Optimizing the enzymatic synthesis of β-d-galactopyranosyl-d-xyloses for their use in the evaluation of lactase activity in vivo

Article abstract of DOI:10.1016/j.bmc.2007.04.067

Disaccharides 2-O-, 3-O-, and 4-O-β-d-galactopyranosyl-d-xyloses (2, 3, and 1, respectively) were obtained by β-galactosidase-catalyzed reactions for their use in the evaluation of intestinal lactase activity in vivo. Their administration to suckling rats

Full text of DOI:10.1016/j.bmc.2007.04.067

Bioorganic & Medicinal Chemistry 15 (2007) 4836–4840
Optimizing the enzymatic synthesis of b-D-galactopyranosyl-
D-xyloses for their use in the evaluation of lactase activity in vivo
Carmen Hermida,a,b Guillermo Corrales,a Francisco J. Canada,a,
˜
b,
a,
*
*
´
´
Juan J. Aragon and Alfonso Fernandez-Mayoralas
aDepartamento de Quı´mica Orga´nica Biolo´gica, Instituto de Quı´mica Orga´nica General, CSIC, Juan de la Cierva 3,
28006 Madrid, Spain
bDepartamento de Bioquı´mica and Instituto de Investigaciones Biome´dicas Alberto Sols UAM-CSIC,
Facultad de Medicina, Universidad Auto´noma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain
Received 26 February 2007; revised 17 April 2007; accepted 27 April 2007
Available online 6 May 2007
Abstract—Disaccharides 2-O-, 3-O-, and 4-O-b-D-galactopyranosyl-D-xyloses (2, 3, and 1, respectively) were obtained by b-galac-
tosidase-catalyzed reactions for their use in the evaluation of intestinal lactase activity in vivo. Their administration to suckling rats
followed by determination of the derived D-xylose in the urine and measurement of lactase activity in intestinal homogenates showed
1 to be the most suitable disaccharide for a potential test of the deficiency of intestinal lactase. The synthesis of 1 was further studied
by evaluating the effect of different variables on the yield and regioselectivity of the enzymatic galactosylation, and the purification
process was optimized.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
anosyl-D-xylose (1, Scheme 1), a structural analog of
lactose.4 This compound was shown to be a substrate
of the enzyme in vivo,4 as its administration to suckling
rats led to urinary elimination of D-xylose and to the
accumulation of this pentose in plasma, which increased
in both fluids in a dose-dependent manner.5 In addition
to 1, we reported that the regioisomers 2- and 3-O-b-D-
galactopyranosyl-D-xyloses (2 and 3, respectively) were
also substrates of the enzyme in vitro.6 In fact, the ki-
netic parameters for compounds 2 and 3 showed that
the intestinal lactase from lamb hydrolyzes more effi-
ciently these disaccharides than 1. Therefore, it was nec-
essary to have a procedure to obtain pure preparations
of the three disaccharides in amounts enough to perform
in vivo experiments, so that their individual characteris-
tics could be later examined under these conditions.
Lactose intolerance is caused by deficiency of the en-
zyme lactase (hypolactasia), which is normally produced
by the cells that line the small intestine. Common symp-
toms of lactose intolerance include bloating, abdominal
pain or cramps, flatulence, and diarrhea.1 The most
commonly used test for lactose intolerance in clinic is
the hydrogen breath test.2 This technique requires high
doses of administered lactose, which can produce con-
siderable disturbances in the patients, and frequently
produce both false-negatives and false-positives, due to
individual variations in endogenous gas production
capacity.3
Over the last years, our group has been working at
developing a new diagnostic method to evaluate lactase
activity in vivo based on the use of 4-O-b-D-galactopyr-
We previously reported7 the synthesis of different en-
riched mixtures of disaccharides 1, 2, and 3 by galac-
tosylation of D-xylose (7) using a b-galactosidase
enzyme and o-nitrophenyl b-D-galactopyranoside (6)
(Scheme 1). The ratio of 1, 2, and 3 was dependent on
the source of the b-galactosidase used. We describe here
several modifications of the described enzymatic proce-
dure in order to obtain pure disaccharides in a gram
scale. The results of evaluating the efficiency of each of
Keywords: Disaccharides; Enzymatic synthesis; Galactosidases;
Intestinal lactase.
*
Corresponding authors. Fax: +34 91 4975353 (J.J.A.), tel.: +34 91
562 29 00; fax: +34 91 564 48 53 (A.F.-M.); e-mail addresses:
juanjose.aragon@uam.es; mayoralas@iqog.csic.es
´
Present address: Centro de Investigaciones Biologicas, CSIC, Ramiro
de Maeztu 9, 28040 Madrid, Spain.
