Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-d-galactopyranoside derivatives

Article abstract of DOI:10.1016/j.carres.2011.01.024

α-d-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-d- galactopyranoside was the most potent inhibitor compared to the oth

Full text of DOI:10.1016/j.carres.2011.01.024

Carbohydrate Research 346 (2011) 602–605
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Activity of Debaryomyces hansenii UFV-1
-D-galactopyranoside derivatives
a-galactosidases against
a
Pollyanna A. Viana a, Sebastião T. de Rezende a, Arianne de A. Alves b, Rozângela M. Manfrini b,
Ricardo J. Alves b, Marcelo P. Bemquerer c, Marcelo M. Santoro d, Valéria M. Guimarães a,
⇑
a BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil
b Laboratório de Química Farmacêutica Medicinal da Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
c EMBRAPA Recursos Genéticos e Biotecnologia, PqEB, Brasília, DF, Brazil
d Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
a
-
D
-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hanse-
nii UFV-1 extracellular and intracellular -galactosidases were evaluated. Methyl -galactopyranoside
was the most potent inhibitor compared to the others tested, with K0i values of 0.82 and 1.12 mmol Lꢀ1
for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a
Received 14 October 2009
Received in revised form 10 January 2011
Accepted 20 January 2011
Available online 28 January 2011
a
a-D
,
hydroxyl group in the C-6 position of
a
-
D-galactopyranoside derivatives is important for the recognition
by D. hansenii UFV-1 -galactosidases.
a
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
a
-Galactosidases
Debaryomyces hansenii UFV-1
-Galactopyranoside derivatives
a-D
Inhibition
1. Introduction
a
-galactosyl residues from various
a
-galactosides.13 The produc-
-galactosidases from
tion of intracellular and extracellular
a
Numerous glycosidases have been isolated and fully character-
ized, and they have been classified into families and groups accord-
ing to their amino acid sequences. Enzymes of the same group
usually have common catalytic mechanisms.1 The anomeric speci-
ficity of the glycosidases is usually rigid, but the regioselective
specificity for the other glycosidic positions is more variable
among the glycosidases. Several studies have reported that hydro-
lytic reactions with analogous deoxy derivatives of the appropriate
Debaryomyces hansenii UFV-1 cultivated in a medium containing
galactose as the carbon source was recently reported.14 The sub-
strate specificities of these enzymes were investigated by using
synthetic substrates, galactose-containing oligosaccharides, and
polymers. D. hansenii UFV-1 intracellular and extracellular
tosidases were highly selective, showing absolute specificity for
the -galactosyl bond. These enzymes hydrolyzed pNP Gal but
showed no activity for other synthetic substrates, such as gluco-
sidic derivatives or for b-linked glycosides. -Galactose inhibited
the intracellular enzyme uncompetitively, but was shown to be a
noncompetitive inhibitor for the extracellular
-galactosidase.14,15
Since, galactose was an inhibitor for both D. hansenii UFV-1
-galactosidase isoforms,14,15 we decided to synthesize some of
a-galac-
a
a
glycon can be catalyzed by
D
-glycosidases, but the tolerance of the
D
active site to the position of deoxygenation varies between
enzymes from different sources.2–5 Despite the widespread use of
iminosugars as transition-state analogous inhibitors of glycosi-
dases, inhibitors based on modifications of conventional carbohy-
drates are still valuable.6
a
a
its derivatives to search for new possible lead compounds as
a-Galactosidases (a-D-galactoside galactohydrolase EC 3.2.1.22)
reversible inhibitors of the extracellular and intracellular
have gained considerable attention as industrial catalysts due to
their potential biotechnological applications since they can be iso-
lated from organisms, such as yeasts that are generally recognized
as safe (GRAS).7,8 These glycosyl hydrolases are widely distributed
a
-galactosidases.
The aim of the present study was to evaluate the influence of
substituents in the pyranosidic ring of some a-D-galactopyranoside
derivatives, coupled to some variation in the aglycon moiety on the
hydrolytic activity of extracellular and intracellular D. hansenii
in microorganisms, plants, and animals.9–12
a-D
-Galactoside galac-
tohydrolase catalyzes the hydrolysis of the terminal nonreducing
UFV-1
a
-galactosidase isoforms. Structure and inhibitory activity
-galactopyranoside derivatives,
-galactopyranoside derivatives are
relationships concerning these
as well as 4-nitrophenyl
discussed here.
a
-D
a-D
⇑
Corresponding author. Tel.: +55 31 3899 2374; fax: +55 31 3899 2373.
E-mail address: vmonteze@ufv.br (V.M. Guimarães).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.01.024

