Transglycosylations employing recombinant α- And β-galactosidases and novel donor substrates

Article abstract of DOI:10.1016/j.carres.2014.05.005

Recombinant α- and β-galactosidases could be prepared in larger amounts for chemoenzymatic syntheses of glycosylated oligosaccharides relevant in nutrition approaches. α-Galactosidase RafA from Escherichia coli, another thermophilic α-galactosidase AgaB from Geobacillus stearothermophilus KVE39, and also a thermophilic β-galactosidase BglT from Thermus thermophilus TH 125 could be employed in α- and in β-glycosylations, respectively. With model structures as well as sucrose, isomaltitol, and isomaltulose the stereo- and regiospecificities were studied. Further, a number of modified donor structures with structural variation and different leaving groups were synthesized, employed, and compared to classical donors for these transglycosylations.

Full text of DOI:10.1016/j.carres.2014.05.005

Carbohydrate Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Transglycosylations employing recombinant
a- and b-galactosidases
and novel donor substrates
Sven Schröder a, Lars Kröger a, Ralf Mattes b, Joachim Thiem a,
⇑
a University of Hamburg, Faculty of Science, Department of Chemistry, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
b University of Stuttgart, Institute for Industrial Genetics, Allmandring 31, D-70569 Stuttgart, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Recombinant
syntheses of glycosylated oligosaccharides relevant in nutrition approaches.
Escherichia coli, another thermophilic -galactosidase AgaB from Geobacillus stearothermophilus KVE39,
and also a thermophilic b-galactosidase BglT from Thermus thermophilus TH 125 could be employed in
- and in b-glycosylations, respectively. With model structures as well as sucrose, isomaltitol, and
a
-
and b-galactosidases could be prepared in larger amounts for chemoenzymatic
Received 21 March 2014
Received in revised form 7 May 2014
Accepted 10 May 2014
Available online xxxx
a-Galactosidase RafA from
a
a
isomaltulose the stereo- and regiospecificities were studied. Further, a number of modified donor
structures with structural variation and different leaving groups were synthesized, employed, and
compared to classical donors for these transglycosylations.
Keywords:
Chemoenzymatic glycosylation
Recombinant galactosidases
Galactooligosaccharide synthesis
Ó 2014 Published by Elsevier Ltd.
1. Introduction
2. Results and discussion
In human nutrition galactooligosaccharides represent an inter-
esting group of bioactive compounds in the search for prebiotic
nutrition additives. Some galactooligosaccharides support the pro-
liferation of bifidobacteria and are thus of vital importance to the
intestinal flora.1,2 As a consequence of increasing malnutrition
there is a strong demand for human health promoting additives
to nourishment. Increased significance is given to galactooligosac-
charides that are structural analogs of known mono-, di-, and
trisaccharides. These structural analogs are supposed to be poten-
tially prebiotic nutrition additives.
It was of interest to devise simple and non-toxic synthetic
approaches to such galactooligosaccharides. The often high
regioselectivity of enzymes allows the synthesis of a particular sac-
charide structure without the disadvantages of classical organic
synthesis, such as long synthetic pathways with extended applica-
tion of protecting groups. Further, in the course of classical organic
reactions often toxic substances such as reagents, solvents, and
catalysts are used, however, even small traces of toxic contami-
nants may rule out a product for nutrition purposes. In contrast,
enzymatic syntheses are beneficial in that respect and to date,
substantial advances have been made in synthesis employing
b-galactosidases.3,4
2.1. Preparation of enzymes
Previously, we have cloned the genes of the thermophilic
a
-galactosidase AgaB from Geobacillus stearothermophilus KVE39,5
the b-galactosidase BglT from Thermus thermophilus TH1256 and
the mesophilic
a
-galactosidase RafA from Escherichia coli.7 The
enzymes were overexpressed in E. coli harboring the pBTac plas-
mid derivatives pAMG22 for expression of the agaB gene,
pHWG543 for the bglT gene, and pREM1128 for rafA gene expres-
sion. The recombinant enzymes were produced in high amounts
in E. coli RM448. After disruption of the cells by French press, the
thermophilic enzymes AgaB5 and BglT6 were enriched to 80%
homogeneity by heat precipitation of the host protein. Extracts of
E. coli/pREM1128 were purified for RafA preparation as described
by Spangenberg et al.7
2.2. Glycosylations employing RafA
Attractive donor substrates for
anosyl fluorides. In contrast to b-glycopyranosyl fluorides, the
-anomers are stable even in aqueous medium and easy to handle.
a-galactosidases are glycopyr-
a
Additionally, these fluorides are non-toxic derivatives, and thus
they meet the requirements for food-additive syntheses. For initial
studies concerning regioselectivity and preparative capability of
modified enzymes glycosyl fluorides 1–3 were incubated as donor
substrates for glycosylation of methyl
pH 6.5 and 37 °C for 24 h in phosphate buffer to neutralize the
a-D-glucopyranoside (4) at
⇑
Corresponding author. Tel.: +49 40 42838 4241; fax: +49 40 42838 4325.
E-mail address: thiem@chemie.uni-hamburg.de (J. Thiem).
http://dx.doi.org/10.1016/j.carres.2014.05.005
0008-6215/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

2
S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
released HF. Separation and purification of the reaction products
were done on Biogel P2 to give the corresponding disaccharides.
For structure assignments these were generally peracetylated in
anhydrous pyridine with acetic anhydride.
Except for the monosaccharide model the regioselectivity
observed for RafA catalyzed reactions was remarkably high to give
predominantly the (a1–6)-galactosylated products. This transfer
was largely also possible with various donor substrates such as
the glycopyranosyl fluorides, the p-nitrophenyl glycopyranosides
and the disaccharide melibiose. Further, unusual compounds such
Treatment of 4 with
19% of a mixture of the (
ride (8) in a ratio of 71:29. Assignment by MS and 1H as well as 13
a-
D
-galactopyranosyl fluoride (1)8 led to
1–6)- (5) and the ( 1–4)-linked disaccha-
a
a
C
as the D-fucopyranosyl and the L-arabinopyranosyl fluorides could
NMR of the peracetylated regioisomeric derivatives 5Ac and 8Ac
was unequivocal. It was further of interest to check, whether the
enzyme could tolerate modified donor structures. Thus, under
be employed as donor substrates. Thus, RafA represents a synthet-
ically interesting hydrolase for formation of various derivatives of
interest in the context of novel food components.
corresponding condition
employed, and in reaction with 4 lead to 16% of a disaccharide mix-
ture having the ( 1–6)- (6) and the ( 1–4)-linked disaccharide (9)
a-D
-fucopyranosyl fluoride (2)9 was
2.3. Syntheses of 4-pyridinyl hexopyranosides
a
a
in a ratio of 69:31. As before, the assignment was done spectro-
scopically with the peracetylated regioisomers 6Ac and 9Ac.
Even though para-nitrophenyl (pNP) groups show good leaving
properties and are well suited for monitoring the hydrolytic reac-
tion of glycosidases, their toxicity makes use critical for nutrition
products. In search of alternative donor substrates excellent trans-
fer properties as well as good water solubility are required. Again
nitrophenyl glycopyranosides drop behind in this regard and fol-
lowing previous studies16 pyridinyl glycosides became of interest.
Finally,
L-arabinose as the pentose analog of D-galactose was of
interest to be linked since there are reports for this sugar in animal
feed10,11 as well as dietary food.12 Thus, the b-
L-arabinopyranosyl
fluoride (3) was prepared13 and reacted under similar conditions
with 1 to give in 12% yield a mixture of the three disaccharides
with (
a
1–6)- (7) (
a
1–4)- (10) and (
a1–3)-linkage (11) in a ratio
Thus, along classical glycosylation
was glycosylated with silver 4-pyridoxide18 in acetonitrile/
dimethyl formamide 10:1 to give the b- -galactopyranoside 24,
which after Zemplén deacetylation led to the desired donor
substrate 4-pyridinyl b- -galactopyranoside (27). In a correspond-
ing approach
-acetobromofucose (22)19 gave the glycoside 25 and
a
-acetobromogalactose (21)17
of 65:23:12. Their assignment was clearly achieved employing
the peracetylated disaccharide derivatives 7Ac, 10Ac, and 11Ac
(Scheme 1).
Following these promising results the studies were extended to
synthesize glycosylated sucrose (14). Indeed, by incubation of this
D
D
a
acceptor with RafA and
a
-
D
-galactopyranosyl fluoride (1) the
after deacetylation the b-D-fucopyranoside donor 28. To round up
(a
1–6)-galactosylated trisaccharide 1714 resulted as the single reg-
the series b-acetobromoarabinose (23)20 led to glycoside 26, and
ioisomer in 15.4% yield. With a slightly modified buffer system the
after transesterification according to Zemplén the structurally
corresponding reaction of 14 and the donor p-nitrophenyl
related donor substrate
(Scheme 3).
a-L-arabinopyranoside 29 was obtained
a-D
-galactopyranoside (12)15 could be performed to give 17 in
17% yield. Finally, it was checked whether melibiose (13) could
function as donor substrate, and indeed this was possible to give
the trisaccharide 17 in 9% yield. Improved structural assignments
again could be made by use of the undecaacetate 17Ac. In case of
2.4. Glycosylations employing AgaB
It was of interest to study the thermophilic
a-galactosidase
the donor
saccharide 18 resulted, and with the b-
(3) no transfer could be observed. Apparently, the required cooper-
ative binding of substrates in the enzyme pockets is notably
influenced by the donor structure.
a
-
D
-fucopyranosyl fluoride (2) a yield of only 2% of tri-
-arabinopyranosyl fluoride
AgaB for transfer reactions to mono and disaccharides at 65 °C.
Thus, treatment of methyl -glucopyranoside (4) with the
-galactopyranosyl fluoride (1) in phosphate buffer and the
enzyme AgaB gave selectively the ( 1–6)-linked derivative 5
(methyl -melibioside) in 9% yield. Employing the donor substrate
p-nitrophenyl -galactopyranoside (12) enhanced the yield of 5
L
a-D
a-D
a
a
Another interesting acceptor substrate was palatinitol [15,
a-D
Glcp(
a
1–6)Glcol/Manol], which under the above conditions reacted
to 22%, and with melibiose (13) as donor the yield of 5 amounted
to 15%. In attempts for fucosylation or arabinosylation with donors
2 and 3, respectively, no transfer could be observed and apparently
the enzyme exhibits enhanced donor specificity. With sucrose (14)
as acceptor and 1 as donor compound 17 resulted in 3.4%, and with
melibiose (13) the yield of trisaccharide 17 was 9%. Then palatini-
tol (15) was treated with 1 to give trisaccharide 19 in 6%, and with
melibiose (13) in 12% yield. Finally, isomaltulose (16) was treated
with 1 catalyzed by RafA to give selectively the (
a1–6)-linked
trisaccharide 19 in 17% yield. By reaction with melibiose (13) again
the same structure 19 resulted in 9% yield. Finally the enzymatic
transfer was performed with isomaltulose (16) and fluoride 1 to
give the (a1–6)-linked trisaccharide 20 in 12% yield. With melibiose
(13) compound 20 was obtained in 8% yield. As before the perace-
tate 20Ac could be nicely structurally assigned (Scheme 2).
