25: ꢀ29.4 (c 1.03,
release of UDP during the galactosyltransferase-catalyzed
reaction.28 In these cases, activity assays were run at 25 1C
1
CH2Cl2). H NMR (400 MHz, CDCl3): d 7.42–7.23 (m, 5H),
5.56 (d, 1H, J = 8.2 Hz), 5.12 (s, 2H), 5.09 (t, 1H, J = 7.8 Hz),
4.90–4.81 (m, 2H), 4.51 (m, 1H), 4.48 (d, 1H, J = 6.5 Hz), 4.22
(dd, 1H, J = 2.7, 10.1 Hz), 4.03 (dd, 1H, J = 4.7, 12.1 Hz),
3.79 (dd, 1H, J = 3.1, 10.1 Hz), 3.76 (s, 3H), 3.34 (dd, 1H, J =
7.8, 12.1 Hz,), 2.04 (s, 3H), 2.03 (s, 3H) 2.02 (s, 3H). 13C NMR
(100 MHz, CDCl3): d 170.3, 170.2, 170.1, 169.6, 156.1, 136.3,
128.8, 128.5, 128.4, 100.7, 70.7, 70.2, 69.2, 68.6, 67.4, 61.7,
54.4, 53.0, 21.0, 20.9, 20.8. MS (ES) m/z (calc. 511.2): 534.2
(M + 23). Anal. calc. for C23H29NO12: C 54.01, H 5.71,
N 2.74. Found: C 53.88, H 5.71, N 2.74%.
following the decrease of absorbance at 340 nm (eNADH =
6220 Mꢀ1 cmꢀ1) for 30 minutes in a 1 mL reaction mixture
containing sodium acetate buffer (25 mM, pH 5.0), NADH
(0.2 mM), PEP (0.7 mM), KCl (50 mM), MnCl2 (20 mM), PK
(6 U) and LDH (6 U). Substrate concentrations were varied
between 0.01 and 10 mM. Activity with p-NO2Ph-b-D-Xyl (1)
was also measured by this method and the obtained results
were consistent with those obtained by HPLC.
Inhibition of galactosyltransferase activity by the different
xylopyranoside derivatives was analyzed in a 1 mL reaction
mixture containing sodium acetate buffer (25 mM, pH 5.0),
KCl (50 mM), MnCl2 (20 mM), UDP-gal (50 mM), 1 (10 mM)
and increasing concentrations of inhibitor. Accumulation of
disaccharides was monitored by HPLC as described above.
Oc-b-D-Xylopyranosyl-N-Cbz-L-serine-methyl-ester (13). To
a mixture of KCN (6.5 mg, 0.09 mmol) and MeOH (10 mL)
was added 24 (100 mg, 0.19 mmol). The mixture was stirred at
room temperature until complete conversion into deacetylated
xylopyranoside 13 (1 h), as indicated by TLC (AcOEt–MeOH
25 : 1, v/v). After this time an equimolar quantity of mixed
Dowexs 50X4 (Na+) and Dowexs 1X8 (HCO3ꢀ) resins were
added, then the mixture was filtered and concentrated. The
colorless residue was purified by column chromatography
(AcOEt–MeOH 25 : 0 - 25 : 1) to give 13 (49.8 mg, 70%)
Analysis of kinetic data
Kinetic constants were obtained using the built-in nonlinear
regression tools in SigmaPlot 8.0. For the determination of
apparent kinetic constants (variation of only one substrate),
initial velocities (Vi) were fitted to the Michaelis–Menten
equation (eqn (1)):
1
as a solid. Mp: 42–46 1C. [a]D25: ꢀ23.4 (c 1.13, MeOH). H
app
max
app
M;S
NMR (400 MHz, CD3OD): d 7.40–7.23 (m, 5H), 5.09 (s, 2H),
4.44 (m, 1H), 4.26 (dd, 1H, J = 3.7 Hz, J = 10.1 Hz), 4.16
(d, 1H, J = 7.7 Hz), 3.81 (dd, 1H, J = 5.3 Hz, J = 11.3 Hz),
3.74–3.67 (m, 4H), 3.43 (m, 1H), 3.27 (t, 1H, J = 8.8 Hz),
3.19–3.09 (m, 2H). 13C NMR (100 MHz, CD3OD): d 172.2,
158.7, 138.1, 129.47, 129.0, 128.9, 105.1, 77.6, 74.8, 71.0, 70.5,
67.8, 67.0, 55.7, 53.0. Anal. calc. for C17H23NO9: C 52.98,
H 6.02, N 3.63. Found: C 53.03, H 5.96, N 3.56%.
V
ꢃ ½Sꢄ
Vi ¼
ð1Þ
K
þ ½Sꢄ
Due to substrate inhibition, the apparent kinetic constants for
UDP-Gal and xylopyranoside 14 were determined using the
Michaelis–Menten equation for a mechanism that involves
substrate inhibition (eqn (2)):
app
max
V
ꢃ ½Sꢄ
Vi ¼
ð2Þ
2
app
M;S
½Sꢄ
Enzyme activity assays
K
þ ½Sꢄ þ
app
iS;S
k
Galactosyltransferase activity of the recombinant b-1,4-GalT
7 was determined following the method previously described
by Higuchi et al. with minor modifications.27 Formation of
disaccharide 2 was followed by HPLC with A300nm monitoring
in reaction mixtures of 1 ml containing sodium acetate buffer
(25 mM, pH 5.0), MnCl2 (20 mM), KCl (50 mM), UDP-Gal
(50 mM), and 1 (0.5 mM). Aliquots were taken each 5 min and
analyzed by HPLC using a Lichrosorb RP18 column. The
mobile phase employed was TFA (0.1% in H2O)–CH3CN
17 : 3 (v : v) and the chromatography was run under isocratic
conditions at 1 ml minꢀ1 flow rate. Measurements of kinetic
parameters for UDP-Gal were performed with 11–14 mg of
purified protein at fifteen different UDP-Gal concentrations in
the range 0.025–20 mM and with a fixed concentration of 1 of
10 mM. Assays to determine the kinetic parameters for
p-Nph-b-D-Xyl 1 were performed with 7–8 mg of purified
app
iS
where
K
is the excess substrate apparent inhibition
constant.