P. A. Viana et al. / Carbohydrate Research 346 (2011) 602–605
603
assay contained 650
l
L of 0.1 mol Lꢀ1 NaOAc buffer, pH 5.0,
2. Experimental
100
2 mmol Lꢀ1 pNP
for the extracellular and intracellular
tively. The amount of pNP released was determined at 25 °C at
l
L of enzyme solution (1.02
Gal. The mixture was incubated at 60 and 55 °C
-galactosidases, respec-
l lL of
g protein mLꢀ1), and 250
a
2.1. General methods
a
Solvents were dried and distilled before use. All reactions were
monitored by TLC on Silica Gel 60 (0.25 mm) followed by spraying
the plates with 15% ethanolic H2SO4 and heating at ca. 200 °C. Col-
umn chromatography was performed on silica gel (0.035–
0.070 mm). 1H and 13C NMR spectra were recorded with a Bruker
DPX200 instrument at 200 and 50 MHz, respectively. High-resolu-
tion electrospray-ionization mass spectra were recorded on a
MicroTOF Bruker instrument of the Mass Spectrometry Service of
the University of São Paulo.
410 nm and calculated according to a standard curve (0–0.16 lmol
pNP).
One enzyme unit (U) was defined as the amount of enzyme that
released 1.0
conditions.
lmol of product per minute under the assay
2.4. Inhibitory activity determination
The inhibition constants (Ki or K0) ofD. hansenii UFV-1 extracellu-
lar and intracellular a-galactosidases for each galactoside derivative
The pNP
obtained from Sigma–Aldrich Chemical Co. Methyl
ranoside (1) was purchased from Sigma–Aldrich. Methyl 6-O-(4-
toluenesulfonyloxy)- -galactopyranoside (2), methyl 6-azido-6-
deoxy- -galactopyranoside (3) and 4-(acetylamino)phenyl
aGal (4-nitrophenyl
a
-D
-galactopyranoside) was
i
a
-D
-galactopy-
were calculated from the Dixon plot. The pNPaGal concentrations
were 0.4, 0.8, 1.2, 1.6, and 2.0 mmol Lꢀ1 and the concentrations of
the inhibitors (galactoside derivatives) were 0.5, 1.0 and
a-D
a
-D
2.0 mmol Lꢀ1
.
a-D-galactopyranoside (4) were prepared according to described
procedures.16–18
2.5. Determination of protein concentration
2.2. 4-Nitrophenyl 6-azido-6-deoxy-
a
-
D
-galactopyranoside (5)
-galactopyrano-
Protein concentration in the enzymatic preparations was deter-
mined by the Coomassie Blue binding method (Bio-Rad Protein
Assay) with bovine serum albumin (BSA) as the standard.21
To a solution of 4-nitrophenyl 6-O-tosyl-a-D
side19 (0.12 g, 0.26 mmol) in 5 mL of dry pyridine, 2 mL of Ac2O
was added dropwise. The reaction mixture was kept at 0 °C for
18 h. The mixture was poured onto ice and concd HCl solution
was added until pH 1. The resulting aqueous solution was
extracted with EtOAc (3 ꢁ 30 mL). The combined organic phase
was then washed with satd aq Na2CO3 and then with distilled
water, dried with anhyd Na2SO4, and after filtration, concentrated
to dryness in a rotatory evaporator. The crude peracetate was dis-
solved in 3 mL of dry DMF and sodium azide (0.14 g, 2.15 mmol)
was added. The reaction mixture was heated at 65 °C for 72 h
and then the solvent was removed by evaporation. The crude prod-
uct was dissolved in a two-phase system of 20 mL of cold water
and 20 mL of EtOAc. The resulting solution was transferred to a
separatory funnel, and after shaking, the organic layer was col-
lected. The aqueous phase was further extracted with EtOAc
(2 ꢁ 20 mL), the combined organic phase was treated with dry
Na2SO4, filtered, and concentrated in a rotatory evaporator. The
residue was stirred overnight at room temperature with a solution
of NaOMe prepared by adding a catalytic amount of sodium to
