Preparation of α-galactooligoglycosides by cell walls from Cryptococcus laurentii using a novel α-galactosyl donor

Article abstract of DOI:10.1016/j.tetasy.2017.07.007

The cell walls of an acapsular strain of the yeast Cryptococcus laurentii catalyze the regioselective formation of α-galactooligosaccharides through self-condensation of 4-nitrophenyl α-D-galactopyranoside and of a novel activated α-galactosyl donor 2,2,2-trifluoroethyl α-D-galactopyranoside. The latter substance can be easily prepared by several methods and is highly soluble in water and therefore can be used in higher initial concentrations suppressing secondary product hydrolysis. The preparative reaction catalyzed by cell walls provided 17.4% and 2% of corresponding 2,2,2-trifluoroethyl galactobioside and galactotrioside, respectively, while the reaction with 4-nitrophenyl α-D-galactopyranoside provided the corresponding 4-nitrophenyl galactobioside and galactotrioside in 6.6 and 2.5% yields, respectively. The reactions proceeded with strict α-(1 → 6)-regioselectivity.

Full text of DOI:10.1016/j.tetasy.2017.07.007

Tetrahedron: Asymmetry 28 (2017) 1089–1094
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Preparation of
a-galactooligoglycosides by cell walls from
Cryptococcus laurentii using a novel a-galactosyl donor
⇑
Vladimír Mastihuba , Mária Mastihubová, Miroslav Belák, Jana Dudíková, Elena Karnišová Potocká,
Ladislav Petruš
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
The cell walls of an acapsular strain of the yeast Cryptococcus laurentii catalyze the regioselective forma-
Received 8 July 2017
Accepted 20 July 2017
Available online 7 August 2017
tion of
a
-galactooligosaccharides through self-condensation of 4-nitrophenyl
a-D-galactopyranoside and
of a novel activated
a
-galactosyl donor 2,2,2-trifluoroethyl -galactopyranoside. The latter substance
a-D
can be easily prepared by several methods and is highly soluble in water and therefore can be used in
higher initial concentrations suppressing secondary product hydrolysis. The preparative reaction cat-
alyzed by cell walls provided 17.4% and 2% of corresponding 2,2,2-trifluoroethyl galactobioside and galac-
totrioside, respectively, while the reaction with 4-nitrophenyl
corresponding 4-nitrophenyl galactobioside and galactotrioside in 6.6 and 2.5% yields, respectively.
The reactions proceeded with strict -(1 ? 6)-regioselectivity.
a-D-galactopyranoside provided the
a
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
glycosidic links between monosaccharide units.11 The use of gly-
cosidases in the synthesis of oligosaccharides offers several advan-
tages in view of their regioselectivity and anomeric specificity, thus
avoiding the chain of protection and deprotection steps typical for
standard chemical procedures.12,13 Glycosidases can catalyze the
formation of a glycosidic bond by reverse hydrolysis, providing
that the enzyme is not inhibited by high concentrations of sub-
strates14,15 and is free of contaminating glycosidases with opposite
anomer specificity. The syntheses, however, more frequently
employ the ability of glycosidases to promote glycosyl transfer.
The level of transferase activity differs from enzyme to enzyme
and depends also on the reaction conditions and on the glycosyl
donor. Low regioselectivity and product yields of glycosidases have
prompted extensive studies focusing especially on reaction condi-
tions, new enzyme sources and substrate modifications. Activated
substrates, such as glycosyl fluorides,16–19 azides20 and 1-O-
acetates21 or nitrophenyl,22–24 vinyl,25,26 4-pyridyl19 and nitrated
2-pyridyl glycosides27 may be used as glycosyl donors. The prob-
lems encountered with the use of activated donors usually come
from their stability, water solubility, cost, toxicity and enzyme
inhibition by released aglycon. The search for new types of glycosyl
donors is therefore still an important topic for scientists working
on the enzymatic synthesis of glycosides and oligosaccharides.
a-D
-Galactooligosaccharides, especially various positional iso-
mers of -galactobiose, are structural components of molecules
a
involved in many biological processes, including immunity
response, transplant rejection and pathogen-to-cell adhesion.1–5
Oligosaccharides containing
a-(1,6)-galactosidic linkages (raffi-
nose, stachyose, verbascose and ajugose) are considered to be pre-
biotic non-digestible oligosaccharides widely distributed in
legume seeds and can significantly stimulate the growth of benefi-
cial Bifidobacteria and Lactobacilli.6 Furthermore, there have been
reported anti-allergic and anti-inflammatory effects of
galactooligosaccharides.7–9 This indicates that
-(1,6)-linked
galactobiosides and triosides are useful starting building blocks
for the synthesis of more complex -galactooligosaccharides,
which can help to clarify their immunomodulation effects and to
compare them with similar effects of other indigestible oligosac-
charides.10 As a consequence of increasing malnutrition, there is
a growing demand for ingredients promoting human health and
nourishment. The development of relatively simple and non-toxic
a-(1,6)-D-
a
a-D
methods for the rapid synthesis of
precursors is therefore still important.