RO
OH
A
O
O
RO
RO
HO
HO
1.
A
HO
A
A
RO
1
O
O
O
HO
RO
1
4
RO
RO
OR
1
OMe
OMe
O
HO
6
RO
4
OR
O
3
RO
+
+
HO
α-Galase (RafA)
O
O
RO
RO
O
O
F
RO
RO
2.Ac2O, pyridine
RO
RO
OR
R= H R= Ac
OMe
OMe
A
R= H R= Ac
R= H R= Ac
5
6
7
5Ac
6Ac
7Ac
8
8Ac
1
2
3
-
-
-
-
CH2OH
CH3
H
9
9Ac
10 10Ac
11 11Ac
Scheme 1. Glycosylations of methyl
11Ac].
a-D-glucopyranoside (4) employing a-galactosidase (RafA) [free compounds (R = H): 5–11; peracetylated compounds (R = Ac): 5Ac–
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
HO
HO
3
HO
OH
A
O
O
HO
HO
HO
X
F
1
2
12 X=
A= CH2OH
A= CH3
O
NO2
O
O
HO
HO
13
X=
HO
OH
OH
OH
OH
O
O
O
HO
HO
HO
HO
HO
α-Galase
α-Galase
α-Galase
HO
(RafA)
(RafA)
(RafA)
HO
HO
HO
HO
O
O
O
OH
O
O
HO
HO
OH
16
HO
HO
OH
1.
1.
OH
OH
14
OH
OH
OH
15
2. Ac2O, pyridine
2. Ac2O, pyridine
RO
OR
RO
A
HO
HO
OH
O
O
O
RO
RO
RO
HO
HO
O
O
O
O
O
O
RO
RO
RO
RO
HO
HO
RO
RO
HO
RO
O
O
O
OR
O
O
RO
RO
OH
OH
HOH
OH
O
OR
A= CH2OH, R= H
OR
OR
OR
19
17
20
R= H
17Ac A= CH2OAc, R= Ac
18 A= CH3, R= H
20Ac
R= Ac
Scheme 2. Glycosylations of sucrose (14), palatinitol (15), and isomaltulose (16) employing
a
-galactosidase (RafA).
AcO
AcO
A
HO
A
AgO
N
A
O
O
NaOMe
O
O
O
AcO
AcO
HO
AcO
AcO
24-26
HO
27-29
Br
N
N
21-23
A
A
21 24
,
27 CH2OH
CH2OAc
22 25
,
28 CH3
CH3
29
H
23, 26
H
Scheme 3. Syntheses of 4-pyridinyl hexopyranosides.
with 1 to give the (
a
1–6)-galactosylated trisaccharide structure 20
from bovine testis.22,23 However, isolation of the protein from life
stock is laborious and the activity generally varies affected by var-
ious factors. Thus, the genetically modified b-galactosidase BglT
from Thermus thermophilus TH 125, a thermophilic enzyme with
a maximum efficiency at 65 °C, was of interest for synthetic
applications.6 This enzyme with a preference for establishing
(b1–3)-linkages was to be tested regarding the stereo- and regio-
specificity in transgalactosylations employing a number of novel
as well as established donor and acceptor substrates. As novel
donor substrates the three 4-pyridinyl hexopyranosides 27–29
in 7% and with melibiose (13) in 13% yield (Scheme 4).
These data show that the product spectrum of AgaB corre-
sponds to that of RafA. Yields employing melibiose as donor sub-
strate are higher in this application. Also, AgaB showed enhanced
donor specificity, and is altogether more selective, thus less univer-
sally applicable.
2.5. Glycosylations employing BglT
Excellent transgalactosylations for formation of (b1–3)-linked
oligosaccharides21 can be achieved employing raw b-galactosidase
were used and compared to the known p-nitrophenyl b-
D-galacto-
pyranoside (30)24 as well as lactose (31).
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4
S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
1
X= F
X=
HO
HO
OH
O
12
O
NO2
HO
O
X
O
HO
HO
13 X=
HO
OH
α-Galase
α-Galase
α-Galase
(AgaB)
OH
α-Galase
(AgaB)
(AgaB)
(AgaB)
OH
OH
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
OH
HO
O
O
O
OH
O
O
O
HO
HO
HO
16
OH
HO
HO
HO
HO
OH
HO
OMe
OH
OH
4
OH 14
15
OH
OH
HO
HO
OH
OH
O
HO
HO
HO
OH
OH
O
O
O
HO
HO
HO
HO
O
HO
HO
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
OMe
OH
5
O
O
HO
OH
HO
HO
HO
OH
OH
OH
OH
OH
OH
17
19
20
Scheme 4. Glycosylations of methyl a-D-glucopyranoside (4), sucrose (14), palatinitol (15), and isomaltulose (16) employing a-galactosidase (AgaB).
In galactosylation of the model structure 4 the new donor 27
3. Conclusion
showed to be advantageous and led to the (b1–3)-disaccharide gly-
coside (isolactoside, 32) in 27% yield. With the p-nitrophenyl donor
30 a yield of 13%, and with lactose as donor 23% yield was
obtained. Structure assignments were facilitated after transfer to
the heptaacetate 32Ac.
In this contribution recombinant
a-(RafA and AgaB) and
b-galactosidases (BglT) could be prepared in large amounts and
employed in chemoenzymatic syntheses of glycosylated oligosac-
charides, several of which are relevant in nutrition approaches.
Corresponding experiments were done in the case of sucrose
(14) as acceptor structure. With the 4-pyridinyl b-D-galactopyran-
oside (27) the trisaccharide 33 resulted as the single stereo- and
regioisomer in 31% yield, whereas lactose gave only 12% yield.
Again the NMR assignment was favorably done for the peracetate
As donor substrates hexopyranosyl fluorides of
-fucose and -arabinose, pNP -galactopyranoside and melibiose
proved to be efficient with acceptor structures methyl -gluco-
pyranoside, sucrose, palatinitol, and isomaltulose to give stereo-
and regiospecifically the terminally glycosylated oligosaccharides.
D-galactose,
D
L
a-D
a-D
33Ac.The enzyme BglT showed also to recognize the
donor 28, which led to the modified trisaccharide 34 in 2.6% yield.
On top of this the -arabinose donor 29 could be used to give the
D-fucose
In addition to pNP b-D-galactopyranoside and lactose a number of
novel 4-pyridinyl hexopyranosides turned out to be efficient
donor substrates in transglycosylations of the mentioned acceptor
substrates to give further terminally glycosylated oligosaccharides.
L
corresponding trisaccharide product 35 in 3% yield.
As in the case of sucrose (14) palatinitol (15) could be galactosy-
lated with the enzyme BglT employing the 4-pyridinyl derivative
27 and lactose (31) to yield the (b1–3)-galactosylated trisaccharide
36 in 11.2% and 8% yield, respectively. Finally, isomaltulose (16)
was treated under similar conditions, which led to the terminally
galactosylated trisaccharide 37 in 13% and 8%, respectively. As
above, the structure assignment was performed on the peracetate
37Ac (Scheme 5).
In all cases stereospecific glycosylations were observed, and the
regiospecificity was (1–3) exclusively to the terminal glucopyran-
osyl unit. With these features the novel b-galactosidase BglT
proved to be superior to the one isolated from bovine testis, which
opens up synthetic pathways for a large number of different, ter-
minally (b1–3)-galactosylated oligosaccharides.
4. Experimental
4.1. General methods
Commercially available starting materials were used without
further purification. Solvents were dried according to standard
methods. TLC was performed on precoated aluminium plates
(Silica Gel 60 F254, Merck 5554), and charring was with 10%
H2SO4 in ethanol for visualization. For column chromatography
Silica Gel 60, 230–400 mesh, 40–63 l
m (Merck) was used. 1H
NMR and 13C NMR spectra were recorded on Bruker AMX-400
(400 MHz for 1H, 100.6 MHz for 13C) or on Bruker DRX-500
(500 MHz for 1H, 125.8 MHz for 13C) at 300 K. Chemical shifts
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S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
5
A
X
27
28
29
CH2OH
O
O
N
CH3
H
HO
A
O
X
HO
NO2
30 CH2OH
HO
O
O
HO
HO
31
CH2OH
HO
OH
β-Galase
β-Galase
β-Galase
(BglT)
OH
β-Galase
(BglT)
(BglT)
(BglT)
OH
OH
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
OH
O
O
O
OH
O
HO
HO
O
O
16
OH
HO
OH 14
HO
HO
HO
OH
HO
1.
1.
OMe
OH
OH
1. OH
4
15 OH
2. Ac2O,
pyridine
2. Ac2O,
pyridine
2. Ac2O,
pyridine
OR
RO
HO
HO
OH
OH
A
O
O
O
O
RO
HO
O
O
RO
RO
RO
HO
HO
OMe
O
32
A= CH2OH, R= H
36
OH
OH
OR
HO
HO
32Ac A= CH2OAc, R= Ac
OH
RO
RO
OR
OR
O
RO
A
O
O
O
RO
RO
RO
O
O
RO
RO
RO
O
RO
RO
O
OR
O
O
33
37
37Ac
A= CH2OH, R= H
RO
OR
R= H
R= Ac
RO
33Ac A= CH2OAc, R= Ac
OR
OR
OR
34
35
A= CH3, R= H
A= H, R= H
Scheme 5. Glycosylations of methyl a-D-glucopyranoside (4), sucrose (14), palatinitol (15), and isomaltulose (16) employing b-galactosidase (BglT).
were calibrated to solvent residual peaks. Signals were assigned by
H,H-COSY, HSQC, and HMBC experiments. Optical rotations were
measured using Perkin–Elmer polarimeter 341 (589 nm) at 20 °C.
Melting points were measured on a Reichert heating microscope
or on ST-apotec and are uncorrected. MALDI-TOF-MS was per-
formed on a Bruker Biflex III with dihydroxybenzoic acid as matrix
in positive reflector mode. HRFAB-MS was performed on a VG 70S
mass spectrometer in positive ion mode with a xenon FAB-gun and
m-nitrobenzyl alcohol as matrix at 5000 resolution.
water/ethanol 1:5 as eluent. Version A for glycopyranosyl fluoride
donors: 0.3 M KH2PO4/K2HPO4, pH 6.5. Version B for other donors:
0.1 M K2HPO4/K2HPO4, pH 6.5.
4.2.2. Transgalactosylation with
The donor component (1 equiv), the acceptor substrate
(2.5 equiv), and -galactosidase AgaB (10 U/mmol acceptor) were
dissolved in potassium phosphate buffer (100 L, version A or ver-
a-galactosidase AgaB (GP 2)
a
l
sion B) and incubated in a thermomixer for 24 h at 65 °C. For ter-
mination the temperature was enhanced to 95 °C for 15 min.
Separation and purification of the products were done either on
Biogel P2 with water as eluent or on Sephadex LH-20 with
water/ethanol 1:5 as eluent. Version A for glycopyranosyl fluoride
donors: 0.3 M KH2PO4/K2HPO4, pH 6.5. Version B for other donors:
0.1 M K2HPO4/K2HPO4, pH 6.5.
4.2. General procedures
4.2.1. Transgalactosylation with
The donor component (1 equiv), the acceptor substrate
(2 equiv), and -galactosidase RafA (20 U/mmol acceptor) were
dissolved in potassium phosphate buffer (100 L, version A or
a-galactosidase RafA (GP 1)
a
l
version B) and incubated in a thermomixer for 24 h at 37 °C. For
termination the temperature was enhanced to 95 °C for 10 min.