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.067

C. Hermida et al. / Bioorg. Med. Chem. 15 (2007) 4836–4840
4837
O
OH
HO
HO
RO
OR
RO
RO
RO
OR
O
O
OR
RO
RO
O
O
O
O
RO
HO
O
OH
O
OR
HO
HO
RO
RO
RO
1
2 R=H
4 R=Bz
3 R=H
5 R=Bz
OH
HO
HO
NO2
β
-galactosidase
O
O
HO
HO
O
1 + 2 + 3
+
OH
HO
HO
NO2
6
7
HO
Scheme 1. Synthesis of disaccharides 1, 2, and 3 by galactosidation of D-xylose (7) using a b-galactosidase.
the three disaccharides in detecting the changes of rat
lactase activity in vivo are discussed.
the acidic proton at C-2. In view of these difficulties,
we changed the route to obtain disaccharide 3, and the
enzymatic galactosylation was carried out on a xylose
derivative with a protecting group at the anomeric posi-
tion. In previous studies on the regioselectivity of disac-
charide synthesis using b-galactosidase from Escherichia
coli,10 we showed that the presence of an aromatic ring
at the anomeric position of b-D-xylopyranoside accep-
tors drives the reaction toward the formation of 3-O-
b-D-galactopyranosyl derivatives. Thus, when the galac-
tosylation was performed on benzyl b-D-xylopyranoside
as substrate (8, Scheme 3) the disaccharide 9 was ob-
tained as main product, which after hydrogenolysis
afforded 3.
2. Results and discussion
Following our previous results,7 the galactosylation of
D-xylose using b-galactosidase from Aspergillus oryzae
gave a mixture of 2 and 3 as main products, accompa-
nied by some minor amount of 1 (5:49:46 ratio of 1, 2,
and 3, respectively, determined by gas chromatography).
To separate the disaccharide products, the mixture was
benzoylated and fractionated on silica gel column chro-
matography, to give benzoyl derivatives 4 and 5 (see
Scheme 1) separately. Debenzoylation of 4 afforded 2.
However, attempts to obtain 3 from 5 under a variety
of conditions (NaOMe/MeOH; guanidine/NaOMe/
MeOH8; KCN/MeOH9) led to a partial cleavage of the
glycosidic bond with formation of galactose. The easy
cleavage of the glycosidic bond can be explained by con-
sidering that debenzoylation at the anomeric position
takes place more rapidly due to electronic factor. Hemi-
acetal I (Scheme 2) equilibrates with the acyclic carbonyl
species II, which leads to elimination by abstraction of
For the synthesis of 1, the galactosylation of D-xylose
was carried out in the presence of b-galactosidase from
E. coli at 25 °C, pH 7.0, to give a mixture of 1, 2, and
3 in a ratio of 8.0:1.5:0.5, respectively. After purification
using a carbon–Celite column compound 1 was
obtained.
Galactosylation at the anomeric position of xylose was
not observed in any of the enzymatic reactions investi-
gated. The lack of formation of 1-O-b-D-galactopyrano-
syl-a,b-D-xylopyranosides could be due to the low
nucleophilicity of the anomeric hydroxyl group.
Base
O
BzO
5
O
OH
Gal
BzO
6
O
HO
HO
I
OCH Ph
2
HO
β
-galactosidase
from E. coli
8
:B
H
OH
BzO
OH
BzO
elimination
reaction
HO
HO
O
H
2
O
Gal
O
HO
O
O
OCH Ph
3
2
HO
II
HO
Pd/C
9
Scheme 2. Proposed mechanism for the cleavage of the glycosidic
bond during the debenzoylation of 5.
Scheme 3. Synthetic pathway for disaccharide 3.