The main problem in the chemical preparation of oligosaccha-
rides is the control of the formation of stereo- and regioselective
a-D-galactooligosaccharides
a
-Galactosidases13,28,29 (E.C. 3.2.1.22) hydrolyze the
a-1,6-link-
age of galactosyl residues on the non-reducing end of the oligo-
and polysaccharides such as melibiose, raffinose, stachyose, and
galactomannan. These enzymes have found use in the food
⇑
Corresponding author.
E-mail address: chemvrma@savba.sk (V. Mastihuba).
http://dx.doi.org/10.1016/j.tetasy.2017.07.007
0957-4166/Ó 2017 Elsevier Ltd. All rights reserved.

1090
V. Mastihuba et al. / Tetrahedron: Asymmetry 28 (2017) 1089–1094
industry for the hydrolysis of raffinose to improve crystallization of
sucrose or hydrolysis of -galactooligosaccharides in soybean
milk. Their main synthetic potential is however in the opposite
of better solubility and less complex mixtures after evaporation
of the released alcohol. The rate of transglycosylation with these
glycosides may be markedly low. Glycosyl fluorides, despite their
preparation under toxic conditions, are also known to be used
and well accepted as substrates by glycosidases as well as by their
a-D
type of reaction – the regioselective preparation of structured
a-
D-galactooligosaccharides or
a
-galactosides.30,31 Contrary to
b-galactosidases, only a limited number of
a
-galactosidases is
mutants, glycosynthases.16 However, the -galactosyl fluoride46
a
commercially available. New sources of their inexpensive stable
variants are therefore in high demand.
used as a donor under our enzymatic transgalactosylation condi-
tions failed. We have mainly obtained hydrolysis products.
To circumvent the problems with substrate solubility, low reac-
Several wild
a-galactosidases of plant or microbial origin, as
well as their mutants, have been reported to catalyze the enzy-
tivity and product separation, we prepared 2,2,2-trifluoroethyl
a-
matic synthesis of
a
-
D
-galactooligosaccharides through transgalac-
D
-galactopyranoside 1 as a novel type of activated glycosyl donor
tosylations.17–19,22,23,32–38 When the donor and acceptor of
transglycosylation are distinct structures, the self-condensation
of activated donor occurs as a competitive reaction.39,40 On the
other hand, the regioselective self-condensation of suitable donor
for a-transgalactosylations catalyzed by the cell walls of C. lauren-
tii. We expected that the trifluoroethoxy moiety would be a better
leaving group in tranglycosylations in comparison with the non-
substituted alkoxy moieties, while possessing all the other advan-
tages of alkyl glycosides as donors in transglycosylations. Substrate
1 can be prepared enzymatically, as reported by Símerská et al.30
provides access to a-D
-galactooligosaccharides.17,22,23,32,38,40
Previously,41 we reported the synthesis on a scale of b-fructofu-
ranosides by crude cell walls from an acapsular strain of Cryptococ-
cus laurentii. Besides b-fructofuranosidase, the cell walls also
The reported reaction started from 4-nitrophenyl a-D-galactopyra-
noside 2 and the yield of 1 was only 7%. We used our preparation of
cell walls from C. laurentii to test economically more favorable
trangalactosylation from melibiose to 2,2,2-trifluoroethanol, but
obtained 1 in only 12% yield (Scheme 1).
display
transglycosylation potential of the cell wall material in regioselec-
tive synthesis of -galactooligosaccharides and their respective
glycosides. For this purpose, we introduced a novel activated
-galactosyl donor, 2,2,2-trifloroethyl -galactopyranoside and
compared its usability with 4-nitrophenyl -galactopyranoside,
a-galactosidase activity. Herein we report the preparative
a
-D
a
a-D
OH
CF3CH2OH
a
-D
HO
α-galactosidase
O
a compound standardly used in enzymatic transgalactosylations.
(cell walls C. laurentii)
pH 4.8, 37°C, 6 h, 12 %
OH
HO
HO
HO
HO
O
O
2. Results and discussion
HO
O
HO
HO
OCH2CF3
D
-glucose
1
HO
OH
2.1. Preparation of the biocatalyst
Scheme 1. Preparation of 2,2,2-trifluoroethyl
cell walls from C. laurentii from melibiose.
a-D-galactopyranoside 1 catalyzed by
The cell wall material from Cryptococcus laurentii CCY 17-3-6
used as the biocatalyst was prepared using an identical procedure
to that previously reported method for the production of cell walls
comprising b-fructofuranosidase.41 During our preliminary testing,
we observed that the cell wall material hydrolyses melibiose,
The chemical synthesis of substrate 1 was accessible by stan-
dard Fisher glycosylation. -Galactose was refluxed with 2,2,2-tri-
D
fluoroethanol in the presence of Lewis acid catalyst, boron
trifluoride diethyl etherate (BF3ꢀOEt2) over a long reaction time
(15 h) until products equilibrium was reached and the most of
the thermodynamically stable a-pyranoside 1 was formed. Product
1 was isolated in 26% yield (section 4.4., Method A).