Separation and purification of the products were done either on
Biogel P2 with water as eluent or on Sephadex LH-20 with
4.2.3. Transgalactosylation with b-galactosidase BglT (GP 3)
The donor component (1 equiv), the acceptor substrate (2 equiv),
and b-galactosidase BglT (10 U/mmol acceptor) were dissolved in
potassium phosphate buffer (0.1 M K2HPO4/K2HPO4, pH 6.5) and
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

6
S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
incubated in a thermomixer for 24 h at 65 °C. For termination the
temperature was enhanced to 95 °C for 15 min. Separation and
purification of the products were done either on Biogel P2 with
water as eluent or on Sephadex LH-20 with water/ethanol 1:5 as
eluent.
J1,2 = 3.5 Hz, H-1), 4.45 (t, 1H, J2,3 = J3,4 = 11.2 Hz, H-3), 4.23–4.16
(m, 3H, H-40, H-6a, H-6b), 4.09 (mc, 1H, H-60a), 4.04 (mc, 1H,
H-60b), 4.01 (m, 1H, H-50), 3.85 (ddd, 1H, J4,5 = 2.5, J5,6a = 6.5,
J5,6b = 12.1 Hz, H-5), 3.80 (dd, 1H, J1,2 = 3.5, J2,3 = 11.2 Hz, H-2),
3.42 (s, 3H, OCH3), 2.14, 2.11, 2.09, 2.06, 2.03, 2.01, 1.99 (7s, each
3H, CO-CH3). 13C NMR (C6D6, 100 MHz): d 96.87 (C-1), 96.48
(C-10), 75.17 (C-2), 71.83 (C-4), 70.84 (C-30), 69.84 (C-5), 69.53
(C-20), 68.92 (C-3), 67.89 (C-50), 66.54 (C-40), 62.43 (C-6), 62.29
(C-60), 54.98 (OCH3), 20.41–19.95 CO-CH3.
4.3. Syntheses
4.3.1. Methyl
(5) and methyl
glucopyranoside (8)
(a) Methyl -glucopyranoside (4, 194 mg, 1.0 mmol) and
-galactopyranosyl fluoride (1,8 91 mg, 0.5 mmol) dissolved in
a
-
a
D
-galactopyranosyl-(1?6)-
a-D-glucopyranoside
-D
-galactopyranosyl-(1?4)-
a-
D-
Calcd for 5Ac/8Ac: C27H38O18 (650.20). MALDI-TOF: m/z 673.05
[M+Na]+, 689.19 [M+K]+.
a-D
a-D
4.3.2. Methyl
and methyl
Methyl
a
-
D
-fucopyranosyl-(1?6)-
-fucopyranosyl-(1?4)-
-glucopyranoside (4, 194 mg, 1 mmol) and a-D
a
a-D
-
D
-glucopyranoside (6)
-glucopyranoside (9)
-fuco-
potassium phosphate buffer (1 mL, 0.3 M, KH2PO4/K2HPO4,
pH = 6.5) were incubated according to GP 1, version A. Workup
and chromatography on Biogel P2 gave 33.8 mg (19%) of a
colorless, amorphous solid. 1H NMR showed the mixture to be
5:8 = 71: 19.
a-
D
a
-
D
pyranosyl fluoride (2,9 83 mg, 0.5 mmol) dissolved in potassium
phosphate buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were
incubated according to GP 1, version A. Workup and chromatogra-
phy on Sephadex LH-20 gave 27.2 mg (16%) of a colorless, amor-
phous solid. The mixture of 6 and 9 was peracetylated with
acetic anhydride/anhydrous pyridine at room temperature and
usual workup to give 6Ac:9Ac 69:31 according to 1H NMR.
Peracetate 6Ac: 1H NMR (C6D6, 500 MHz): d 5.91 (t, 1H,
(b) Methyl
a-D-glucopyranoside (4, 242 mg, 1.25 mmol) and
a-D-galactopyranosyl fluoride (1, 91 mg, 0.5 mmol) dissolved in
potassium phosphate buffer (1 mL, 0.3 M, KH2PO4/K2HPO4,
pH = 6.5) were incubated according to GP 2, version A. Workup
and chromatography on Biogel P2 gave 16.0 mg (9%) of a colorless,
amorphous solid.
0
0
0
0
J2,3 = J3,4 = 9.8 Hz, H-3), 5.80 (dd, 1H,, J2 ,3 = 11.3, J3 ,4 = 3.2 Hz,
(c) Methyl
p-nitrophenyl a-D
a
-
D
-glucopyranoside (4, 242 mg, 1.25 mmol) and
H-30), 5.60 (dd, 1H, J1 ,2 = 3.8, J2 ,3 = 11.3 Hz, H-20), 5.55 (d, 1H,
0
0
0
0
-galactopyranoside (12,15 150 mg, 0.5 mmol) dis-
J3 ,4 = 3.2 Hz, H-40), 5.40 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 5.33 (t, 1H,
J3,4 = J4,5 = 9.8 Hz, H-4), 5.09 (d, 1H, J1,2 = 3.8, J2,3 = 10.3 Hz, H-2),
0
0
0
0
solved in potassium phosphate buffer (1 mL, 0.1 M, KH2PO4/
K2HPO4, pH = 6.5) were incubated according to GP 2, version B.
Workup and chromatography on Biogel P2 gave 32.9 mg (22%) of
a colorless, amorphous solid.
4.93 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.11 (q, 1H, J5 ,6 = 5.8 Hz, H-50),
3.96 (ddd, 1H, J4,5 = 9.8, J5,6a = 5.4, J5,6b = 2.2 Hz, H-5), 3.70 (dd,
1H, J5,6a = 5.4, J6a,6b = 11.4 Hz, H-6a), 3.49 (dd, 1H, J5,6b = 2.2,
0
0
(d) Methyl
a-D-glucopyranoside (4, 242 mg, 1.25 mmol) and
J6a,6b = 11.4 Hz, H-6b), 1.05 (d, 3H, J5 ,6 = 5.8 Hz, 60-CH3), 2.14,
2.11, 2.09, 2.06, 2.03, 2.01, (6s, each 3H, CO-CH3). 13C NMR (C6D6,
100 MHz): d 169.76, 169.62, 169.56, 169.49, (C@O), 96.58 (C-1),
96.29 (C-10), 71.35 (C-40), 70.90 (C-3), 70.40 (C-4), 69.30 (C-20),
68.32 (C-5), 68.27 (C-50), 68.01 (C-20), 67.98 (C-30), 65.74 (C-6),
55.32 (OCH3), 15.47 (C-60).
0
0
melibiose (13, 171 mg, 0.5 mmol) dissolved in potassium phos-
phate buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incu-
bated according to GP 2, version B. Workup and chromatography
on Biogel P2 gave 26.7 mg (15%) of a colorless, amorphous solid.
½
a 2D0 +106 (c 0.7, H2O); selected signals of 5: 1H NMR (D2O,
500 MHz):
ꢀ
d
4.93 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 4.76 (d, 1H,
0
0
Peracetate 9Ac: 1H NMR (C6D6, 500 MHz): d 5.76 (t, 1H,
J1 ,2 = 3.8 Hz, H-1), 3.97–3.91 (m, 3H, H-40, H-6a, H-6b), 3.82
0
0
0
0
0
0
J2,3 = J3,4 = 9.2 Hz, H-3), 5.67 (dd, 1H, J2 ,3 = 10.7, J3 ,4 = 3.2 Hz,
(dd, 1H, J2 ,3 = 9.9, J3 ,4 = 3.5 Hz, H-30), 3.77 (mc, 1H, H-20), 3.72–3.67
(m, 3H, H-5, H-60a, H-60b), 3.64–3.59 (m, 1H, H-3), 3.52 (dd, 1H,
J1,2 = 3.8, J2,3 = 9.8 Hz, H-2), 3.46 (t, 1H, H-4, J3,4 = J4,5 = 9.8 Hz, H-
0
0
0
0
H-30), 5.45 (s, 1H, H-40), 5.42 (dd, 1H, J1 ,2 = 3.8, J2 ,3 = 10.7 Hz,
0
0
0
0
H-20), 5.30 (t, 1H, J3,4 = J4,5 = 9.2 Hz, H-4), 5.27 (d, 1H, J1 ,2 = 3.8 Hz,
0
0
H-10), 4.69 (d, 1H, H-1, J1,2 = 3.8 Hz, H-1), 4.38–4.33 (m, 2H, H-6a,
13
4). C NMR (D2O, 100 MHz): d 99.76 (C-1), 98.47 (C-10), 73.68
H-6b), 4.14 (d, 1H, H-50, J5 ,6 = 5.8 Hz, H-50), 3.87 (ddd, 1H,
J4,5 = 2.2, J5,6a = 5.8, J5,6b = 11.1 Hz, H-5), 3.63 (dd, 1H, J1,2 = 3.8,
0
0
(C-3), 71.56 (C-2), 71.35 (C-4), 68.83 (C-20), 66.01 (C-6), 61.48 (C-
60), 55.57 (OCH3). Selected signals of 6: 1H NMR (D2O, 500 MHz):
0
0
J2,3 = 9.2 Hz, H-2), 3.07 (s, 3H, OCH3), 1.00 (d, 3H, J5 ,6 = 5.8 Hz,
60-CH3), 2.13, 2.10, 2.09, 2.05, 2.03, 2.01, (6s, each 3H, CO-CH3).
13C NMR (C6D6, 100 MHz): d 169.29, 169.19, 169.12, 169.01,
168.77 (C@O), 96.58 (C-10), 95.87 (C-1), 75.99 (C-4), 73.56 (C-2),
72.35 (C-20), 71.35 (C-40), 70.97 (C-3), 68.54 (C-30), 67.98 (C-50),
67.74 (C-5), 61.61 (C-6), 55.34 (OCH3), 15.27 (C-60). Calcd for
6Ac/9Ac: C25H36O16 (592.20) MALDI-TOF m/z 615.07 [M+Na]+,
631.25 [M+K]+.
d 5.10 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 4.92 (d, 1H, J1,2 = 3.8 Hz, H-1),
4.18 (dd, 1H, J1,2 = 3.8, J2,3 = 10.2 Hz, H-2), 4.10 (t, 1H,
J2,3 = J3,4 = 10.2, H-3). 13C NMR (D2O, 100 MHz): d 96.96 (C-1),
96.53 (C-10), 71.45 (C-3), 68.97 (C-2).