4838
C. Hermida et al. / Bioorg. Med. Chem. 15 (2007) 4836–4840
The above-described procedures allowed us to obtain
gram quantities of pure disaccharides utilizable for
in vivo experiments. As seen in Table 1, administration
of disaccharides 1–3 separately to suckling rats followed
by determination of the derived D-xylose in the urine
and measurement of lactase activity in intestinal homog-
enates elicited a gradual decrease in the elimination of
urinary pentose as a function of age, that was markedly
different with the three compounds. Thus, although
maximal elimination of D-xylose was similar for each
disaccharide at the 15th day, its decrease with com-
pound 1 was practically similar to the physiologic de-
cline of lactase activity along the weaning age, whereas
the decrease of D-xylose elimination was lower with
compound 2, and much lower with compound 3, as
compared with the enzyme decline. Consequently, corre-
lation between D-xylose elimination and lactase activity
was the highest with 1, lower with 2, and very low with
3. These results agree with our previous data describing
the individual behavior of these disaccharides in greater
details.5 Steady-state kinetics of rat intestinal lactase
in vitro showed the highest Vmax/Km ratio for compound
3 (even greater than that for lactose), intermediate for 2,
and very low for 1.5 Therefore, the high catalytic effi-
ciency with compound 3 accounts for its extensive
hydrolysis in vivo even with low levels of lactase activity,
whereas the low activity for compound 1 accommodates
the changes of D-xylose elimination to those of enzyme
activity. Thus, disaccharide 1 resulted the most effective
compound to evaluate the changes in lactase activity
in vivo, despite being the worst substrate. These obser-
vations are in agreement with previous reports on the
function of the hydroxyl groups of the lactose molecule
in its interaction with lactase.11,12 Thus, the HO-6 was
found important for hydrolysis of the glycosidic
bond,11,12 and so its absence in compound 1 makes this
disaccharide a relatively poor substrate. This position is
occupied by HO-2 in compound 3.13 which improves the
enzyme catalytic efficiency, making it a substrate even
better than lactose. In compound 2, HO-3 is equivalent
to the HO-6 group, but the anomeric nature of HO-1
appears to hinder its interaction with the enzyme. Com-
pound 1 was, therefore, chosen as the most suitable tool
for a potential test of the deficiency of intestinal lactase
in noninvasive way.
Regarding the synthesis of 1, a regiospecific synthesis of
this disaccharide by galactosylation of benzyl a-D-xylo-
pyranoside (the anomer of 8) using b-galactosidase
from E. coli has been reported.14 However, this synthe-
sis requires the preparation of the xylopyranoside
acceptor and subsequent removal of the benzyl group
after glycosylation. We pursued a straightforward syn-
thesis of 1 by optimizing the enzymatic galactosylation
of unprotected xylose. Thus, using the enzyme from
E. coli, we evaluated the effect of addition of cosolvents
(DMF, DMSO, THF, diethyleneglycoldiethylether, and
acetonitrile), pH, and temperature, on the yield and reg-
ioselectivity of disaccharides formed (Table 2). Glyco-
sylation was observed only when DMF and DMSO
were used as cosolvents (20% v/v) (entries 1 and 2).
However, reactions took longer and the yield of disac-
charides decreased. The pH changes from 7.0 to 5.0
or 8.5 (entries 3–5) had also a negative effect on the
yield, although an increase of selectivity toward the for-
mation of 1 was observed at pH 5.0. In the case of tem-
perature, an appreciable change of regioselectivity in
Table 2. Yield and regioselectivity (as the ratio of 1:[2 + 3]) of the
disaccharides 1, 2, and 3 from the b-galactosidase-catalyzed reaction of
6 and 7
Entry Cosolvent T (°C) pH Yield (%) Ratio of 1:(2 + 3)
1
2
3
4
5
6
7
8
9
DMSO
DMF
—
37
37
37
37
37
45
25
5
7.0 35
7.0 38
7.0 50
5.0 22
8.5 36
7.0 48
7.0 45
7.0 48
7.0 46
70:30
72:28
71:29
81:19
68:32
68:32
79:21
80:22
83:17
—
—
—
—
—
—
ꢀ5
Table 1. Elimination of D-xylose in the urine of suckling rats after oral administration of the disaccharides 1, 2, and 3, and intestinal lactase activity
as a function of agea
Age (days)
D-Xylose elimination in urine (%)b
Lactase activity (U/mg protein)
From 1
From 2
From 3
15
18
30
23.66 0.60
6.99 0.55
3.05 0.21
r = 0.98c
23.10 0.28
20.71 0.64
9.43 1.05
r = 0.84c
23.30 0.94
20.63 0.23
19.39 0.28
r = 0.58c
0.172 0.0026
0.070 0.0023
0.015 0.0005
r2 = 0.96c
r2 = 0.71c
r2 = 0.33c
P < 0.0001c
P < 0.001c
P < 0.05c
a Distinct groups of four suckling rats from the same litter and of the indicated age were used for each disaccharide. Rats were fasted for 6 h in
metabolic cages at 30 °C and orally administered 4 mg of the corresponding disaccharide 1–3 in 0.3 mL of water. Urine was collected by
intermittent transabdominal bladder pressure during 6 h after disaccharide administration. D-Xylose elimination during this time was determined in
the urine colorimetrically and intestinal lactase activity was determined post mortem in all animals. Data are means SE.
b
D-Xylose eliminated in the urine is indicated as percentage of the amount of the administered disaccharide.
c Correlation parameters were calculated by linear regression analysis of urinary D-xylose after administration of each disaccharide and intestinal
lactase activity. Statistical significance was set up at P < 0.05.