10 mL of dry MeOH.20 The solution was neutralized with Amberlite
IR-120 (H+) resin, filtered, and concentrated to dryness, and the
residue was submitted to column chromatography (9:1 EtOAc–
hexane) to furnish the desired compound (0.056 g, 65% overall
3. Results and discussion
Based on the results presented in Table 1, modifications on the
galactopyranoside ring had a significant influence in the interac-
tion of these compounds with the D. hansenii UFV-1
a-galactosi-
dases. None of the derivatives analyzed (Fig. 1) were substrates
for the enzymes; all were inhibitors.
Methyl
tor of the compounds studied (Table 1 and Fig. 2). The extracellu-
lar and intracellular -galactosidases were uncompetitively
inhibited by this compound, with K0i values of 0.82 and
1.12 mmol Lꢀ1, respectively. Compound 1 inhibited the
-intra-
cellular galactosidase isoform with an affinity comparable to that
of -galactose (Fig. 1 and Table 1). The monosaccharide -galact-
-galactosidases,
a-D-galactopyranoside (1) was the most potent inhibi-
a
a
D
D
ose, a final product of the reactions catalyzed by
noncompetitively inhibited the extracellular
a
a
-galactosidase
(Ki = 2.7 mmol Lꢀ1 14
)
and uncompetitively (K0i ¼ 0:7 mmol Lꢀ1
)
the intracellular enzyme.15 Comparing the data for methyl
a-
D
-
galactopyranoside with those obtained for
D-galactose suggests
that the hydrogen bond donor capability of the anomeric center
can be removed with no deleterious consequence for the inhibi-
tory effect. Nevertheless, this modification affects the binding
mode for the extracellular isoform, since galactose is a noncom-
yield) as a light-yellow solid: mp 170 °C (decomp.), ½a D21
ꢂ
+125 (c
0.4, MeOH).
1H NMR (CD3OD, 200 MHz): d 8.22 (d, 2H, J = 9.2 Hz), 7.32 (d,
2H, J = 9.2 Hz), 5.74 (d, 1H, J = 2.2 Hz), 4.10–3.88 (m, 3H, H-2, H-
petitive inhibitor while methyl
a-
D-galactopyranoside is an
uncompetitive one.
3, H-4), 3.68–3.49 (m, 2H, H-5, H-6a), 3.23 (dd, 1H,
J
Methyl 6-O-(4-toluenesulfonyl)-a-D-galactopyranoside (2), and
methyl 6-azido-6-deoxy-a-D-galactopyranoside (3), were synthe-
6a,6b = 12 Hz, J 6b,5 = 3.7 Hz, 6b). 13C NMR (CD3OD, 50 MHz): d
163.4 (C-10), 143.8 (C-40), 126.6 (C-30), 117.8 (C-20), 99.1 (C-1),
72.9, 71.1, 70.9, 69.3 (C-2, C-3, C-4, C-5, interchangeables), 52.5
(C-6). HRESIMS calcd for C12H14N4O7Na: m/z 349.0762; found: m/
z 349.0764 [M+Na+].
sized for comparison with the parent methyl galactoside derivative
(1). The derivatives of compound 1 present groups of different
chemical nature in the C-6 position (a bulky lipophilic group and
a smaller hydrophilic group, respectively) (Fig. 1). Thus, the impor-
tance of the hydroxyl group in the C-6 position, as well as the effect
of the groups with distinct properties could be evaluated concern-
ing the recognition of the enzymatic active sites. A significant
reduction of inhibitory effect occurred for compounds 2 and 3 in
comparison with that of compound 1, which indicated that the hy-
droxyl group in C-6 is important for the recognition of the active
2.3. a-Galactosidase assay
D. hansenii UFV-1
matography as previously described14,15 were used in this study.
The hydrolytic activities of the -galactosidases were determined
by measuring the release of 4-nitrophenol (pNP) from 4-nitro-
phenyl -galactopyranoside (pNP Gal). The -galactosidase
a-galactosidases purified by two-step chro-
a
sites of D. hansenii UFV-1
a-galactosidases (Table 1). There are a
a-
D
a
a
few studies in literature concerning inhibition of
a
-galactosidase