0
0
Peracetate 5Ac: 1H NMR (C6D6, 500 MHz): d 5.50 (t, 1H,
J2,3 = J3,4 = 9.8 Hz, H-3), 5.46 (s, 1H, H-40, J < 1), 5.36 (dd, 1H,
J2 ,3 = 11.0, J3 ,4 = 3.5 Hz, H-30), 5.18 (d, 1H, J1 ,2 = 3.5 Hz, H-10),
0
0
0
0
0
0
5.11 (dd, 1H, J1 ,2 = 3.5, J2 ,3 = 11.0 Hz, H-20), 5.02 (dd, 1H,
J2,3 = J3,4 = 9.8 Hz, H-4), 4.91 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.85 (dd,
1H, J1,2 = 3.5, J2,3 = 9.8 Hz, H-2), 4.28 (m, 1H, H-50), 4.12 (dd, 1H,
0
0
0
0
4.3.3. Methyl b-
L
-arabinopyranosyl-(1?6)-
a
-
D
-glucopyranoside
-glucopyranoside
-arabinopyranosyl-(1?3)- -glucopyrano-
J5 ,6 a = 6.5, J6 a,6 b = 11.4 Hz, H-60a), 4.06 (dd, 1H, J5 ,6 b = 6.5 Hz,
0
0
0
0
0
0
(7), methyl b- -arabinopyranosyl-(1?4)-a-D
L
J6 a,6 b = 11.4 Hz, H-60b), 3.95 (ddd, 1H, J4,5 = 2.2, J5,6a = 5.8,
J5,6b = 11.1 Hz, H-5), 3.73 (dd, 1H, J5,6a = 5.8, J6a,6b = 11.1 Hz, H-6a),
3.53 (dd, 1H, J5,6b = 2.2, J6a,6b = 11.1 Hz, H6b), 3.35 (s, 3H, OCH3),
2.14, 2.12, 2.08, 2.06, 2.04, 2.01, 1.99 (7s, each 3H, COACH3). 13C
NMR (C6D6, 100 MHz): d 170.80, 170.59, 170.45, 170.29, 169.93
(C@O), 96.90 (C-10), 96.52 (C-1), 71.22 (C-3), 70.54 (C-2), 69.48
(C-5), 68.53 (C-40), 68.47 (C-20, C-4), 67.85 (C-50), 66.82 (C-6),
66.50 (C-30), 62.20 (C-60), 55.75 (OCH3), 20.41–19.95 (CO-CH3).
Peracetate 8Ac: 1H NMR (C6D6, 500 MHz): d 5.42 (t, 1H,
0
0
(10) and methyl b-
L
a-D
side (11)
Methyl a-D-glucopyranoside (4, 194 mg, 1 mmol) and b-L-arabi-
nopyranosyl fluoride (3,13 76 mg, 0.5 mmol) dissolved in potas-
sium phosphate buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5)
were incubated according to GP 1, version A. Workup and chroma-
tography on Sephadex LH-20 gave 19.5 mg (12%) of a colorless,
amorphous solid. The mixture of 7, 10, and 11 was peracetylated
with acetic anhydride/anhydrous pyridine at room temperature
and usual workup to give 7Ac:10Ac:11Ac 65:23:12 according to
1H NMR.
J3,4 = J4,5 = 9.8 Hz, H-4), 5.32 (d, 1H, J1 ,2 = 3.5 Hz, H-10), 5.25 (dd,
0
0
1H, J1 ,2 = 3.5, J2 ,3 = 10.8 Hz, H-20), 4.97 (m, 1H, H-30), 4.82 (d, 1H,
0
0
0
0
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
7
Peracetate 7Ac: 1H NMR (C6D6, 500 MHz): d 5.90 (t, 1H,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 2, ver-
sion B. Workup and chromatography on Biogel P2 gave 22.7 mg
(9%) of 17 as a colorless, amorphous solid.
0
0
0
0
J2,3 = J3,4 = 10.1 Hz, H-3), 5.74 (dd, 1H,, J2 ,3 = 11.0, J3 ,4 = 1.6 Hz,
H-30), 5.63 (dd, 1H, J1 ,2 = 3.8, J2 ,3 = 11.0 Hz, H-20), 5.55 (dd, 1H,
0
0
0
0
J3 ,4 = J4 ,5 a = 1.9 Hz, H-40), 5.40 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 5.29 (t,
1H, J3,4 = J4,5 = 10.1 Hz, H-4), 5.08 (d, 1H, J1,2 = 3.8, J2,3 = 10.1 Hz,
H-2), 4.90 (d, 1H, J1,2 = 3.8 Hz, H-1), 3.95 (m, 1H, H-5), 3.89 (d,
½ ꢀ
a 2D0
+73 (c 1.0, H2O), lit.14 +122.9 (c 2.0, H2O); selected signals
0
0
0
0
0
0
of 17: 1H NMR (D2O, 500 MHz): d 5.27 (d, 1H, J1 ,2 = 3.8 Hz, H-100),
4.84 (d, 1H, J1,2 = 3.5 Hz, H-1), 3.41 (dd, 1H, J1,2 = 3.8, J2,3 = 9.8 Hz H-
2), 3.26 (t, 1H, H-3 J2,3 = J3,4 = 9.8 Hz). 13C NMR (D2O, 100 MHz): d
104.20 (C-20), 98.87 (C-100), 92.49 (C-1), 72.75 (C-2), 71.79 (C-3).
Calcd for C18H32O16 (504.44) MALDI-TOF m/z 527.51 [M+Na]+,
543.45 [M+K]+.
00 00
1H, J5 a,5 b = 12.9 Hz, H-50a), 3.67 (dd, 1H, J5,6a = 5.7, J6a,6b = 11.8 Hz,
0
0
H-6a), 3.54 (dd, 1H, J4 ,5 b = 1.9, J5 a,5 b = 12.9 Hz, H-50b), 3.45 (dd,
1H, J5,6b = 3.1, J6a,6b = 11.8 Hz, H-6b), 3.11 (s, 3H, OCH3), 2.14,
2.11, 2.09, 2.06, 2.03, 2.01, (6s, each 3H, CO-CH3). 13C NMR (C6D6,
0
0
0
0
100 MHz):
d
170.51, 170.30, 170.15, 170.10, 170.00, 169.91
Peracetate 17Ac: 1H NMR (CDCl3, 500 MHz): d 6.08 (d, 1H,
(C@O), 97.70 (C-1), 97.41 (C-10), 71.71 (C-20), 71.19 (C-2), 70.07
(C-3), 69.68 (C-40), 69.46 (C-4), 69.32 (C-30), 68.05 (C-5), 66.54
(C-6), 61.13 (C-50), 55.40 (OCH3).
J1 ,2 = 3.5 Hz, H-100), 5.97 (d, 1H, J3 ,4 = 1.3 Hz, H-400), 5.95–5.92
(m, 2H, H-300, H-6a), 5.87 (dd, 1H, J5,6b = 2.5, J6a,6b = 11.9 Hz, H-
00 00
00 00
6b), 5.72 (d, 1H, J3 ,4 = 7.9 Hz, H-30), 5.68 (dd, 1H, J1,2 = 3.8,
J2,3 = 10.9 Hz, H-2), 5.62–5.58 (m, 1H, H-500), 5.43 (d, 1H,
J1,2 = 3.8 Hz, H-1), 5.30 (t, 1H, J2,3 = J3,4 = 10.9 Hz, H-3), 5.10 (dd,
0
0
Peracetate 10Ac: 1H NMR (C6D6, 500 MHz): d 5.78 (t, 1H,
0
0
0
0
J2,3 = J3,4 = 10.1 Hz, H-3), 5.74 (dd, 1H, J2 ,3 = 10.9, J3 ,4 = 1.6 Hz, H-
30), 5.54 (d, 1H, J3 ,4 = 1.6 Hz, H-40), 5.42 (m, 1H, H-20), 5.45 (d,
1H, J1 ,2 = 3.8, J2 ,3 = 10.4 Hz, H-200), 4.82 (d, 1H, J3 ,4 = 7.9 Hz, H-
0
0
00 00
00 00
0
0
1H, J1,2 = 3.8, J2,3 = 10.1 Hz, H-2), 5.25 (d, 1H, J1 ,2 = 3.8 Hz, H-10),
40), 4.72–4.64 (m, 5H, H-10a, H-10b, H-60a, H-60b, H-5), 4.52–4.47
0
0
0
0
0
0
0
0
4.64 (d, 1H, H-1, J1,2 = 3.8 Hz, H-1), 4.34 (dd, 1H, J4 ,5 a = 4.4,
(m, 2H, H-6a, H-6b), 4.35 (dd, 1H, J5 ,6 a = 6.9, J5 6 b = 11.1 Hz, H-
J5 a,5 b = 12.3 Hz, H-50a), 4.22 (m, 1H, H-4), 4.10 (dd, 1H,
50), 3.93 (dd, 1H, J5 ,6 a = 6.9, J6 a,6 b = 10.8 Hz, H-600a), 3.70 (dd,
0
0
00 00
00
00
J4 ,5 b = 2.2, J5 a,5 b = 12.3 Hz, H-50b), 3.93 (m, 1H, H-5), 3.60 (dd,
1H, J5,6a = 4.7, J6a,6b = 11.3 Hz, H-6a), 3.48 (dd, 1H, J5,6b = 2.8,
J6a,6b = 11.3 Hz, H-6b), 3.06 (s, 3H, OCH3), 2.12, 2.10, 2.07, 2.05,
2.02, 2.01, (6s, each 3H, CO-CH3). 13C NMR (C6D6, 100 MHz): d
170.55, 170.35, 170.05, 170.00, 169.72, 169.53 (C@O), 97.70
(C-1), 96.42 (C-10), 73.87 (C-4), 73.03 (C-30), 71.88 (C-3), 70.92
(C-2), 69.98 (C-40), 69.92 (C-20), 67.72 (C-5), 62.46 (C-50), 61.65
(C-6), 55.51 (OCH3).
1H, J5 ,6 b = 1.9, J6 a,6 b = 10.8 Hz, H-600b), 2.07–1.96 (several s, 33H,
CO-CH3). 13C NMR (CDCl3, 100 MHz): d 171.06, 170.73, 170.61,
170.47, 170.16, 170.09, 169.81, 169.53 (C@O), 104.14 (C-20),
96.55 (C-100), 91.03 (C-1), 81.17 (C-5), 77.37 (C-3), 77.11 (C-30),
76.15 (C-300), 71.38 (C-200), 70.40 (C-40), 70.15 (C-500), 69.83 (C-400),
69.05 (C-40), 68.55 (C-2), 67.23 (C-50), 66.94 (C-6), 64.19 (C-10),
62.27 (C-60), 62.14 (C-600), 20.77–20.31 (CH3).
