Modular synthesis and immunological evaluation of suspected allergenic galactooligosaccharides

Article abstract of DOI:10.1039/c9ob00108e

Galactooligosaccharides (GOS) are widely used in the food industry as prebiotics and in very rare cases, can lead to an allergic reaction. Due to the microheterogeneity of GOS it is very difficult to extract pure and well defined oligosaccharides to establish which component is responsible for the observed allergenicity. Herein, we report the chemical synthesis of a suspected allergen 4PX and three closely related oligosaccharides based on a modular approach. The fact that synthesized 4PX and a regioisomer did not cause basophil activation in subjects with confirmed GOS-allergy excludes both tetrasaccharides as key-epitopes in GOS-allergenicity in Singapore.

Full text of DOI:10.1039/c9ob00108e

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2019, DOI: 10.1039/C9OB00108E.
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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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DOI: 10.1039/C9OB00108E
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Modular Synthesis and Immunological Evaluation of Suspected
Allergenic Galactooligosaccharides
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b,c
b,c
d
Hidde Elferink , Emiel Rossing , Chiung-Hui Huang , Bee Wah Lee , Linqiu Cao , Dianne J.
d
d
a*
Delsing , Andre Groeneveld , Thomas J. Boltje
DOI: 10.1039/x0xx00000x
www.rsc.org/
Galactooligosaccharides (GOS) are widely used in the food also differ in chain length. GOS have a safe history of use and
1
0,11,12
industry as prebiotics and in very rare cases, can lead to an allergic are Generally Recognized As Safe (GRAS) by the US FDA.
.
reaction. Due to the microheterogeneity of GOS it is very difficult In very rare cases reported from the Southeast Asian region,
to extract pure and well defined oligosaccharides to establish consumption of GOS is reported to lead to an allergic reaction
1
3-16
which component is responsible for the observed allergenicity. in subjects with pre-existing allergies
Herein, we report the chemical synthesis of a suspected allergen microheterogeneity of GOS, it is very difficult to extract pure
PX and three closely related oligosaccharides based on a modular and well defined oligosaccharides to identify the allergen. A
. However, due to the
4
approach. The fact that synthesized 4PX and a regioisomer did not recent study attributes GOS-related allergenicity in Japanese
cause basophil activation in subjects with confirmed GOS-allergy oyster workers to the branched tetrasaccharide Galβ(1-
1
7
excludes both tetrasaccharides as key-epitopes in GOS- 4)Galβ(1-6)-[Galβ(1-4)]-Glc referred to as 4PX (2, Scheme 1) .
allergenicity in Singapore.
Galactooligosaccharides (GOS)
OH
HO
HO
O
Introduction
Galactooligosaccharides (GOS) are used in the food industry as
prebiotics since the 1990’s. GOS are an important component
of infant formula to boost the low oligosaccharide content of
bovine milk (~0.05g/L) compared to human milk (~20g/L) .
Additionally, GOS have been shown to be beneficial to infant
health by promoting the growth of benign micro-organisms
O
OH
OH
O
HO
OH
O
OH
HO
HO
OH
n
O
O
1
OH
O
O
HO
O
HO
OH m
OH
HO
2
,
3
1: m = 0, n = 0
: m = 0, n = 1 (4PX)
3: m = 0, n = 2
4: m = 1, n = 0 (4PY)
such as Bifidobacteria , improving stool consistency and
2
4
, 5
6
frequency , enhancing natural defenses and improving the
uptake of minerals7 . GOS are prepared enzymatically using a
β-galactosidase, an enzyme that hydrolyzes lactose into
, 8
9
galactose and glucose . However, at high lactose
OPMB
O
OBn
O
OAc
O
LevO
BnO
BnO
SEt AcO
concentration, transgalactosylation becomes the dominant
reaction pathway. Under these conditions, the galactose
residue is cleaved and transferred to another lactose molecule
at the galactose and/or glucose residue, giving rise to a
complex mixture consisting of linear- and branched GOS that
HO
BnO
STol
BnO
OAc
OAc
OBn
core module 5
extension module 6 capping module 7
Scheme 1: Structures of the oligosaccharides prepared in this
study from three monosaccharide modules.