Microwave irradiation of acid-catalyzed glycosylation of galac-
tose at 90 °C in a pressurized tube47 accelerated the Fisher reaction
to form the major product 1 in 5 minutes, which is a significant
shortening of the reaction time in comparison with the reaction
in the open system under conventional heating. After purification
of the reaction mixture, product 1 was obtained in 38%. yield (Sec-
tion 4.4., Method B). The capacity of our microwave reactor how-
ever limited the preparation of 1 to 0.25 gram in one batch. The
relatively low yield of the Fisher reaction (under either conven-
tional or microwave heating) can be attributed to the low acceptor
diluted 4-nitrophenyl
a
-D
-galactopyranoside and short-alkyl
a-
galactosides and also catalyzes
a
-galactosyl transfer from meli-
biose to short chain alcohols, while production of galactooligosac-
charides by reversed hydrolysis was negligible (data not shown).
Induction of
observed by Mészárosová et al.42 It is interesting to note that the
expression of the -galactosidase was induced by the presence of
lactose, which in fact bears the b-galactosyl structural fragment.
The presence of -galactosidase activity in the cell walls of acapsu-
a-galactosidase in Cryptococcus laurentii was also
a
a
lar mutant CCY 17-3-6 should be linked with the structure of a
complex extracellular polysaccharide galactoxylomannan with an
a
-(1 ? 6)-galactan backbone, obviously produced by acapsular
mutants of C. neoformans43 and C. laurentii.44,45
nucleophilicity and the insolubility of
trifluoroethanol.
D-galactose in 2,2,2-
2.2. Galactosyl donors
For practical purposes, the donor 1 used in further experiments
was prepared by glycosylation of 2,2,2-trifluoroethanol using per-
The production of oligosaccharides by transglycosylations may
suffer from obvious problems related to the choice of glycosyl
donor. The use of an oligosaccharide as an inexpensive glycosyl
donor brings the problem with product purification due to the for-
mation of a complex combination of starting saccharide, product
oligosaccharides and monosaccharides coming from substrate
hydrolysis. The use of activated donors such as nitrophenyl glyco-
sides, is therefore preferred, taking advantage from faster reaction
velocity. Due to their solubility, these rather expensive donors are
used in low concentrations and therefore a large portion of the
substrate is hydrolyzed.
O-acetylated
moter (Section 4.4., Method C). The thermodynamic product 2,2,2-
trifluoroethyl per-O-acetyl- -galactopyranoside was obtained in
D-galactose as a glycosyl donor and BF3.OEt2 as a pro-
a-D
56% yield after heating for three days. Substrate 1 was obtained
after Zemplén deacetylation in 89% yield (Scheme 2).
2.3. Transgalactosylations catalyzed by cell walls from C.
laurentii
Reactions carried out with 2,2,2-trifluoroethyl
a-D-galactopyra-
Other glycosides, especially those derived from short chain
alkyl alcohols, may serve as glycosyl donors, with the advantages
noside proved its applicability as an activated donor for
1

V. Mastihuba et al. / Tetrahedron: Asymmetry 28 (2017) 1089–1094
1091
CF3CH2OH
OH
HO
HO
Δ
BF3.OEt2,
15 h, 26 %
,
OH
HO
HO
O
O
or
HO
HO
OH
OCH2CF3
1
CF3CH2OH
Dowex 50WX8
MW, 5 min, 38 %
MeONa
MeOH
Ac2O/I2
CF3CH2OH
BF3.OEt2, CH2Cl2
Δ, 72 h, 59 %
OAc
OAc
AcO
AcO
AcO
AcO
O
O
AcO
AcO
OAc
OCH2CF3
Scheme 2. Preparation of 1 by conventional chemical methods.
Figure 1. Production of 2,2,2-trifluoroethyl galactosides 3 and 4 by transgalacto-
sylations catalyzed by cell walls from Cryptococcus laurentii. Starting concentrations
transgalactosylations (Scheme 3). The reaction in the initial period
proceeds as a self-condensation, where the glycosyl donor also
serves as the acceptor. Later, all products of hydrolysis and transg-
lycosylation can serve as acceptors and form another free or glyco-
of 2,2,2-trifluoroethyl
a-D-galactopyranoside 1 are 83 mM (j, h), 166 mM (d, s)
and 332 mM (N, 4). Solid markers stand for production of 3, empty markers for
production of 4.
sylated
galactooligosaccharides.
The
concentration
of
galactotrioside reached its plateau above 6% between 6–7 h.
(Fig. 2). It is therefore reasonable to run the reaction over longer
reaction periods if galactotriosides are the target products.