0
0
0
0
0
00
00
00
Peracetate 11Ac: 1H NMR (C6D6, 500 MHz): d 5.58 (d, 1H,
0
4.3.5.
fructofuranoside (18)
Sucrose (14, 342 mg, 1 mmol) and
a-
D
-Fucopyranosyl-(1?6)-
a
-D
-glucopyranosyl-(1M2)-b-
D-
J1 ,2 = 3.8 Hz, H-10), 5.49 (m, 1H, H-20), 5.32 (d, 1H, J3 ,4 = 1.8 Hz,
H-40), 5.01 (d, 1H, H-1, J1,2 = 3.5 Hz, H-1), 4.85 (d, 1H, J1,2 = 3.5,
J2,3 = 9.9 Hz, H-2), 4.46 (t, 1H, J2,3 = J3,4 = 9.9 Hz, H-3), 3.84 (ddd,
1H, J4,5 = 2.2, J5,6a = 4.7, J5,6b = 12.3 Hz, H-5), 2.97 (s, 3H, OCH3),
2.10, 2.08, 2.05, 2.02, 1.99, 1.97, (6s, each 3H, CO-CH3). 13C NMR
0
0
0
a-D-fucopyranosyl fluoride
(2, 83 mg, 0.5 mmol) dissolved in potassium phosphate buffer
(1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated accord-
ing to GP 1, version A. Workup and chromatography on Biogel
(C6D6, 100 MHz):
d
170.50, 170.32, 170.03, 169.80, 169.72,
P2 gave 5.1 mg (2%) of 18 as a colorless, amorphous solid. ½a D20
ꢀ
169.43 (C@O), 99.51 (C-1), 97.91 (C-10), 76.26 (C-3), 71.45 (C-40),
+41 (c 0.2, MeOH); 1H NMR (D2O, 500 MHz): d 5.38 (d, 1H,
71.02 (C-2), 69.91 (C-20), 62.53 (C-50), 55.19 (OCH3). Calcd for
J1 ,2 = 3.5 Hz, H-100), 4.82 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.22 (m, 1H,
00 00
H-300), 4.16 (d, 1H, J3 ,4 = 8.4 Hz, H-30), 4.05–3.98 (m, 3H, H-400,
H-3, H-4), 3.93 (dd, 1H, J1,2 = 3.5, J2,3 = 9.1 Hz, H-2), 3.86–3.83
(m, 2H, H-200, H-500), 3.80 (m, 1H, H-5), 3.77–3.73 (m, 4H, H-10a,
H-10b, H-60a, H-60b), 3.66–3.63 (m, 2H, H-6a, H-6b), 3.42 (t, 1H,
0
0
7Ac/10Ac/11Ac
[M+Na]+, 618.65 [M+K]+.
C
24H34O18 (578.13) MALDI-TOF m/z 600.71
4.3.4.
b- -fructofuranoside (17)
(a) Sucrose (14, 342 mg, 1 mmol) and
a-D-Galactopyranosyl-(1?6)-a-D-glucopyranosyl-(1M2)-
D
J3 ,4 = 8.4 Hz, H-40), 1.16 (d, 3H, J5 ,6 = 6.3 Hz, 600-CH3). 13C NMR
0
0
00 00
a
-D
-galactopyranosyl
(D2O, 100 MHz): d
104.53 (C-20), 98.65 (C-100), 96.83 (C-1),
fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 1, version A. Workup and chromatography on Bio-
gel P2 gave 38.8 mg (15.4%) of 17 as a colorless, amorphous solid.
81.71 (C-50), 77.49 (C-5), 73.53 (C-300), 72.37 (C-40), 72.32 (C-3),
72.24 (C-30), 72.15 (C-500), 72.00 (C-2), 71.19 (C-200), 69.84 (C-
400), 68.54 (C-6), 67.39 (C-4), 62.69 (C-60), 61.76 (C-10), 15.65
(C-600). Calcd for C18H32O15 (488.44) MALDI-TOF m/z 511.45
[M+Na]+, 527.47 [M+K]+.
(b) Sucrose (14, 342 mg, 1 mmol) and p-nitrophenyl a-D-galac-
topyranoside (12,15 150 mg, 0.5 mmol) dissolved in potassium
phosphate buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were
incubated according to GP 1, version B. The formed p-nitro-phenol
was extracted with ethyl acetate (5 mL). Further workup and chro-
matography on Biogel P2 gave 42.9 mg (17%) of 17 as a colorless,
amorphous solid.
4.3.6.
-glucitol/mannitol (19)
(a) Palatinitol (15, 344 mg, 1 mmol) and
a-D-Galactopyranosyl-(1?6)-a-D-glucopyranosyl-(1?6)-
D
a-D-galactopyranosyl
fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 1, version A. Workup and chromatography on Bio-
gel P2 gave 43.0 mg (17%) of 19 as a colorless, amorphous solid.
(b) Palatinitol (15, 344 mg, 1 mmol) and melibiose (13, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 1, ver-
sion B. Workup and chromatography on Biogel P2 gave 22.8 mg
(9%) of 19 as a colorless, amorphous solid.
(c) Sucrose (14, 342 mg, 1 mmol) and melibiose (13, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 1, ver-
sion B. Workup and chromatography on Biogel P2 gave 17.6 mg
(7%) of 17 as a colorless, amorphous solid.
(d) Sucrose (14, 427 mg, 1.25 mmol) and
a-D-galactopyranosyl
fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 2, version A. Workup and chromatography on Bio-
gel P2 gave 8.6 mg (3.4%) of 17 as a colorless, amorphous solid.
(e) Sucrose (14, 427 mg, 1.25 mmol) and melibiose (13, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
(c) Palatinitol (15, 344 mg, 1 mmol) and
a-D-galactopyranosyl
fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 2, version A. Workup and chromatography on Bio-
gel P2 gave 15.3 mg (6%) of 19 as a colorless, amorphous solid.
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

8
S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
(d) Palatinitol (15, 344 mg, 1 mmol) and melibiose (13, 171 mg,
purified by column chromatography with petroleum ether (50–70)/
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 1,
version B. Workup and chromatography on Biogel P2 gave
30.5 mg (12%) of 19 as a colorless, amorphous solid.
ethyl acetate 1:1 to give 1.96 g (66%) of 24 as amorphous solid.
½
a 2D0 +54 (c 0.5, CHCl3); 1H NMR (CHCl3, 500 MHz): d 8.51 (m,
ꢀ
2H, Ar-2, Ar-6), 6. 92 (m, 2H, Ar-3, Ar-5), 5.49 (dd,1H, J1,2 = 7.6,
J2,3 = 10.7 Hz, H-2), 5.45 (m, 1H, H-4), 5.18 (d, 1H, J1,2 = 7.6 Hz,
H-1), 5.11 (dd, 1H, J2,3 = 10.7, J3,4 = 3.6 Hz, H-3), 4.20–4.09 (m, 3H,
H-5, H-6a, H-6b), 2.16, 2.05, 2.04, 2.00 (4s, 12H, COCH3). 13C
NMR (100.6 MHz, CDCl3): d 170.71, 170.52, 170.45, 169.66 (C@O),
163.54 (Ar-4), 151.05 (2 C, Ar-2, Ar-6), 112.27 (2C, Ar-3, Ar-5),
98.29 (C-1), 71.89 (C-5), 71.02 (C-3), 68.61 (C-2), 67.08 (C-4),
61.74 (C-6), 21.09, 21.05, 21.03, 20.96 (COCH3).
½
a 2D0 + 141 (c 1.0, H2O); 1H NMR (D2O, 500 MHz): d 4.94 (d, 1H,
ꢀ
J1 ,2 = 3.8 Hz, H-100), 4.88 (m, 2H, CH2-1). 13C NMR (D2O, 100 MHz):
d 98.70, 98.57 (C-100), 98.53 (C-1), 73.68 (C-500), 73.49 (C-3), 73.27
(C-300), 72.15 (C-5), 71.97 (C-2), 71.88 (C-200), 69.84 (C-4), 68.80
(C-60), 69.58 (C-6), 68.89 (C-400), 63.64 (C-10, Man-ol), 62.79 (C-10,
Glc-ol), 61.61 (C-600). Calcd for C18H34O16 (506.18): C, 42.69; H,
6.77; found: C, 37.98; H, 6.66. MALDI-TOF m/z 529.35 [M+Na]+,
545.53 [M+K]+.
00 00
4.3.9. (4-Pyridinyl) 2,3,4-tri-O-acetyl-b-
D-fucopyranoside (25)
2,3,4-Tri-O-acetyl- -fucopyranosyl bromide (22, 19 430 mg,
a-D
1.22 mmol) and silver 4-pyridoxide18 (246 mg, 1.22 mmol) dis-
solved in acetonitrile (50 mL) and dimethyl formamide (5 mL)
were heated at 80 °C under stirring for 5 h. After cooling the mix-
ture was filtered through Celite, washed with saturated aqueous
sodium hydrogen carbonate and water, dried over Mg2SO4, and
purified by column chromatography with toluene/acetone 10:1
4.3.7.
b- -fructofuranose (20)
(a) Isomaltulose (16, 342 mg, 1 mmol) and
a-D-Galactopyranosyl-(1?6)-a-D-glucopyranosyl-(1?6)-
D
a-D-galactopyrano-
syl fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 1, version A. Workup and chromatography on Bio-
gel P2 gave 30.3 mg (12%) of 20 as a colorless, amorphous solid.
(b) Isomaltulose (16, 342 mg, 1 mmol) and melibiose (13,
171 mg, 0.5 mmol) dissolved in potassium phosphate buffer
(1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incubated according
to GP 1, version B. Workup and chromatography on Biogel P2 gave
20.2 mg (8%) of 20 as a colorless, amorphous solid.
to give 183 mg (41%) of 25 as slightly yellow syrup. ½a D20
ꢀ
+35.7 (c
0.3, CHCl3); 1H NMR (C6D6, 500 MHz): d 8.48 (m, 2H, Ar-2, Ar-6),
6.92 (m, 2H, Ar-3, Ar-5), 5.49 (dd, 1H, J1,2 = 7.8, J2,3 = 10.4 Hz, H-
2), 5.33 (d, 1H, J3,4 = 3.4 Hz, H-4), 5.19 (d, 1H, J1,2 = 7.8 Hz, H-1),
5.14 (dd, 1H, J2,3 = 10.4, J3,4 = 3.4 Hz, H-3), 4.05 (d, 1H,
J5,6 = 6.3 Hz, H-5), 2.20, 2.05, 2.02 (3s, 9H, COCH3), 1.28 (d, 3H,
J5,6 = 6.3 Hz, 6-CH3). 13C NMR (100.6 MHz, CDCl3):
d 170.53,
(c) Isomaltulose (16, 427 mg, 1.25 mmol) and
a-D-galactopyr-
170.10, 169.34 (C@O), 151.14 (Ar), 111.62 (Ar), 97.78 (C-1), 71.05
(C-3), 69.91 (C-5), 69.82 (C-4), 68.37 (C-2), 20.69, 20.63, 20.59
(COCH3), 16.08 (C-6).
anosyl fluoride (1, 91 mg, 0.5 mmol) dissolved in potassium phos-
phate buffer (1 mL, 0.3 M, KH2PO4/K2HPO4, pH = 6.5) were
incubated according to GP 2, version A. Workup and chromatogra-
phy on Biogel P2 gave 17.7 mg (7%) of 20 as a colorless, amorphous
solid.
4.3.10. (4-Pyridinyl) 2,3,4-tri-O-acetyl-a-L-arabinopyranoside
(26)
(d) Isomaltulose (16, 427 mg, 1.25 mmol) and melibiose (13,
171 mg, 0.5 mmol) dissolved in potassium phosphate buffer
(1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incubated according
to GP 2, version B. Workup and chromatography on Biogel P2 gave
32.8 mg (13%) of 20 as a colorless, amorphous solid.