a. Cluster for Molecular Chemistry, Institute for Molecules and Materials, Radboud
University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
Department of Paediatrics, Yong Loo Lin School of Medicine, National University
4PX was extracted as a 2-aminopyridine(PA) conjugate after
extensive purification by size exclusion of GOS-mixtures,
chemical conversion to PA-conjugates by chemical
modification and again HPLC purification, emphasizing the
effort needed to obtain pure GOS components. 4PX was
recently synthesized by Saito and coworkers using a chemo-
b.
of Singapore
c.
Department of Paediatrics, National University Hospital, Singapore
FrieslandCampina , Amersfoort, The Netherlands
d.
*Corresponding Author: Thomas J. Boltje Tel: +31 (0)2436 52331,
E-mail: t.boltje@ru.nl
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1
8
enzymatic approach . However, this approach does not allow Regioselective opening of the p-methoxybenzylidene to the C-
View Article Online
DOI: 10.1039/C9OB00108E
.
for the synthesis of a broad collection of GOS derivatives.
4 hydroxyl using Bu
2 3
BOTf/BH THF under previously reported
1
9
conditions at -78°C afforded 5 in excellent yield (89%) .
Herein we report the modular chemical synthesis of suspected Extension module 6 was designed to enable the synthesis of
allergenic GOS component 4PX and three closely related the β-1,4-galactose repeats. C-2 O-acetyl participation will
oligosaccharides. The use of orthogonally protected modules ensure β-selective glycosylation while the C-4 Lev ester allows
in our approach is potentially applicable to the synthesis of a for orthogonal deprotection and subsequent extension. C-3
much larger collection of GOS. To test their allergenicity, two and C-6 benzyl ethers were used to exclude the possibility of
of the synthesized GOS structures were tested as pure protecting group migration towards the C-4 position during
2
0
tetrasaccharides in subjects with confirmed GOS-related glycosylation. Starting from known compound 12 , selective
allergy in Singapore.
benzylation provided compound 13 in moderate yield (60%).
Acetylation of the C-2 alcohol followed by regioselective
3
opening of the 4,6-benzylidene using NaCNBH /TFA afforded
Results and Discussion
alcohol 15. Esterification with levulinic acid afforded module 6
in excellent yield (92%). Finally, galactose capping module 7
The galactooligosaccharides shown in Scheme 1 are made-up
of a lactose core carrying β-1,4-galactose extensions at C-4 of
the galactose and/or C-6 of the glucose residue. We therefore
designed three monosaccharide modules to enable the
synthesis of such GOS components (1-4) in a modular fashion.
The synthesis of the glucose core module 5 was achieved in
four steps starting from glucose (Scheme 2). Fischer
glycosylation using BnOH/AcCl proceeded uneventfully to
2
1
was prepared from known galactoside 16 by regioselective
opening of the 4,6-benzylidene followed by acetylation of the
primary alcohol (Scheme 2).
OAc
BnO
OPMB
NIS, TfOH
71%)
O
O
5
5
+
+
7
6
AcO
O
BnO
(
AcO
BnO
18
OBn
afford -benzylglucoside 9. Installation of
methoxybenzylidene was carried out next, followed by
benzylation of the 2,3-diol to provide 11.
a
4,6-p-
OBn
O
LevO
BnO
OPMB
O
NIS, TfOH
43%)
O
A
OH
OH
BnO
(
AcO
BnO
O
BnOH, AcCl
(60%)
O
19
OBn
HO
HO
HO
HO
HO OH
HO
OBn
9
Scheme 3: Synthesis of lactoside precursors 18 and 19.
8
PMP(OMe)2
pTSA (70%)
With the building blocks in hand, the modular assembly of 1-4
was explored next. Two lactose derivatives 18 and 19 were
prepared to enable the synthesis of 1-3 and 4, respectively.