The reaction was then run on a preparative scale at the same
concentration. The isolated yields of free and glycosylated oli-
galactobioside 3 reached a maximum of 11 and 18% in 3 and 4 h,
respectively, upon the respective initial donor concentrations 83
and 166 mM. At the initial donor concentration of 332 mM, the for-
mation of galactobioside 3 slowed down after 5 h, but its hydroly-
sis did not prevail within 8 h (Fig. 1). Galactobioside 3 was not the
final product of the transgalactosylation, since the process contin-
ued to form galactotrioside 4. Only the reaction with the highest
initial donor concentration produced 4 with a reasonable level of
conversion (Fig. 1) Samples from the reaction mixture were ana-
lyzed by HPLC using an RI-detector. According to the method used,
only glycosides could be quantified. TLC analysis however con-
firmed the formation of free galactose and nonglycosylated galac-
tobiose 5 and galactotriose 6. Preparative reaction starting from
0.3 M of 1 provided 17% 3 as the main product within 6.5 h and
2% of the corresponding galactotrioside 4. Free oligogalactoses 5
and 6 were isolated in 11% and 3% yields, respectively. No tetrasac-
charides, either free or in glycosylated form, were found in the
reaction mixture. Structural analysis of all products revealed strict
regioselectivity of the transgalactosylation catalyzed by the cell
gogalactosides were 7% of 4-nitrophenyl
of 4-nitrophenyl -galactotrioside 8, and 14% of
5. The yield of -galactotriose 6 was however higher (12%) than
its yield in the reaction with 1. Structural analysis of the products
a-
D-galactobioside 7, 3%
a-
D
a-D
-galactobiose
a
-D
walls. Only
reaction.
a-(1 ? 6)-galactosyl bonds were formed in the
Due to its lower solubility, kinetic studies of transgalactosyla-
tions starting from 4-nitrophenyl -galactopyranoside 2 were
a-D
only carried out at the initial concentration of 166 mM to compare
its reactivity with the reactivity of 1. The synthesis of galacto-
bioside 7 peaked at 11% in 3 h, after which the concentration
slowly decreased, while the concentration of the corresponding
Figure 2. Production of 4-nitrophenyl
-galactotrioside (s) by cell walls from Cryptococcus laurentii at initial
concentration of 4-nitrophenyl
a-D-galactobioside 7 (d) and 4-nitrophenyl
a-
D
8
a-
D
-galactopyranoside 166 mM.
OH
OH
HO
HO
HO
O
O
HO
+
HO
HO
HO
HO
OH
HO
HO
O
O
+
O
α-galactosidase
O
O
HO
HO
HO
(cell walls C. laurentii)
HO
HO
HO
OR
O
OR
OH
HO
HO
3
7
,
1, 2
O
O
HO
HO
HO
HO
OH
HO
OR
O
1, 3, 4
7 8
R = -CH2CF3
R =
4, 8
O
O
+
2
,
,
NO2
HO
HO
HO
HO
HO
HO
O
O
O
O
6
5
HO
HO
HO
HO
OH
OH
Scheme 3. Synthesis of a-oligogalactosides and a-galactooligosaccharides catalyzed by cell walls from C. laurentii.

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V. Mastihuba et al. / Tetrahedron: Asymmetry 28 (2017) 1089–1094
confirmed the regioselectivity of the reaction to the primary posi-
tion of the acceptor galactoside.
minutes at 750 rpm and 37 °C. The enzymatic reaction was
terminated by the addition of 6 volumes of a saturated solution
of borax. The solution was centrifuged and the formation of
nitrophenol was measured at 410 nm. One unit of enzyme activity
was defined as the amount of enzyme that catalyzed the formation
of 1 mmol of nitrophenol per minute under the described
conditions.
3. Conclusion
Cell walls from an acapsular strain of Cryptococcus laurentii cat-
alyzed regioselective
duced galactobiosides, galactotriosides and the corresponding
free galactooligosaccharides from 4-nitrophenyl -galactopyra-
noside 2 and 2,2,2-trifluoroethyl -galactopyranoside 1. The lat-
ter substance served as a novel type of activated -galactosyl
a-(1 ? 6) transgalactosylations and pro-
4.3. Preparation of 2,2,2-trifluoroethyl
by cell walls
a-D-galactopyranoside 1
a
-D
a-D
a
The standard reaction mixture (75 mL overall) containing meli-
biose (5 g, 14.6 mmol), 2,2,2-trifluoroethanol (5 mL) and cell wall
preparation (10% dry mass) in 0.1 M McIlvaine buffer (pH 4.8)
was shaken on VIBRAMAX 100 shaker (Heidolf) at 750 rpm and
37 °C. The course of the reaction was monitored by TLC (chloro-
form/methanol, 3:1, v/v). After 6 h, the reaction was stopped by fil-
tration on Celite to remove the biocatalyst, concentrated by
evaporation under reduced pressure, applied to a column of Dowex
1x4 in OHꢁ cycle and eluted with water to remove most of the
reducing saccharides. Fractions containing 1 were pooled and
evaporated to dryness. The crude product was purified by column
chromatography on silicagel eluted with chloroform/methanol
(3:1) and crystallized from 2-propanol to give 460.7 mg (1.8 mmol,
12%) of 1.
donor with better performances than 2. Its good solubility in water
allowed for a higher starting concentration and therefore sup-
pressed the redundant hydrolysis of the products, which resulted
in higher yields of the main galactobioside product. 2,2,2-Trifluo-
roethyl glycosides may therefore serve as an inexpensive, less toxic
alternative to the generally used nitrophenyl glycosides as glycosyl
donors in enzymatic transglycosylations, by taking advantage of
the high reactivity, good water solubility, easy product purification
(2,2,2-trifluoroethanol is easily removed by distillation) and possi-
bility to be prepared directly in the laboratory.