2,3,4-Tri-O-acetyl-b-L
-arabinopyranosyl bromide (23,20 4.41 g,
13.2 mmol) and silver 4-pyridoxide18 (2.6 g, 13.2 mmol) dissolved
in acetonitrile (200 mL) and dimethyl formamide (60 mL) were
heated at 80 °C under stirring for 5 h. After cooling the mixture
was filtered through Celite, washed with saturated aqueous
sodium hydrogen carbonate and water, dried over Mg2SO4, and
purified by column chromatography with toluene/acetone 10:1
½
a 2D0
ꢀ
+145 (c 1.0, H2O); 1H NMR (D2O, 500 MHz): d 4.80 (d, 1H,
J1,2 = 3.5 Hz, H-1), 4.75 (d, 1H, J1 ,2 = 3.1 Hz, H-100), 3.98 (m, 1H,
H-6a), 3.90 (mc, 1H, H-6b). 13C NMR (D2O, 100 MHz): d 102.11
(C-20), 98.73 (C-100), 98.42 (C-1), 69.88 (C-6).
00 00
to give 1.76 g (38%) of 26 as slightly yellow syrup. ½a D20
ꢀ
+115.0
1
(c 1.0, CHCl3); H NMR (C6D6, 500 MHz): d 8.49 (m, 2H, Ar-2, Ar-6),
6.98 (m, 2H, Ar-3, Ar-5), 5.41 (dd, 1H, J1,2 = 5.7, J2,3 = 8.2 Hz, H-2),
5.34 (ddd, 1H, J3,4 = 3.5, J4,5a = 4.7, J4,5b = 2.2 Hz, H-4), 5.25 (d, 1H,
J1,2 = 5.7 Hz, H-1), 5.19 (dd, 1H, J2,3 = 8.2, J3,4 = 3.5 Hz, H-3), 4.10
(dd, 1H, J4,5a = 4.7, J5a,5b = 12.6 Hz, H-5a), 3.79 (dd, 1H, J4,5b = 2.2,
J5a,5b = 12.6 Hz, H-5b), 2.14, 2.09, 2.05 (3s, 9H, COCH3). 13C NMR
(100.6 MHz, CDCl3): d 170.10, 169.99, 169.30 (C@O), 151.39 (Ar),
111.61 (Ar), 96.91 (C-1), 69.24 (C-3), 68.58 (C-4), 66.57 (C-2),
62.27 (C-5), 20.84, 20.72, 20.68 (COCH3).
Calcd for C18H36O16 (504.44): C, 42.86; H, 6.39; found: C, 38.60;
H, 6.53. MALDI-TOF m/z 527.45 [M+Na]+, 543.35 [M+K]+.
Peracetate 20Ac: 1H NMR (C6D6, 500 MHz): d 5.85–5.76 (m, 2H,
H-300, H-3), 5.73 (d, 1H, J3 ,4 = 2.2 Hz, H-400), 5.58 (dd, 1H, J1,2 = 3.5,
00 00
J2,3 = 9.6 Hz, H-2), 5.51 (m, 1H, H-500), 5.20 (d, 1H, J1,2 = 3.5 Hz, H-1),
5.13 (d, 1H, J1 ,2 = 3.8 Hz, H-100), 5.05 (dd, 1H, J1 ,2 = 3.8,
00 00
00 00
J2 ,3 = 8.9 Hz, H-20), 5.00 (d, 1H, J3 ,4 = 8.9 Hz, H-30), 4.67 (t, 1H,
00 00
0
0
J3 ,4 = J4 ,5 = 8.9 Hz, H-40), 4.58 (m, 1H, H-50), 4.53 (m, 1H, H-6a),
4.45 (m, 1H, H-6b), 4.32–4.22 (m, 4H, H-10a, H-10b, H-60a, H-60b),
3.70 (m, 1H, H-5), 3.64–3.59 (m, H-600a, H-600b), 1.91–1.78, (several
s, 33H, CO-CH3). 13C NMR (C6D6, 125 MHz): d 170.95–169.55
(C@O), 103.56 (C-20), 96.79 (C-1), 95. 97 (C-100), 82.26 (C-50),
79.87 (C-40), 77.60 (C-500), 74.84 (C-30), 71.60 (C-5), 71.10 (C-4),
69.88 (C-300), 69.20 (C-3), 69.07 (C-400), 68.95 (C-2), 68.38 (C-200),
67.39 (C-6), 62.38 (C-60), 62.27 (C-10), 61.96 (C-600), 21.19–20.74
(CH3).
0
0
0
0
4.3.11. (4-Pyridinyl) b-D-galactopyranoside (27)
Compound 24 (1.24 g, 2.91 mmol) dissolved in anhydrous
methanol (70 mL) was treated with NH3 in methanol (1.5 mL) for
6 h at room temperature then concentrated and purified by column
chromatography (ethyl acetate/methanol 6:1) to give 717 mg
(96%) of 27 as amorphous solid. ½a D20
ꢀ
+94.0 (c 0.1, MeOH); 1H
NMR (DMSO-d6, 400 MHz): d 8.43 (m, 2H, Ar-2, Ar-6), 7.04 (m,
2H, Ar-3, Ar-5), 5.02 (d, 1H, J1,2 = 8.1 Hz, H-1), 3.72 (d, 1H,
J3,4 = 3.5 Hz, H-4), 3.65 (dt, 1H, J3,4 = 3.5, J5,6a = 5.7, J5,6b = 6.6 Hz,
H-5), 3.60 (dd, 1H, J1,2 = 8.1, J2,3 = 9.5 Hz, H-2), 3.55 (dd, 1H,
J5,6a = 5.7, J6a,6b = 11.0 Hz, H-6a), 3.49 (dd, 1H, J5,6b = 6.6,
J6a,6b = 11.0 Hz, H-6b), 3.41 (dd, 1H, J2,3 = 9.5, J3,4 = 3.5 Hz, H-3).
13C NMR (100.6 MHz, DMSO-d6): d 163.59 (Ar-4), 151.30 (Ar-2,
Ar-6), 111.97 (Ar-3, Ar-5), 100.14 (C-1), 76.06 (C-5), 73.56 (C-3),
70.35 (C-2), 68.43 (C-4), 60.66 (C-6).
4.3.8. (4-Pyridinyl) 2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside
(24)
2,3,4,6-Tetra-O-acetyl-a-D
-galactopyranosyl bromide (21,17
2.88 g, 7.0 mmol) and silver 4-pyridoxide18 (1.63 g, 8.07 mmol)
were refluxed in toluene (30 mL) for 1 h. After cooling the mixture
was filtered through Celite, washed with saturated aqueous
sodium hydrogen carbonate and water, dried over Mg2SO4, and
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
9
J5 ,6 b = 6.5 Hz, J6 a,6 b = 11.0 Hz, H-60b), 3.95 (ddd, 1H, J4,5 = 2.5,
0
0
0
0
4.3.12. (4-Pyridinyl) b-
D-fucopyranoside (28)
0
0
0
0
Compound 25 (183 mg, 0.5 mmol) dissolved in anhydrous
methanol (10 mL) was treated with sodium methoxide in metha-
nol (0.1 M, 0.5 mL) for 6 h at room temperature, then neutralized
with Amberlite IR 120 (H+) to give 110 mg (92%) of 28 as slightly
J5,6a = 5.0, J5,6b = 12.1 Hz, H-5), 3.27 (t, 1H, J5 ,6 a = J5 ,6 b = 6.5 Hz, H-
50), 3.35 (s, 3H, OCH3), 2.16, 2.14, 2.10, 2.06, 2.04, 1.98, 1.96 (7s,
each 3H, CO-CH3). 13C NMR (C6D6, 125 MHz): d 170.62, 170.58,
170.31, 170.10, 169.94, 169.83, 169.55 (C@O), 101.69 (C-10),
97.24 (C-1), 76.59 (C-4), 73.65 (C-2), 71.89 (C-30), 71.14 (C-50),
69.76 (C-20), 69.27 (C-3), 68.24 (C-5), 67.30 (C-40), 62.70 (C-6),
60.75 (C-60), 54.96 (OCH3), 20.71, 20.44, 20.21, 20.03, 20.00,
19.98, 19.95 (CO-CH3). Calcd for 32Ac: C27H38O18 (650.20).
MALDI-TOF: m/z 673.17 [M+Na]+, 689.25 [M+K]+.
yellow, amorphous solid. ½a D20
ꢀ
+124.0 (c 0.2, H2O); 1H NMR
(MeOH-d4, 500 MHz): d 8.41 (m, 2H, Ar), 7.05 (m, 2H, Ar), 5.03
(d, 1H, J1,2 = 7.5 Hz, H-1), 3.84 (m, 1H, H-5), 3.57 (dd, 1H,
J1,2 = 7.5, J2,3 = 9.8 Hz, H-2), 3.51 (d, 1H, J3,4 = 3.2 Hz, H-4), 3.44
(dd, 1H, J2,3 = 9.8, J3,4 = 3.2 Hz, H-3), 1.18 (d, 3H, J5,6 = 6.3 Hz,
6-CH3). 13C NMR (MeOH-d4, 100.6 MHz): d 151.29 (Ar), 111.86
(Ar), 99.75 (C-1), 73.67 (C-3), 71.19 (C-4), 70.86 (C-5), 69.98
(C-2), 16.83 (C-6).
4.3.15. b-
b- -fructofuranoside (33)
(a) Sucrose (14, 342 mg, 1 mmol) and (4-pyridinyl) b-D-galacto-
D-Galactopyranosyl-(1?3)-a-D-glucopyranosyl-(1M2)-
D
Calcd for C11H15NO5 (241.24): C, 54.77; H, 6.27; N, 5.81; found:
C, 54.26; H, 6.39; N, 5.49.
pyranoside (27, 78 mg, 0.3 mmol) dissolved in potassium phos-
phate buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were
incubated according to GP 3. Workup and chromatography on Bio-
gel P2 gave 46.9 mg (31%) of 33 as a colorless, amorphous solid.
(b) Sucrose (14, 342 mg, 1 mmol) and lactose (31, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 3.
Workup and chromatography on Biogel P2 gave 30.2 mg (12%) of
33 as colorless, amorphous solid.
4.3.13. (4-Pyridinyl) a-L-arabinopyranoside (29)
Compound 26 (1.76 g, 5.0 mmol) dissolved in anhydrous meth-
anol (100 mL) was treated with sodium methoxide in methanol
(0.1 M, 5.0 mL) for 6 h at room temperature, then neutralized with
Amberlite IR 120 (H+) to give 932 mg (89%) of 29 as orange, amor-
phous solid. ½a 2D0
ꢀ
+5.0 (c 0.15, H2O); 1H NMR (MeOH-d4, 500 MHz):
d 8.42 (m, 2H, Ar), 7.05 (m, 2H, Ar), 5.03 (d, 1H, J1,2 = 6.9 Hz, H-1),
3.76–3.71 (m, 2H, H-4, H-5a), 3.68–3.62 (m, 2H, H-2, H-5b), 3.48
(m, 1H, H-3). 13C NMR (MeOH-d4, 100.6 MHz): d 151.31 (Ar),
111.90 (Ar), 99.81 (C-1), 72.67 (C-3), 70.34 (C-4), 67.74 (C-2),
66.10 (C-5).