Glycosylation of 5 with 7 was achieved using the NIS/TfOH
promoter system affording 18 in good yield. Under the same
conditions, glycosylation of 5 with 6 afforded lactoside 19 in a
fair yield.
OPMB
O
.
BH THF,
3
PMP
O
Bu BOTf, -78°C
2
O
HO
BnO
O
RO
(
89%)
BnO
5
RO
10: R = H
OBn
OBn
BnBr, NaH (85%)
1
1: R = OBn
Ph
Ph
B
To prepare 1-3, lactoside 18 was extended and the C-6 position
of the glucose residue. To this end, DDQ oxidation was used to
deprotect the PMB ether and reveal the C-6 alcohol.
Disaccharide 20 was obtained in a moderate yield and a more
polar byproduct was also recovered. NMR analysis showed
that the C-4 benzyl on the galactose residue had also been
1
) Bu SnO, MeOH
O
2
O
O
O
2) BnBr, CsF
O
O
HO
SEt
(60%)
BnO
SEt
OH
OR
12
13: R = H
Ac O, Pyr (quant.)
2
14: R = OAc
NaCNBH3
TFA (93%)
2
2
removed which is a known side reaction in literature . By
limiting the amount of DDQ an improved yield of 61% was
OBn
OBn
LevO
BnO
HO
BnO
DCC, LevOH,
DMAP
obtained. Subsequent glycosylation with
6
proceeded
O
O
smoothly to afford trisaccharide 21 which after removal of the
Lev ester using hydrazine acetate afforded 22 in good yield.
Further deprotection by deacetylation and hydrogenation
afforded trisaccharide 1. An additional cycle of glycosylation
with 6 and Lev deprotection afforded tetrasaccharide 23. Lev
deprotection using the aforementioned conditions gave rise to
acceptor 24 in good yield. Global deprotection of 24 afforded
SEt
SEt
(92%)
OAc
OAc
6
15
C
Ph
O
.
OR
O
BH THF
BnO
AcO
3
O
Bu2BOTf, 0°C
O
AcO
STol
STol
(75%)
OAc
OAc
17: R = H
: R = Ac
4
PX tetrasaccharide 2 which was in perfect agreement with
16
Ac O, Pyr (93%)
2
18
the synthesized structure by Saito and coworkers . Although
the deacetylation as final deprotection step in Saito’s study
7
Scheme 2: Synthesis of the monosaccharide modules.
2
| J. Name., 2012, 00, 1-3
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gave a higher yield, our strategy allows for access of various isolated as a mixture with one other saccharide present, 4PY
View Article Online
DOI: 10.1039/C9OB00108E
other GOS-epitopes.
(4). However, initial comparison of our 4PX (2) NMR-data
showed a mismatch with the proposed characterization of 4PX
by van Leeuwen et al. Extensive 2D-NMR studies of our
synthesized 4PX (2) confirmed the regiochemistry and a
RO OBn
O
BnO OAc
OR
6
BnO
O
O
NIS, TfOH
BnO OAc
AcO
O
AcO
O
correct
structural
assignment
(see
supplementary
BnO
O
AcO
(68%)
O
BnO
AcO
O
OBn
BnO
information), suggesting the isolated compound by Van
Leeuwen et al. was erroneously assigned as 4PX. The
misassignment of isolated compounds from crude Vivinal®
GOS underlines the difficulty of characterizing complex
mixtures of closely related oligosaccharides and emphasizes
the need for the chemical preparation of analytical standards.
DDQ, H2O
18: R = PMB
20: R = H
AcO
BnO
OBn
(71%)
.