604
P. A. Viana et al. / Carbohydrate Research 346 (2011) 602–605
Table 1
Inhibitory activity (Ki or K0 ) of
a-D-galactopyranoside derivatives against Debaryomyces hansenii UFV-1
a
-galactosidases
i
Ki or K0i (mmol Lꢀ1
)
Compound
Inhibition
Extracellular
Intracellular
Extracellular
Intracellular
0.82 0.01 (K0i)
6.00 0.02
9.35 0.02 (K0i)
3.03 0.03
>20
1.12 0.03 (K0i)
2.33 0.02
2.45 0.01
5.53 0.03
>20
1
2
3
4
Uncompetitive
Competitive
Uncompetitive
Competitive
NDa
Uncompetitive
Competitive
Competitive
Noncompetitive
NDa
5
0.7 0.02 (K0i)
Galactose
Noncompetitive
Uncompetitive
2.7 0.01
a
ND: not determined under assay conditions used.
OH
OH
OH
N3
OH
OTs
O
O
O
HO
OH
HO
HO
OH
OMe
OH
3
OMe
1
OMe
OH
2
OH
OH
N
3
O
O
O
HO
HO
HO
4
N
H
OH
O
CH
3
5
NO
O
2
Figure 1.
a-
D-Galactopyranoside derivatives: Methyl
a-
D
-galactopyranoside (1); methyl 6-O-(4-toluenesulfonyl)-a-D-galactopyranoside (2); methyl 6-azido-6-deoxy-a-D-
galactopyranoside (3); 4-(acetylamino)phenyl
a-
D-galactopyranoside (4); 4-nitrophenyl 6-azido-6-deoxy-a-D
-galactopyranoside (5).
Figure 2. Inhibition of Debaryomyces hansenii UFV-1 extracellular a-galactosidase by methyl a-D
-galactopyranoside at different concentrations (0, 0.5 and 1.0 mmol Lꢀ1). (A)
Dixon plots in different substrate concentrations (mmol Lꢀ1): 0.4 (d); 0.8 (s); 1.2 (N) and 1.6 (4); (B) Cornish–Bowden plots in different substrate concentrations
(mmol Lꢀ1): 0.4 (d); 0.8 (s); 1.2 (N) and 1.6 (4).
activity using synthetic compounds. The presence of three hydro-
xyl groups (at the C-3, C-4, and C-6 positions) in the pNP Gal sub-
strate is necessary for its derivatives to be substrates of Aspergillus
of the glycosyl hydrolases isoforms (Fig. 1). The aromatic galacto-
side 4 is a derivative of 4-nitrophenyl -galactopyranoside in
which the nitro group was reduced and acetylated with the aim
of evaluating the capability of the -galactosidases to hydrolyze
aryl -galactopyranosides with electron-releasing groups
a
a-D
niger
a-galactosidase, but coffee bean
a-galactosidase tolerates
a
both the 2- and 6-deoxy derivatives.2 Compound 2 was a slightly
more potent inhibitor than compound 3, especially for the extra-
a-D
instead of electron-withdrawing ones. Finally, compound 5 was
cellular
a-galactosidase isoform (Fig. 1 and Table 1), but a direct
prepared to evaluate whether the presence of the azido group at
comparison must be viewed with care since the inhibition mode
is different for the two compounds. This difference could be attrib-
uted to the more lipophilic character of compound 2 and more effi-
cient desolvation when this galactoside derivative is transferred to
the enzymatic binding site. Thus, synthesis of glycosyl derivatives
with hydrophobic substituents at the anomeric carbon could be a
good strategy to search for more potent inhibitors. Other modifica-
tions in the C-6 position are necessary to furnish information about
the possibility of substituting hydrophobic moieties for the C-6 hy-
droxyl group.
C-6 influences the
the parent substrate 4-nitrophenyl
ary experiments carried out with a partially purified b-glycosidase
from Biomphalaria glabrata (unpublished data) showed that identi-
cal substitution in 4-nitrophenyl b-
corresponding 6-azido derivative that was an enzymatic inhibitor
with a Ki in the range of 1.0 mmol Lꢀ1
In the series of the aromatic galactosides, it was observed that a
variation of the substituent groups promoted significant influence
a
-galactosidase activity in comparison with
a-D-glucopyranoside. Prelimin-
D-glucopyranoside led to the
.
on the behavior of those derivatives when compared with pNPa-
The compounds 4-(acetylamino)phenyl
(4) and 4-nitrophenyl 6-azido-6-deoxy-
were prepared to be initially assayed as substrates or inhibitors
a
-
D
-galactopyranoside
Gal. Galactoside 4 was an inhibitor, though less potent than 1, con-
firming the previous suggestion (Fig. 1 and Table 1). Galactoside 4
is an inhibitor while pNPaGal is a substrate, probably because the
a
-D-galactopyranoside (5)

P. A. Viana et al. / Carbohydrate Research 346 (2011) 602–605
605
aglycon of 4 is a poorer leaving group (4-acetylaminophenol) than
that of pNP Gal (4-nitrophenol) due to its electron-releasing char-
acter. However, it is important to consider that the binding of the
N-acetyl group to the enzymatic active site may also contribute to
its recognition as an inhibitor.
nação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES,
and Conselho Nacional de Desenvolvimento Científico e Tecnológ-
ico—CNPq, Brazil.
a
References
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bind to the -galactosidases. Considering that it was not possible
to determine the Ki values of both -galactosidase isoforms for
compound 5 under the assay conditions, it seems plausible to as-
sume that this substance is not recognized by the enzymes, evi-
dencing once more the poor binding of azido group in the C-6
position already observed for derivative 3.
aGal, did not show the ability to
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-
D
-galactopyranoside derivatives, in addition to the active site. It
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for each -galactosidase isoform through further chemical
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Acknowledgments
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This study was supported by grants from the Fundação de
Amparo à Pesquisa do Estado de Minas Gerais—FAPEMIG, Coorde-