½
a 2D0
ꢀ
+49 (c 1.0, H2O), lit.:4 +45 (c 0.5, H2O); selected signals of
30: 1H NMR (D2O, 500 MHz): d 5.46 (d, 1H, J1,2 = 4.0 Hz, H-1),
4.67 (d, 1H, J1 ,2 = 7.9 Hz, H-100), 4.24 (d, 1H, J2 ,3 = J3 ,4 = 8.7 Hz,
00 00
0
0
0
0
H-30), 4.08 (t, 1H, J3 ,4 = J4 ,5 = 8.7 Hz, H-40), 4.02 (t, 1H,
J2,3 = J3,4 = 9.3 Hz, H-3), 3.93–3.89 (m, 1H, H-5), 3.84 (m, 2H, H-
0
0
0
0
Calcd for C10H13NO5 (227.27): C, 52.86; H, 5.77; N, 6.16; found:
C, 51.98; H, 5.89; N, 6.29.
60a, H-60b), 3.79 (d, 1H, J1,2 = 4.0 Hz, H-2), 3.65 (dd, 1H, J1 ,2 = 7.9,
00 00
13
J2 ,3 = 9.3 Hz, H-200). C NMR (D2O, 125 MHz): d 104.07 (C-100),
00 00
4.3.14. Methyl b-
D
-galactopyranosyl-(1?3)-
a-
D-glucopyranoside
103.56 (C-2), 92.50 (C-1), 82.14 (C-40), 81.77 (C-3), 76.94 (C-30),
74.40 (C-50), 62.74 (C-60). Calcd for C18H32O16 (504.44). MALDI-
TOF m/z 527.33 [M+Na]+, 543.45 [M+K]+.
(32)
(a) Methyl
a-
D-glucopyranoside (4, 194 mg, 1.0 mmol) and
(4-pyridinyl)b-
D
-galactopyranoside (27, 129 mg, 0.5 mmol) dis-
Peracetate 33Ac: 1H NMR (C6D6, 500 MHz): d 5.93 (d, 1H,
J1,2 = 3.8 Hz, H-1), 5.88 (d, 1H, J3 ,4 = 6.3 Hz, H-30), 5.75 (t, 1H,
0
0
solved in potassium phosphate buffer (1 mL, 0.1 M, KH2PO4/
K2HPO4, pH = 6.5) were incubated according to GP 3. Workup and
chromatography on Biogel P2 gave 47.9 mg (27%) of 32 asa color-
less, amorphous solid.
J3 ,4 = J4 ,5 = 6.3 Hz, H-40), 5.65 (dd, 1H, J1 ,2 = 8.0, J2 ,3 = 10.4 Hz,
0
0
0
0
00 00
00 00
H-200), 5.60 (d, 1H, J3 ,4 = 2.8 Hz, H-400), 5.30 (dd, 1H, J2 ,3 = 10.4,
00 00
00 00
J3 ,4 = 2.8 Hz, H-300), 5.24 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4), 5.16 (dd,
00 00
1H, J1,2 = 3.8, J2,3 = 10.0 Hz, H-2), 4.94 (d, 1H, J1 ,2 = 8.0 Hz, H-100),
00 00
(b) Methyl
p-nitrophenyl b-
a
D
-
D
-glucopyranoside (4, 194 mg, 1.0 mmol) and
-galactopyranoside (30,24 150 mg, 0.5 mmol) dis-
4.62–4.56 (m, 3H, H-5, H-6a, H6b), 4.54 (t, 1H, J2,3 = J3,4 = 10.0 Hz,
solved in potassium phosphate buffer (1 mL, 0.1 M, KH2PO4/
K2HPO4, pH = 6.5) were incubated according to GP 3. The formed
p-nitrophenol was extracted with ethyl acetate (5 mL). Further
workup and chromatography on Biogel P2 gave 40.8 mg (23%) of
32 as a colorless, amorphous solid.
H-3), 4.50–4.41 (m, 4H, H-10a, H-10b, H-60a, H-60b), 4.34 (dd, 1H,
H-50, J5 ,6 a = 6.9, J5 ,6 b = 11.3 Hz, H-50), 4.29 (dd, 1H, J5 ,6 a = 6.9,
0
0
0
0
00 00
J6 a,6 b = 11.0 Hz, H-600a), 4.24 (dd, 1H, J5 ,6 b = 11.3, J6 a,6 b = 11.0 Hz,
00
00
00 00
00
00
H-600b), 3.67 (dd, 1H, J5 ,6 a = 6.9, J5 ,6 b = 11.3 Hz, H-500), 2.02–2.68
(several s, 33H, CO-CH3). 13C NMR (C6D6, 125 MHz): d 170.57,
170.55, 170.48, 170.29, 170.25, 170.06, 169.94, 169.83, 169.35,
169.12 (C@O), 104.98 (C-20), 102.01 (C-100), 91.21 (C-1), 79.68
(C-50), 77.01 (C-30), 76.46 (C-3), 76.10 (C-40), 73.26 (C-2), 72.00
(C-300), 71.18 (C-500), 70.00 (C-200), 69.63 (C-5), 69.25 (C-4), 67.71
(C-400), 64.43 (C-10), 63.76 (C-600), 62.75 (C-60), 61.40 (C-6), 20.85,
20.65, 20.51, 20.43, 20.35, 20.22, 20.17, 20.05, 19.98, 19.95, 19.91
(CO-CH3).
00 00
0
0
(c) Methyl a-D-glucopyranoside (4, 242 mg, 1.25 mmol) and lac-
tose (31, 171 mg, 0.5 mmol) dissolved in potassium phosphate buf-
fer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 3. Workup and chromatography on Biogel P2 gave
23.1 mg (13%) of 32 as colorless, amorphous solid.
½
a 2D0 +97 (c 1.0, H2O); selected signals of 32: 1H NMR (D2O,
500 MHz): d 4.77 (d, 1H, J1 ,2 = 3.8 Hz, H-1), 4.56 (d, 1H, J1 ,2 = 7.9 -
ꢀ
0
0
0
0
Hz, H-10), 3.87 (d, 1H, J3 ,4 = 3.1 Hz, H-40), 3.81 (m, 1H, H-3), 3.82
0
0
(dd, 1H, J2 ,3 = 9.8, J3 ,4 = 3.1 Hz, H-30), 3.55 (dd, 1H, J1 ,2 = 7.9,
4.3.16. b-
-fructofuranoside (34)
Sucrose (14, 342 mg, 1 mmol) and (4-pyridinyl) b-
D
-Fucopyranosyl-(1?3)-a-D-glucopyranosyl-(1M2)-b-
0
0
0
0
0
0
13
J2 ,3 = 9.8 Hz, H-20). C NMR (D2O, 125 MHz): d 103.28 (C-10),
99.41 (C-1), 82.98 (C-3), 71.72 (C-20), 71.31 (C-30), 71.06 (C-4).
Calcd for C13H24O11 (356.33): C, 43.82; H, 6.79; found: C, 40.72;
H, 6.85.
D
0
0
D-fucopyr-
anoside (28, 121 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 3. Workup and chromatography on Biogel P2 gave
Peracetate 32Ac: 1H NMR (C6D6, 500 MHz): d 5.48 (dd, 1H,
J1 ,2 = 8.0, J2 ,3 = 10.7 Hz, H-20), 5.40 (d, 1H, J3 ,4 = 3.1 Hz, H-40),
6.5 mg (2.6%) of 34 as colorless, amorphous solid. ½a D20
ꢀ
+45 (c 0.2,
0
0
0
0
0
0
0
0
5.16 (t, 1H, J2,3 = J3,4 = 10.1 Hz, H-3), 5.06 (dd, 1H, J2 ,3 = 10.7,
H2O); 1H NMR (D2O, 500 MHz): d 5.39 (d, 1H, J1,2 = 3.8 Hz, H-1),
J3 ,4 = 3.1 Hz, H-30), 5.01 (dd, 1H, J1,2 = 3.8, J2,3 = 10.1 Hz, H-2),
4.56 (d, 1H, J1 ,2 = 7.9 Hz, H-100), 4.15 (d, 1H, J3 ,4 = 8.8 Hz, H-30),
0
0
00 00
0
0
4.78 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.62 (d, 1H, J1 ,2 = 8.0 Hz, H-10),
4.35 (dd, 1H, J2,3 = J3,4 = 10.1 Hz, H-4), 4.25 (dd, 1H, J5,6a = 5.0,
J6a,6b = 12.1 Hz, H-6a), 4.16 (dd, 1H, J5,6b = 2.5, J6a,6b = 12.1 Hz, H-
4.01 (t, 1H, J3 ,4 = J4 ,5 = 8.8 Hz, H-40), 3.95 (dd, 1H, J1,2 = 3.8,
J2,3 = 9.2 Hz, H-2), 3.90 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 3.83 (m, 1H,
H-5), 3.74–3.73 (m, 5H, H-300, H-10a, H-10b, H-60a, H-60b), 3.71
0
0
0
0
0
0
6b), 4.08 (dd, 1H, J5 ,6 a = 6.5, J6 a,6 b = 11.0 Hz, H-60a), 4.01 (dd, 1H,
(m, 1H, H-50), 3.67 (d, 1H, J3 ,4 = 2.1 Hz, H-400), 3.63–3.60 (m, 3H,
0
0
0
0
00 00
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005

10
S. Schröder et al. / Carbohydrate Research xxx (2014) xxx–xxx
H-500, H-6a, H-6b), 3.57 (dd, 1H, J1 ,2 = 7.9, J2 ,3 = 10.2 Hz, H-200),
+43 (c 1.2, H2O). Calcd for C18H32O18 (504.15). MALDI-TOF m/z
527.45 [M+Na]+, 543.35 [M+K]+. Selected signals of 34: 1H NMR
00 00
00 00
3.50 (t, 1H, J2,3 = J3,4 = 9.2 Hz, H-3), 1.19 (d, 3H, J5 ,6 = 6.5 Hz, 600-
00 00
13
CH3). C NMR (D2O, 100 MHz): d 104.43 (C-20), 103.08 (C-100),
(D2O, 500 MHz): d 4.92 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.57 (d, 1H,
13
92.43 (C-1), 82.57 (C-3), 81.71 (C-50), 76.79 (C-30), 74.30 (C-40),
73.09 (C-300), 72.43 (C-5), 71.63 (C-500), 71.27 (C-2), 71.23 (C-200),
71.08 (C-4), 70.91 (C-400), 62.69 (C-10), 61.79 (C-60), 60.43 (C-6),
15.84 (C-600).
J1 ,2 = 7.9 Hz, H-100). C NMR (D2O, 100 MHz): d 103.72 (C-100),
00 00
103.25 (C-20), 98.57 (C-1), 82.72 (C-3), 68.31 (C-60).
Peracetate 37Ac: 1H NMR (C6D6, 500 MHz): d 5.59 (d, 1H,
J3 ,4 = 2.5 Hz, H-400), 5.56 (dd, 1H, J2 ,3 = 9.2, J3 ,4 = 2.5 Hz, H-300),
00 00
00 00
00 00
Calcd for C18H32O15 (488.17). MALDI-TOF m/z 510.25 [M+Na]+,
526.51 [M+K]+.