21: R = Lev
H2NNH2 AcOH (81%)
22: R = H
1
1
) KOtBu, MeOH
2) H2, Pd/C
6
(61%, two steps)
BnO OAc
O
NIS, TfOH
(84%)
AcO
O
OBn
O
RO OBn
AcO
BnO
O
OBn
O
O
OBn
AcO
BnO
BnO
O
O
AcO
O
BnO OAc
7
BnO
O
AcO
BnO OAc
O
O
BnO OAc
O
BnO OAc
AcO
R2O OBn
O
7
NIS, TfOH
OR1
O
AcO
OBn
O
O
NIS, TfOH
O
AcO
O
O
AcO
AcO
O
AcO
O
BnO
O
BnO
BnO
BnO
AcO
BnO
AcO
AcO
O
BnO
OBn
BnO
OBn
AcO
82%
O
O
BnO
OBn
BnO
AcO
3
25
.
23: R = Lev
4: R = H
BnO
OBn
1
(
2
) KOtBu, MeOH
14%, two steps)
) H2, Pd/C (90%)
H2NNH2 AcOH (78%)
DDQ, H O (61%)
1
9: R1 = PMB, R2 = Lev
2
2
2
1) KOtBu, MeOH
2) H2, Pd/C
.
26: R1 = H, R2 = Lev
27: R1 = H, R2 = H
H2NNH2 AcOH (89%)
1) KOtBu, MeOH
) H2, Pd/C
2
(48%, two steps)
28
4
(26%, two steps)
Scheme 4: Synthesis of GOS components 1-3 using a modular
approach.
Scheme 5: Synthesis of 4PX regioisomer 4PY (4) using a double
coupling approach.
Alternatively, glycosylation of 24 with 7 using NIS/TfOH
afforded pentasaccharide 25. Though the glycosylation went Next, we set out to prepare regioisomer 4PY (4, Scheme 5).
smoothly, inseparable trisaccharide and trehalose derived Because GOS exists as a microheterogeneous mixture, it is very
byproducts complicated purification. The impurities were difficult to reliably establish which component is responsible
removed upon deprotection of 25 using KOtBu/MeOH and for biological activity. This is especially true for isobaric
subsequent column chromatography. Final deprotection by components that are regioisomeric. Due to our modular
hydrogenation using Pd/C,
H
2
afforded the pure approach, tetrasaccharide 4 was accessible by simply switching
pentasaccharide 3.
the order of assembly without the need to prepare additional
A study by Van Leeuwen et al. aimed to identify the monosaccharide building blocks. Furthermore, to shorten the
oligosaccharides present in a mixture of commercial Vivinal® synthesis procedure we opted to install the β-1,4-galactose
2
3
extensions on the lactose scaffold in a single step. To this end
GOS up to the pentasaccharide . Through extensive
chromatographical separation and NMR analysis, over 40 we first prepared the appropriate acceptor diol 27 by a
structures were identified, including 4PX (referred to as stepwise deprotection of precursor 19 (Scheme 5). Subsequent
DP4f1a). The tetrasaccharide in van Leeuwen’s study was glycosylation with 7 and NIS/TfOH afforded the desired
Figure 1: 4PX and 4PY do not induce basophil activation. Expression of CD203c and CD63 on IgE-high cells after stimulating with GOS, 4PX or
4
PY were analyzed by using flow cytometry. Results were expressed as mean±SEM. (n=5 in case of 4PX, n=3 in case of 4PY)
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tetrasaccharide 28. Final deprotection as described earlier There are no conflicts to declare.
afforded tertrasaccharide 4 with small aromatic impurities
present related to the hydrogenation. The pure compound 4
was obtained after C18-chromatography.
View Article Online
DOI: 10.1039/C9OB00108E
Allergenicity Data
Acknowledgements
With the synthesized suspected allergenic 4PX (2) and
We would like to thank Paul B. White for his help with the
NMR analyses of 1-4.
regioisomer 4PY (4) in hand we set out to study their
allergenicity on blood samples from adult subjects with
previously confirmed GOS-related allergy in Singapore. The
effect of 4PX and 4PY on basophil activation was determined
by measuring the expression of the activation markers CD203c
and CD63 for each subject. Figure 1 shows that the positive 1.
control Vivinal GOS (blue line) induces dose-dependent
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1
1.
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Conflicts of interest
4
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