4. Experimental
4.1. General
4.4. Preparation of 2,2,2-trifluoroethyl
by chemical synthesis
a-D-galactopyranoside 1
The reactions were performed with commercial reagents pur-
chased from Sigma–Aldrich, Acros Organics, Merck. Melibiose
¯
was product from Production Department, Institute of Chemistry,
SAS, Bratislava. Solvents were refluxed for 2 h over an appropriate
drying agent (dichloromethane—P2O5, methanol–Mg/I2) under an
argon atmosphere and subsequently distilled. The Fisher reaction
was carried out in a commercial microwave reactor Discover
(CEM-SP model, CEM Corporation, USA) equipped with a non-con-
tact infra-red continuous feed-back temperature system. TLC was
performed on alumina sheets pre-coated with silica gel 60 F254
(Merck). Spots were visualized by UV lamp (kmax = 254 nm) and
charred with 5% sulfuric acid in ethanol (1% orcinol). Column chro-
matography was performed on Silica gel 60 (0.035–0.070 mm, pore
4.4.1. Method A
D
-Galactose (9 g, 50 mmol) and BF3ꢀOEt2 (0.05 mL) were
refluxed in 2,2,2-trifluoroethanol (100 mL) for 15 h. After cooling
the reaction mixture, the unreacted galactose (4 g) was filtered
off; the filtrate was neutralized by anhydrous sodium acetate, fil-
tered again and concentrated under reduced pressure. The residue
(7 g) was dissolved in distilled water (32 ml), and 1mg of Fungal
lactase (Shin Nihon) was added. The mixture was stirred overnight,
then concentrated under reduced pressure and purified by chro-
matography on silica gel (CHCl3/MeOH from 9:1 to 5:1). The pro-
duct (3.34 g, 26%) was crystallized from 2-propanol.
diameter ca. 6 nm, Acros Organics), Amberlite XAD-4, Dowex 50
¯
Wx8, Dowex 1x4 (Sigma) and Biogel P-2 (Bio-Rad). High-perfor-
mance liquid chromatography was performed on an Agilent 1200
Series apparatus consisting of quarternary pump, RI and UV–VIS
detectors, column thermostat and Rheodyne injector with a 20 mL
loop. Melting points were recorded with a Kofler hot-block and are
uncorrected. The structures of products were determined by a
combination of 1H and 13C NMR spectroscopy as well as by two-
dimensional homonuclear and heteronuclear techniques (COSY,
HSQC, HMBC, TOCSY) recorded with a Varian 400 MHz and
600 MHz spectrometers. Optical rotations were measured on Per-
kin–Elmer 241 and Jasco P2000 polarimeters at 20 °C. High resolu-
tion mass determination was performed on Orbitrap Velos PRO
spectrometer (Thermo Fisher Scientific). The microorganism Cryp-
tococcus laurentii CCY 17–3–6 (acapsular strain) was provided by
the Culture Collection of Yeasts (Institute of Chemistry, SAS, Brati-
slava, Slovakia). The cell walls were prepared according to the pro-
cedure reported previously.41 The material was standardized to dry
4.4.2. Method B
To a pressure tube (15 mL) sealed with a Teflon septum and
equipped with a Teflon-coated magnetic stirring bar, D-galactose
(0.45 g, 2.5 mmol) and 0.25 g Dowex 50WX8 in H+ form (50–100
mesh) were suspended in 2,2,2-trifluoroethanol (10 mL). The mix-
ture was stirred and irradiated with microwaves (150 W) at 90 °C
for 5 min. The reaction suspension was cooled down and filtered,
neutralized by anhydrous sodium acetate, filtered again and the fil-
trate was concentrated under reduced pressure. The residue was
purified by chromatography on silica gel (CHCl3/MeOH, 9:1). The
product 1 (0.25 g, 38%) was obtained and crystallized from 2-
propanol.
4.4.3. Method C
Per-O-acetyl-a,b-D
-galactopyranose48 (10.96 g, 28 mmol) and
2,2,2-trifluoroethanol (10 mL) were dissolved in dry CH2Cl2
(150 mL) after which BF3ꢀOEt2 (10.82 mL) was added to the stirred
reaction mixture at 0 °C. The reaction was then stirred under an
argon atmosphere at 40 °C for 72 h. Water (100 mL) was then
added to the mixture and the reaction was stirred for 15 min, then
diluted with CH2Cl2 (100 mL). The organic layer was separated,
washed with water (2 ꢂ 100 mL) and brine (2 ꢂ 100 mL), dried
over Na2SO4 and concentrated. The crude mixture was purified
by column chromatography (toluene:EtOAc, 3:1). Per-O-acetylated
mass 0.5 g/mL, and had activity 32 U
dry mass.
a-galactosidase per gram of
4.2. Assay of
a
-galactosidase in the preparation of cell walls
An amount of 50
lL of diluted and sonicated suspension of cell
walls was mixed with 1 mL of 5 mM solution of 2 in McIlvain
buffer (0.1 M, pH 4.8). The reaction mixture was incubated for 10

V. Mastihuba et al. / Tetrahedron: Asymmetry 28 (2017) 1089–1094
1093
trifluoroethyl-
a-
D-galactopyranoside (6.78 g, 56%) was obtained as
4.9. -galactopyranosyl-(1 ? 6)-
2,2,2-Trifluoroethyl
a
-
D
a-D-
a white solid. 2,2,2-Trifluoroethyl 2,3,4,6-tetra-O-acetyl-
a
-D
-galac-
galactopyranosyl-(1 ? 6)-a-D-galactopyranoside 4
topyranoside (5.955 g, 13.8 mmol) was suspended to methanol
(69 mL) and 0.5 M sodium methoxide (13.8 mL) was added. The
reaction was stirred for 2 h, then neutralized by Dowex 50WX8, fil-
tered, evaporated to dryness (2.46 g, 89%) and crystallized from 2-
propanol.