C. Hermida et al. / Bioorg. Med. Chem. 15 (2007) 4836–4840
4839
favor of 1 was also observed as the reaction tempera-
ture was lowered (entries 7–9). However, at 5 and
ꢀ5 °C a greater amount of enzyme had to be added
in order to proceed the reactions until completion (6
and 12 times more enzyme, respectively). The best re-
sults in terms of enzyme efficiency and yield were ob-
tained at 37 °C in buffer pH 7.0 (entry 3), under
which the disaccharide 1 was formed with satisfactory
regioselectivity. Therefore, these were the conditions
of choice for the synthesis of 1.
procedure allows to obtain multi-gram quantities of 1 in
only two working days.
3. Conclusions
The first synthesis of disaccharide 1 was carried out
using a multi-step approach15 requiring toxic chemical
reagents, which was unpractical regarding a possible
industrial application. The synthesis procedure de-
scribed in this work involves a simple enzymatic process
using readily available substrates, and is presently being
adapted for production at industrial scale. The synthesis
of 1 optimized herein, in conjunction with the recent
optimization of the measurement of the derived D-xylose
in either urine or blood from animals,5 as an estimate of
the overall lactase activity in vivo, provides a new meth-
od for the noninvasive diagnosis of hypolactasia which
is now ready for clinical trials.
We next focused on the isolation of 1 from the reaction
mixture. Fractionation on a carbon–Celite column re-
quired large volumes of isopropanol–water that may
be troublesome for industrial application. We quanti-
fied the adsorption preference on carbon of the different
sugars present in the reaction mixture, in order to min-
imize the amount of carbon added and, therefore, the
volume of solvent during the elution step. To a typical
crude reaction mixture (66 mL volume), carbon was
added in 0.5–1 g portions. Aliquots from the solution
were analyzed by gas chromatography. As can be seen
in Figure 1, the relative selectivity of the adsorption
on carbon is o-nitrophenyl b-D-galactopyranoside
(6) > disaccharides (1, 2, 3) > xylose (7), galactose.
About 90% of disaccharides were adsorbed after addi-
tion of 10 g of carbon to the reaction mixture, while
around 80% of xylose and galactose remained in solu-
tion. In view of these results, we modified the procedure
in the following manner. After the reaction was fin-
ished, carbon (0.12 g/mL of solution) was added, then
the solution, containing the monosaccharides and buffer
salts, was filtered, and the carbon washed with 5% iso-
propanol in water. Washing fractions containing the
disaccharide 1 were concentrated and crystallized from
acetone–water. This modified procedure has several
advantages. The volume of fractions containing the
disaccharide is reduced in more than tenfold. In addi-
tion, heating the mixture to stop the reaction by deacti-
vation of the enzyme is not required. Application of this
4. Experimental
4.1. General remarks
b-Galactosidases were purchased from a commercial
source and used without further purification. o-Nitro-
phenyl b-D-galactopyranoside, D-xylose, and other
chemicals were from commercial sources. Melting points
are uncorrected. TLC was performed on silica gel GF254
1
with detection by charring with H2SO4. H NMR spec-
tra were measured at 300 or 400 MHz. 13C NMR were
obtained at 125 MHz. Gas–liquid chromatographic
analysis was carried out on a chromatograph with
FID detector using fused SE-54 capillary column (5%
Ph Me silicone, 12 m, 0.2 mm id, and 0.33 lm film). A
flow rate of 1 mL/min of nitrogen was utilized. Samples
were previously silylated. D-Xylose was determined col-
orimetrically with phloroglucinol.16
4.1.1. 2-O-b-D-Galactopyranosyl-D-xylose (2). To a solu-
tion of 6 (10 g, 50 mM) and D-xylose (7, 50 g, 500 mM)
in buffer (0.2 M KH2PO4, pH 5.5) was added b-galacto-
sidase from A. oryzae (85 mg, 450 U), and the mixture
was incubated at 30 °C for 2.5 h. When all the substrate
was consumed (2.5 h, TLC isopropanol/NH3/H2O,
7.5:0.5:2.5) the reaction was stopped by heating at
100 °C for 10 min, washed with CH2Cl2, and concen-
trated. The residue was fractionated on a carbon–Celite
column (H2O/isopropanol, 100:0 ! 95:5) to give a sepa-
rated fraction containing the mixture of disaccharides 1,
2, and 3 (3.6 g). This mixture was dissolved in CH2Cl2
(30 mL) and treated with pyridine (7.0 mL), N,N-di-
methyl-4-aminopyridine (cat.), and benzoyl chloride
(10.0 mL, 86.0 mmol), stirring at 5 °C for 24 h. After
this time, the mixture was diluted with CH2Cl2, washed
with H2O, dried (Na2SO4), and concentrated. The resi-
due was eluted through a silica gel column chromatogra-
phy (toluene/AcOEt, 10:1 ! 10:1). Fractions containing
4 were crystallized from CH2Cl2/Et2O/hexane (1:20:20).