5.49 (m, 1H, H-4), 5.25 (t, 1H, J3 ,4 = J4 ,5 = 6.7 Hz, H-40), 5.04 (d,
0
0
0
0
00 00
00 00
1H, J1,2 = 3.8 Hz, H-1), 4.76 (dd, 1H, J1 ,2 = 7.8, J2 ,3 = 9.2 Hz, H-
200), 4.62 (ddd, 1H, J4 ,5 = 6.7, J5 ,6 a = 6.2, J5 ,6 b = 3.3 Hz, H-50), 4.58
0
0
0
0
0
0
00 00
4.3.17.
b- -fructofuranoside (35)
Sucrose (14, 342 mg, 1 mmol) and (4-pyridinyl)
a
-
L
-Arabinopyranosyl-(1?3)-
a
-
D
-glucopyranosyl-(1M2)-
(dd, 1H, J1,2 = 3.8, J2,3 = 8.2 Hz, H-2), 4.52 (d, 1H, J1 ,2 = 7.8 Hz,
D
H-100), 4.46 (t, 1H, J2,3 = J3,4 = 8.2 Hz, H-3), 4.40–4.36 (m, 2H, H-30,
H-5), 4.30 (m, 1H, H-500), 4.23–4.18 (m, 4H, H-6a, H-6b, H-600a, H-
600b), 4.10 (m, 1H, H-10a), 4.05 (m, 1H, H-10b), 3.79 (dd, 1H,
a-L-arabinopyr-
anoside (29, 113 mg, 0.5 mmol) dissolved in potassium phosphate
buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were incubated
according to GP 3. Workup and chromatography on Biogel P2 gave
J5 ,6 a = 6.2, J6 a,6 b = 10.7 Hz, H-60a), 3.73 (dd, 1H, J5 ,6 b = 3.3,
0
0
0
0
0
0
J6 a,6 b = 10.7 Hz, H-60b), 2.23–2.00 (several s, 33H, CO-CH3). 13C
NMR (C6D6, 100 MHz): d 170.52, 170.45, 170.41, 170.37, 170.27,
170.21, 170.17, 170.14, 170.07, 170.01, 169.95 (C@O), 102.88
(C-20), 101.71 (C-100), 96.60 (C-1), 83.12 (C-3), 81.54 (C-50), 79.33
(C-500), 78.39 (C-300), 76.90 (C-40), 76.79 (C-30), 76.31 (C-300), 73.61
(C-4), 73.53 (C-5), 71.89 (C-400), 71.75 (C-2), 71.71 (C-200), 68.55
(C-60), 61.75 (C-600), 61.26 (C-10), 61.14 (C-6), 20.78, 20.74, 20.72,
20.67, 20.63, 20.58, 20.56, 20.48, 20.42 (CO-CH3).
0
0
7.2 mg (3.0%) of 35 as colorless, amorphous solid. ½a D20
ꢀ
+100 (c 1.0,
H2O); 1H NMR (D2O, 500 MHz): d 5.36 (d, 1H, J1,2 = 3.8 Hz, H-1),
4.53 (d, 1H, J1 ,2 = 6.5 Hz, H-100), 4.15 (d, 1H, J3 ,4 = 8.9 Hz, H-30),
00 00
0
0
3.98 (t, 1H, J3 ,4 = J4 ,5 = 8.9 Hz, H-40), 3.92–3.87 (m, 2H, H-3, H-
400), 3.82 (m, 2H, H-5, H-50), 3.77–3.74 (m, 4H, H-10a, H-10b, H-60a,
H-60b), 3.67 (dd, 1H, J1,2 = 3.8, J2,3 = 9.8 Hz, H-2), 3.46 (dd, 1H,
0
0
0
0
J2 ,3 = 7.8, J3 ,4 = 2.5 Hz, H-300), 3.62–3.58 (m, 4H, H-500a, H-500b,
00 00
00 00
H-6a, H-6b), 3.54 (dd, 1H, J1 ,2 = 6.5, J2 ,3 = 7.8 Hz, H-200), 3.49 (t,
1H, J3,4 = J4,5 = 9.5 Hz, H-4). 13CNMR (D2O, 100 MHz): d 104.02
(C-20), 103.77 (C-100), 92.48 (C-1), 82.52 (C-3), 82.10 (C-50), 76.85
(C-30), 74.32 (C-40), 72.60 (C-500), 72.52 (C-5), 71.56 (C-200), 71.51
(C-2), 70.98 (C-4), 68.61 (C-400), 66.53 (C-300), 62.69 (C-10), 61.80
(C-6), 60.46 (C-60). Calcd for C17H30O15 (474.15). MALDI-TOF m/z
497.25 [M+Na]+, 513.09 [M+K]+.
00 00
00 00
Acknowledgements
Partial support of this study by the Federal Ministry of Econom-
ics and Technology (BMWi) via German Federation of Industrial
Research Associations (AIF) and Research Association of the Ger-
man Food Industry (FEI) within Project No. 12401N is gratefully
acknowledged.
4.3.18. b-
glucitol/mannitol (36)
(a) Palatinitol (15, 344 mg, 1 mmol) and (4-pyridinyl) b-
D-Galactopyranosyl-(1?3)-a-D-glucopyranosyl-(1?6)-
References
D
-galac-
1. Onishi, N.; Yamashiro, A.; Yokozeki, K. Appl. Environ. Microbiol. 1995, 61, 4022–
4025.
topyranoside (27, 78 mg, 0.3 mmol) dissolved in potassium phos-
phate buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5) were
incubated according to GP 3. Workup and chromatography on Bio-
gel P2 gave 17.0 mg (11.2%) of 36 as a colorless, amorphous solid.
(b) Palatinitol (15, 344 mg, 1 mmol) and lactose (31, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 3.
Workup and chromatography on Biogel P2 gave 22.2 mg (8.8%) of
2. Fransen, C. T. M.; van Laere, K. M. J.; van Wijk, A. A. C.; Brüll, L. P.; Dignum, M.;
Thomas-Oates, J. E.; Haverkamp, J.; Schols, H. A.; Voragen, A. G. J.; Kamerling, J.
P.; Vliegenthart, J. F. G. Carbohydr. Res. 1998, 314, 101–114.
3. Farkas, E.; Schmidt, U.; Thiem, J.; Kowalczyk, J.; Kunz, M.; Vogel, M. Synthesis
2003, 699–706.
4. Schröder, S.; Thiem, J.; Kowalczyk, J.; Kunz, M.; Vogel, M. Tetrahedron 2004, 11,
2601–2608.
5. Foucault, M.; Watzlawick, H.; Mattes, R.; Haser, R.; Gouet, P. Acta Crystallogr.
2006, 62, 100–103.
36 as a colorless, amorphous solid. ½a D20
ꢀ
+54 (c 1, H2O); lit.:4 +52
6. Fridjonsson, O.; Watzlawick, H.; Mattes, R. Extremophiles 2000, 4, 23–33.
7. Spangenberg, P.; André, C.; Dion, M.; Rabiller, C.; Mattes, R. Carbohydr. Res.
2000, 329, 65–73.
(c 0.6, H2O); 1H NMR (D2O, 500 MHz): d 4.90 (m, 2H, CH2-1), 4.45
(d, 1H, J1 ,2 = 7.8 Hz, H-100). 13C NMR (D2O, 100 MHz): d 103.68
00 00
8. Barnett, J. E. G. Carbohydr. Res. 1969, 9, 21–31.
(C-100), 98.47 (C-1), 82.37 (C-3), 75.43 (C-500), 72.88 (C-300), 71.40
(C-2), 71.13 (C-200), 69.91/69.63 (C-4/400), 68.99/68.94 (C-60), 63.63
(C-10, Man-ol), 62.78 (C-10, Glc-ol), 61.36 (C-600), 60.86 (C-6). Calcd
for C18H34O16 (506.18). MALDI-TOF m/z 529.13 [M+Na]+, 545.34
[M+K]+.
9. Steinmann, A.; Thimm, J.; Matwiejuk, M.; Thiem, J. Macromolecules 2010, 43,
3606–3612.
10. Schuttle, J. B.; de Jong, J.; van Weerden, E. J.; Tamenga, S. Br. J. Nutr. 1992, 1,
195–207.
11. Osaki, S.; Kimura, T.; Sugimoto, T.; Hizukuri, S.; Iritani, N. J. Nutrition 2001, 3,
796–799.
12. Nisikawa, Y.; Yoshikawa, G.; Tanaka, H.; Morimoto, A.; Mukai, K. Unitika Ltd,
WO0157230A1, 2001; Chem. Abstr. 2001, 135, 136725.
13. Kent, P. W.; Barnett, J. E. G. J. Chem. Soc. 1964, 6196–6204.
14. Kuhn, R.; Grassner, H. Liebigs Ann. Chem. 1957, 55–67.
15. Helferich, B.; Jung, K.-H. Liebigs Ann. Chem. 1954, 589, 77–81; Helferich, B.;
Jung, K.-H. Liebigs Ann. Chem. 1955, 595, 242.
16. Yasukochi, T.; Inaba, C.; Fukase, K.; Kusumoto, S. Tetrahedron Lett. 1999, 40,
6585–6589.
17. Lemieux, R. U. Methods Carbohydr. Chem. 1963, 2, 221–222.
18. Hanessian, S.; Saavedra, O. M.; Mascitti, V.; Marterer, W.; Oehrlein, R.; Mak, C.-
P. Tetrahedron 2001, 57, 3267–3280.
4.3.19. b-
b- -fructofuranose (37)
(a) Isomaltulose (16, 342 mg, 1 mmol) and (4-pyridinyl)
-galactopyranoside (27, 78 mg, 0.3 mmol) dissolved in potas-
D-Galactopyranosyl-(1?3)-a-D-glucopyranosyl-(1?6)-
D
b-
D
sium phosphate buffer (1 mL, 0.1 M, KH2PO4/K2HPO4, pH = 6.5)
were incubated according to GP 3. Workup and chromatography
on Biogel P2 gave 19.7 mg (13.2%) of 37 as a colorless, amorphous
solid.
19. Nicolaou, K. C.; Schreiner, E. P.; Stahl, W. Angew. Chem., Int. Ed. 1991, 30, 585–
590.
(b) Isomaltulose (16, 342 mg, 1 mmol) and lactose (31, 171 mg,
0.5 mmol) dissolved in potassium phosphate buffer (1 mL, 0.1 M,
KH2PO4/K2HPO4, pH = 6.5) were incubated according to GP 3.
Workup and chromatography on Biogel P2 gave 19.5 mg (7.9%) of
20. Smiatacz, Z.; Zygfryd, M. Carbohydr. Res. 1990, 196, 167–174.
21. Kröger, L.; Scudlo, A.; Thiem, J. Adv. Synth. Catal. 2006, 348, 1217–1227.
22. Distler, J. J.; Jourdan, G. W. J. Biol. Chem. 1973, 248, 6772–6780.
23. Hedbys, L.; Johannson, E.; Mosbach, K.; Larsson, P.-O.; Gunnarsson, A.;
Svensson, S. Carbohydr. Res. 1989, 186, 217–223.
37 as a colorless, amorphous solid. ½a D20
ꢀ
+44 (c 0.97, H2O); Lit.:4
24. Heyworth, R.; Walker, P. G. Biochem. J. 1962, 83, 331–335.
Please cite this article in press as: Schröder, S.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.005