Amorphous colourless solid; [a]
20 = +129.3 (c 0.5, H2O); 1H NMR
D
(600 MHz, D2O) d 5.05 (d, J = 3.8 Hz, 1H, H-1), 4.96 (d, J = 3.6 Hz,
1H, H-100), 4.94 (d, J = 3.4 Hz, 1H, H-10), 4.20–4.05 (m, 2H, CH2-
CF3), 4.14 (dd, J = 8.1, 4.4 Hz, 1H, H-50), 4.14 (dd, J = 7.2, 4.3 Hz,
1H, H-5), 4.01–3.99 (m, 2H, H-4, H-40), 3.98–3.96 (m, 2H, H-500,
H-400), 3.88 (dd, J = 10.5, 3.2 Hz, 1H, H-3), 3.87 (dd, J = 10.6,
7.3 Hz, 1H, H-6a), 3.84–3.81 (m, 2H, H-30, H-300), 3.83 (dd, J = 10.3,
3.6 Hz, 1H, H-2), 3.81 (dd, J = 10.8, 7.2 Hz, 1H, H-60a), 3.80 (dd,
J = 10.8, 3.6 Hz, 1H, H-20), 3.79 (dd, J = 10.3, 3.6 Hz, 1H, H-200),
3.71 (bd, J = 6.3 Hz, 2H, H-600a, H-600b), 3.69 (dd, J = 10.8, 4.9 Hz,
1H, 60b), 3.66 (dd, J = 10.8, 4.6 Hz, 1H, 6b); 13C NMR (151 MHz,
D2O) d 126.8 (q, J = 277.7 Hz, CF3), 102.0 (C-1), 100.9 (C-100), 100.9
(C-10), 73.8 (C-500), 72.3, 72.3, 72.2, 72.1, 72.1, 72.1, 71.9, 71.5,
71.1, 71.0, 70.9 (C-5, C-50 C-4, C-40, C-400, C-3, C-30, C-300, C-2, C-20,
C-200), 69.3, 69.2 (C-6, C-60), 67.4 (q, J = 34.4 Hz, CH2CF3), 64.0 (C-
600); HRMS (ESI) m/z calcd for C20H33F3O16Na [M+Na]+,
609.16184; found, 609.16169.
4.5. 2,2,2-Trifluoroethyl
White crystals (2-propanol); Mp = 118–119 °C; [a]
20 = +156.0 (c
D
a-D-galactopyranoside 1
1.0, CH3OH); 1H NMR (400 MHz, CD3OD) d 4.93 (d, J = 3.6 Hz, 1H,
H-1), 4.18–3.95 (m, 2H, CH2CF3), 3.91 (dd, J = 3.2, 1.2 Hz, 1H, H-
4), 3.83 (dd, J = 10.2, 3.6 Hz, 1H, H-2), 3.81 (dd, J = 6.2, 4.8 Hz, 1H,
H-5), 3.75 (dd, J = 10.1, 3.0 Hz, 1H, H-3), 3.76–3.65 (m, 2H, H-6a,
H-6b); 13C NMR (101 MHz, CD3OD) d 125.6 (q, J = 277.1 Hz, CF3),
101.1 (C-1), 73.2 (C-5), 71.1, 71.0 (C-3, C-4), 69.9 (C-2), 65.6 (q,
J = 34.6 Hz, CH2CF3), 62.7 (C-6); HRMS (ESI) m/z calcd for C8H13F3-
O6Na [M+Na]+, 285.05619; found, 285.05576.
4.6. Time course of transgalactosylation with 2,2,2-
trifluoroethyl
a-
D-galactopyranoside 1 as the donor
4.10. Time course of transgalactosylation with 4-nitrophenyl
-galactopyranoside 2 as the donor
a-
D
The reaction mixtures (1.5 mL) comprising cell wall preparation
(10% dry mass) and 1 (83, 166 or 332 mM) in McIlvain buffer
(0.1 M, pH 4.8) were incubated and shaken in 5 mL glass vials on
VIBRAMAX 100 shaker (Heidolf) at 750 rpm and 37 °C. The course
of reactions was monitored every hour by TLC (butanol/ethanol/
water, 10:8:7, v/v/v) and HPLC. The sample was diluted 10 times
with 0.1 M Na2CO3, centrifuged and supernatant filtered through
syringe filter. The filtrate was applied to AquaPerfect column tem-
perated to 35 °C, eluted with deionized water (1 mL/min.) and
detected with RI detector.