Crystals were dissolved in MeOH (180 mL) and treated
with 0.5 M NaOMe in MeOH (12 mL) at room temper-
ature for 2 h. The solution was neutralized with Amber-
100
80
60
40
20
0
0
2
4
6
8
10
12
Amount of added carbon (g)
Figure 1. Percentage of remaining sugars in solution after addition of
activated carbon to the reaction mixture: d, D-xylose; s, D-galactose;
j, 1; h, 2; ,, 3; ., 6.

4840
C. Hermida et al. / Bioorg. Med. Chem. 15 (2007) 4836–4840
lite IR-120 (H+) and concentrated to give pure 2 (1.2 g).
1H and 13C NMR spectra of 2 were identical to those
previously reported6; [a]D +10° (c 1.0, H2O), lit.6 [a]D
+12° (c 1.0, H2O).
Acknowledgments
´
We thank Dr O.H. Martınez-Costa for critical reading
of the manuscript. Financial support from the Comuni-
dad de Madrid [08.6/0035.2/2001 (to J.J.A.)], Fondo de
Investigaciones Sanitarias [PI050563 (to J.J.A.)], and
4.1.2. 3-O-b-D-Galactopyranosyl-D-xylose (3). To a solu-
tion of 6 (5.6 g, 50 mM) and 815 (9.0 g, 100 mM) in buf-
fer (0.2 M K2HPO4, pH 7.0) was added b-galactosidase
from E. coli (3.6 mg, 1152 U), and the mixture was left
at 37 °C. After 1 and 3.5 h more 6 was added (2.8 g at
the time). When all the substrate was consumed (TLC
isopropanol/NH3/H2O, 7.5:0.5:2.5) the reaction was
stopped by heating at 100 °C for 10 min. The solution
was concentrated, and the residue was fractionated by
silica gel column chromatography (AcOEt/MeOH,
30:1 ! 5:1), to give 9 (1.9 g). Hydrogenation of 9
(1.85 g) with 10% Pd–C (0.5 g) in H2O/MeOH (1:1) at
atmospheric pressure afforded 3 (1.4 g). 1H and 13C
NMR spectra of 3 were identical to those previously re-
ported6; [a]D +22° (c 1.0, H2O), lit.6 [a]D +17° (c 1.0,
H2O).
´
Ministerio de Educacion y Ciencia [CTQ2004-03523/
BQU (to A.F.-M.) and BFU2005-09071 (to J.J.A.)] is
greatly appreciated.
References and notes
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and Molecular Bases of Inherited Disease; Scriber, C. R.,
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4. Aragon, J. J.; Fernandez-Mayoralas, A.; Jimenez-Barbero,
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´
´
´
4.1.3. 4-O-b-D-Galactopyranosyl-D-xylose (1). To
a
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mixture was left at 37 °C for 22 h. After this time, acti-
vated carbon (200 g) was added and the mixture was
stirred for 30 min. Then, the mixture was filtered, and
the solid was washed with H2O/isopropanol
(100:0 ! 95:5). Fractions containing disaccharide
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trated, and the residue was crystallized from acetone–
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Fernandez-Mayoralas, A.; Aragon, J. J. Clin. Chem.
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1
H2O, to give 1 (5.15 g). The H NMR spectrum of 1
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´
´
10. Lopez, R.; Fernandez-Mayoralas, A. J. Org. Chem. 1994,
13
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tained as described4 and homogenized with 10 vol-
umes of 100 mM sodium maleate, pH 6.0. Lactase
activity was determined in the homogenate by measur-
ing the liberated galactose as described.4 One unit of
enzyme activity is defined as the amount of enzyme
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at 25 °C.
´
tın-Lomas, M. Carbohydr. Res. 1995, 271, 31–42.
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Jimenez-Barbero, J. Tetrahedron 1994, 50, 6417–6432.
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