IC50 for the different xylopyranosides was calculated from
the four-parameter logistic regression model, which assumes
symmetry around the inflection point of the standard curve.
To calculate ki for 14, data were fitted to eqn (3) that describe
the competitive inhibition:
app
max
V
ꢃ ½Sꢄ
Vi ¼
ð3Þ
app
M;S
app
ð1 þ ½Iꢄ=k Þ þ ½Sꢄ
K
ic
where [I] denoted concentration of inhibitor and kic the
competitive inhibition.
Antiproliferative activity studies
b-1,4-GalT
7
at 10 concentrations of the substrate
For the determination of the antiproliferative activity, A549
cells were seeded at 3000 cells per well in 96-well plates in
media containing 10% of fetal bovine serum (FBS). After 4 h
of plating, the cells were serum-starved for 24 h. Cells were
then allowed to proliferate in media without FBS, containing
25 ng mlꢀ1 of epidermal growth factor (EGF), in the presence
of different concentrations of xylosides (330, 220, 147, 98, 65,
43, 29, 19 mM). Four days later, the number of viable cells was
determined with the MTT test.29
(0.01–10 mM) under saturating concentrations of UDP-Gal
(0.6 mM).
Activity of the recombinant b-1,4-GalT 7 with xylopyrano-
sides derivatives 11 and 14 and their kinetic parameters were
determined by HPLC as described previously. Activity with
compounds 5–10, 12 and 13 was measured spectrophoto-
metrically in a coupled enzymatic assay, where the decrease
of NADH absorbance at 340 nm is directly proportional to the
c
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Mol. BioSyst., 2011, 7, 1312–1321 1319

View Article Online
8 (a) K. Mani, B. Havsmark, S. Persson, Y. Kaneda, H. Yamamoto,
K. Sakurai, S. Ashikari, H. Habuchi, S. Suzuki, K. Kimata,
A. Malmstrom, G. Westergren-Thorsson and L.-A. Fransson,
¨
Cancer Res., 1998, 58, 1099–1104; (b) K. Mani, M. Belting,
U. Ellervik, N. Falk, G. Svensson, S. Sandgren, F. Cheng and
L.-A. Fransson, Glycobiology, 2004, 14, 387–397.
9 M. Belting, L. Borsig, M. M. Fuster, J. R. Brown, L. Persson,
L.-A. Fransson and J. D. Esko, Proc. Natl. Acad. Sci. U. S. A.,
2002, 99, 371–376.
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Chem., 2006, 49, 1932–1938; (b) R. Johnsson, K. Mani and
U. Ellervik, Bioorg. Med. Chem., 2007, 15, 2868–2877;
(c) F. Cheng, R. Johnsson, J. Nilsson, L.-A. Fransson,
U. Ellervik and K. Mani, Cancer Lett., 2009, 273, 148–154.
11 R. Almeida, S. B. Levery, U. Mandel, H. Kresse, T. Schwientek,
E. P. Bennett and H. Clausen, J. Biol. Chem., 1999, 274,
26165–26171.
12 T. Okajima, K. Yoshida, T. Kondo and K. Furukawa, J. Biol.
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14 A quite completed guide to select a method to produce recombi-
Conclusions
In conclusion, we have shown that it is possible to hetero-
logously express the catalytic domain of the human
b-1,4-GalT 7, soluble and stable enough to undertake
in vitro studies with it. This achievement opens the possibility
of developing an easy-to-use method to test the activity as
decoy acceptors of natural and synthetic xylopyranosides.
Since priming the synthesis of GAG is required but not
enough for the antiproliferative activity of the xylosides,10a,b
it is not possible to establish a direct correlation between the
kinetic parameters of the recombinant b-1,4-GalT 7 and the
antiproliferative activity of the different xylopyranosides
tested. On the other hand, preliminary results obtained in
A549 cell line suggest that xylopyranosides 8, 9, 13 and 14
exhibit a promising antiproliferative activity.
nant proteins can be found in: S. Graslund, P. Nordlund and
¨
J. Weigelt, et al., Nat. Methods, 2008, 5, 135–146.
Acknowledgements
15 G. Hannig and S. C. Makrides, Trends Biotechnol., 1998, 16,
54–60; F. Baneyx, Curr. Opin. Biotechnol., 1999, 10, 411–421;
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E. G-J. and A. F-M. thank the Spanish Ministerio de Ciencia e
Innovacion (Grants CTQ2007-67403/BQU and CTQ2010-
´
15418) and Comunidad de Madrid (Grant S2009/PPQ-1752)
for financial support. J. C. has been supported by the Spanish
Ministerio de Ciencia e Innovacion (Grant SAF2008-00706)
´
and Generalitat de Catalunya (Grant 2009SGR-1072).
J.F. G-G. was supported by a predoctoral I3P fellowship of
the European Social Fund. We thank Mrs Eva Dalmau for
excellent technical assistance.
A. Bastida, A. Ferna
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