The reaction mixtures (1.5 mL) comprising of cell wall prepara-
tion (10% dry mass) and 2 (166 mM) in McIlvain buffer (0.1 M, pH
4.8) were incubated and shaken in 5 mL glass vials on VIBRAMAX
100 shaker (Heidolf) at 750 rpm and 37 °C. The course of the reac-
tion was monitored every hour by TLC (butanol/ethanol/water
10/8/7, v/v/v) and HPLC. The sample was diluted 2000 times with
6% 2-propanol in deionized water and immediately filtered
through syringe filter. The filtrate was applied to LiChrospher
100 RP-18 column temperated to 35 °C, eluted with 6% 2-propanol
in deionized water (1 mL/min.) and detected with UV detector at
k = 313 nm.
4.7. Preparative transgalactosylation with 2,2,2-trifluoroethyl
a-
D
-galactopyranoside 1 as the glycosyl donor
4.11. Preparative transgalactosylation with 4-nitrophenyl
galactopyranoside (2) as the glycosyl donor
a-D-
A reaction mixture (10 mL) comprising of 1 (783 mg, 3 mmol)
and Cryptococcus laurentii cell walls (10% final dry mass) in McIl-
vain buffer (0.1 M, pH 4.8) was incubated at 37 °C. After 6.5 h,
the reaction was terminated by boiling on water bath, filtered
through Celite, applied to a Dowex 50WX8 column in Ca2+ cycle
and eluted with 50% ethanol. Two fractions comprising free sac-
charides and glycosides were obtained and separated individually
on Biogel P2 yielding 110.2 mg (17%) of 3, 11.7 mg (2%) of 4,
58.2 mg (11%) of 5, 13.1 mg (3%) of 6. Unreacted 1 was recovered
from the reaction mixture in amount 186.3 mg (24%). The physic-
ochemical properties and NMR spectra of 5 and 6 were in accor-
dance with literature.49,50
Reaction mixture (15 mL) comprising 2 (750 mg, 2.5 mmol) and
Cryptococcus laurentii cell walls (10% final dry mass) in McIlvain
buffer (0.1 M, pH 4.8) was incubated at 37 °C. After 7 h, the reac-
tion was terminated by boiling on water bath, filtered through
Celite and free 4-nitrophenol was removed from the reaction on
Amberlite XAD-4 column eluted with 20% ethanol. Products were
separated on Biogel P2 yielding 38.2 mg (7%) of 7, 13 mg (3%) of
8, 59 mg (14%) of 5 and 50.6 mg (12%) of 6.
4.12.
4-Nitrophenyl
a-D-galactopyranosyl-(1 ? 6)-a-D-
galactopyranoside 7
4.8.
2,2,2-Trifluoroethyl
a-D-galactopyranosyl-(1 ? 6)-a-D-
galactopyranoside 3
Pale yellow amorphous solid; [a]
20 = +182.7 (c 0.5, CH3OH); 1H
D
NMR (600 MHz, D2O) d 8.29 (d, J = 9.2 Hz, 1H, H-Ar), 7.33 (d,
J = 9.2 Hz, 1H, H-Ar), 5.93 (d, J = 3.8 Hz, 1H, H-1), 4.82 (d,
J = 3.7 Hz, 1H, H-10), 4.16 (dd, J = 9.6, 3.0 Hz, 1H, H-5), 4.14 (dd,
J = 10.4, 3.1 Hz, 1H, H-3), 4.10 (bd, J = 3.1 Hz, 1H, H-4), 4.06 (dd,
J = 10.2, 3.8 Hz, 1H, H-2), 3.87 (bd, J = 3.1 Hz, 1H, H-40), 3.85 (dd,
J = 7.2, 4.5 Hz, 1H, H-50), 3.82 (dd, J = 10.2, 8.0 Hz, 1H, H-6a), 3.71
(ddd, J = 4.7, 7,1, 11.8 Hz, 2H, H-60a, H-60b), 3.68 (dd, J = 10.2,
3.8 Hz, 1H, H-20), 3.65 (dd, J = 10.6, 3.4 Hz, 1H, H-6b), 3.31 (dd,
J = 10.2, 3.3 Hz, 1H, H-30); 13C NMR (151 MHz, D2O) d 164.2 (C-
Ar), 145.2 (C-Ar), 128.8 (CH-Ar), 120.0 (CH-Ar), 100.5 (C-10), 99.2
(C-1), 73.6 (C-50), 73.5 (C-5), 72.3 (C-3), 2x72.2 (C-4, C-30), 71.9
(C-40), 70.9 (C-20), 70.7 (C-2), 69.4 (C-6), 63.8 (C-60); HRMS (ESI)
White solid; Mp = 185–187 °C; [a]
20 = +160.2 (c 0.5, CH3OH); 1H
D
NMR (600 MHz, D2O) d 5.08 (d, J = 3.8 Hz, 1H, H-1), 4.98 (d,
J = 2.8 Hz, 1H, H-10), 4.24–4.07 (m, 2H, CH2CF3), 4.17 (dd, J = 8.2,
5.0 Hz, 1H, H-5), 4.06 (bd, J = 3.1 Hz, 1H, H-4), 4.01–3.97 (m, 2H,
H-50, H-30), 3.92 (dd, J = 10.4, 3.3 Hz, 1H, H-3), 3.87 (dd, J = 10.9,
7.0 Hz, 1H, H-20), 3.87 (dd, J = 10.7, 3.8 Hz, 1H, H-2), 3.85 (dd,
J = 11.9, 2.6 Hz, 1H, H-6a), 3.84–3.83 (m, 1H, H-40), 3.75 (bd, 2H,
H-60a, H-60b), 3.74 (dd, J = 11.8, 4.1 Hz, 1H, H-6b); 13C NMR
(151 MHz, D2O) d 126.8 (q, J = 277.7 Hz, CF3), 101.9 (C-1), 101.1
(C-10), 73.9 (C-50), 72.3 (C-30), 72.1 (C-5, C-4), 72.0 (C-3), 71.9 (C-
40), 71.0 (C-2), 70.9 (C-20), 69.1 (C-6), 67.4 (q, J = 34.4 Hz, CH2CF3),
64.0 (C-60); HRMS (ESI) m/z calcd for C14H23F3O11Na [M+Na]+,
447.10902; found, 447.10883.
m/z calcd for
486.12203.
C
18H25NO13Na [M+Na]+, 486.12236; found,

1094
V. Mastihuba et al. / Tetrahedron: Asymmetry 28 (2017) 1089–1094
9. Abe, C.; Fujita, K.; Kikuchi, E.; Hirano, S.; Kuboki, H.; Yamashita, A.; Hashimoto,
H.; Mori, S.; Okada, M. Int. J. Tissue React. 2004, 26, 65–73.
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250.
4.13.
4-Nitrophenyl
a
-
D
-galactopyranosyl-(1 ? 6)-
a-D-
galactopyranosyl-(1 ? 6)-a-D-galactopyranoside 8
Pale yellow amorphous solid; [a]
20 = +131.3 (c 0.5, H2O); 1H
D
NMR (600 MHz, D2O) d 8.31 (d, J = 9.3 Hz, 1H, H-Ar), 7.34 (d,
J = 9.3 Hz, 1H, H-Ar), 5.94 (d, J = 3.9 Hz, 1H, H-1), 5.03 (d,
J = 3.6 Hz, 1H, H-10), 4.82 (d, J = 3.8 Hz, 1H, H-100), 4.16 (dd, J = 8.5,
2.7 Hz, 1H, H-5), 4.14 (dd, J = 10.3, 3.4 Hz, 1H, H-3), 4.09 (bd,
J = 3.3 Hz, 1H, H-4), 4.06 (dd, J = 10.3, 3.8 Hz, 1H, H-2), 4.04 (dd,
J = 7.5, 4.9 Hz, 1H, H-50), 4.01–3.97 (m, 2H, H-500, H-30), 3.91 (bd,
J = 3.4 Hz, 1H, H-400), 3.88 (bd, J = 3.2 Hz, 1H, H-40), 3.87 (dd,
J = 10.6, 7.3 Hz, 1H, H-6a), 3.85 (dd, J = 10.7, 3.6 Hz, 1H, H-20),
3.84 (dd, J = 10.9, 7.7 Hz, 1H, H-60a), 3.79–3.73 (m, 2H, H-600a, H-
600b), 3.71 (dd, J = 10.7, 4.6 Hz, 1H, H-60b), 3.69 (dd, J = 10.2,
3.8 Hz, 1H, H-200), 3.63 (dd, J = 10.6, 3.0 Hz, 1H, H-6b), 3.32 (dd,
J = 10.2, 3.4 Hz, 1H, H-300); 13C NMR (151 MHz, D2O) d 164.2 (C-
Ar), 145.2 (C-Ar), 128.8 (CH-Ar), 120.1 (CH-Ar), 100.7 (C-10),
100.3 (C-100), 99.3 (C-1), 73.8 (C-500), 73.6 (C-5), 72.4 (C-40), 72.3
(C-4), 72.3 (C-3), 72.2 (C-300), 72.1 (C-30), 72.0 (C-400), 71.2 (C-50
+C-20), 70.9 (C-200), 70.6 (C-2), 69.5 (C-6), 69.1 (C-60), 64.0 (C-600);
HRMS (ESI) m/z calcd for C24H35NO18Na [M+Na]+, 648.17518;
found, 648.17609.
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Acknowledgements
This work was supported by the Slovak Research and Develop-
ment Agency under the contract No. APVV-0846-12. This publica-
tion is also the result of the grants from the Slovak Grant Agency
for Science VEGA under the Project No. 2/0157/16 and the Research
& Development Operational Programmes funded by the ERDF
(‘‘Centre of Excellence on Green Chemistry Methods and Pro-
cesses”, CEGreenI, Contract No. 26240120001 as well as ‘‘Amplifi-
cation of the Centre of Excellence on Green Chemistry Methods
and Processes”, CEGreenII, Contract No. 26240120025). The
authors thank I. Uhliariková for NMR measurements and A. Chyba
for mass measurements.
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