The versatile enzyme Araf51 allowed efficient synthesis of rare pathogen-related β-d-galactofuranosyl-pyranoside disaccharides

Article abstract of DOI:10.1039/c3ob42519c

The preparation of galactofuranosyl-containing disaccharidic parts of natural glycoconjugates was performed according to a chemo-enzymatic synthesis. Our goals were firstly to develop an alternative approach to standard chemical strategies by limiting the number of reaction and purification steps, and secondly to evaluate the scope of the Araf51 biocatalyst to transfer a galactofuranosyl moiety to a set of pyranosidic acceptors differing from each other by the series, the anomeric configuration as well as the conformation. The study of binding mode of the resulting disaccharides was also performed by molecular modeling and showed significant differences between (1→2)- and (1→6)-linked disaccharides.

Full text of DOI:10.1039/c3ob42519c

Organic &
Biomolecular Chemistry
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PAPER
The versatile enzyme Araf51 allowed efficient
synthesis of rare pathogen-related
Cite this: Org. Biomol. Chem., 2014,
12, 3080
β-D-galactofuranosyl-pyranoside disaccharides
Ilona Chlubnová,a,b,c Blanka Králová,c Hana Dvořáková,d Petr Hošek,c
Vojtěch Spiwok,c Dominik Filipp,e Caroline Nugier-Chauvin,*a,b
Richard Daniellou†a,b and Vincent Ferrières*a,b
The preparation of galactofuranosyl-containing disaccharidic parts of natural glycoconjugates was per-
formed according to a chemo-enzymatic synthesis. Our goals were firstly to develop an alternative
approach to standard chemical strategies by limiting the number of reaction and purification steps, and
secondly to evaluate the scope of the Araf51 biocatalyst to transfer a galactofuranosyl moiety to a set of
pyranosidic acceptors differing from each other by the series, the anomeric configuration as well as the
conformation. The study of binding mode of the resulting disaccharides was also performed by molecular
modeling and showed significant differences between (1→2)- and (1→6)-linked disaccharides.
Received 17th December 2013,
Accepted 21st February 2014
DOI: 10.1039/c3ob42519c
www.rsc.org/obc
logical processes.6 While furanosyl-containing oligosacchar-
ides are crucial constituents of surface glycoconjugates in cell
Introduction
The complex heterogeneity of carbohydrates in living systems walls of bacteria,7–9 fungi10 and parasitic11,12 microorganisms,
is a direct result of several carbohydrate characteristics: the including some clinically significant pathogens, with an excep-
ability of different types and numbers of sugar residues to tion of 2-deoxy-D-ribose and D-ribose, furanosides are comple-
form glycosidic bonds with one another, the type of anomeric tely absent from mammals. This makes them interesting
linkage, the position and the absence or presence of branch- targets for development of new therapeutics.
ing, the more or less flexible conformations of the resulting
A major limitation preventing the use of oligosaccharides
oligo-, polysaccharides or glycoconjugates,1,2 and the size of as therapeutics is the difficulty in producing sufficient
the monosaccharidic ring.3,4 Indeed, sugars exhibit significant amounts of these molecules in a desired purity. The isolation
differences depending on whether they are present as pyrano- of these compounds from biological sources tends to be low
sides or as furanosides. It is now well established that the yielding and presents the risk of contamination from infec-
importance of the furanose ring in biology can no longer be tious agents.13 Although progress in the chemical synthesis of
understated. A key characteristic of furanose ring systems oligosaccharides has been made14–20 and synthesis of docosana-
is their higher flexibility compared to that of their pyranosidic arabinofuranoside realized by Lowary’s research team21 is
counterparts,5 and this profoundly influences their role in bio- impressive evidence, this approach still remains a challenge.
The chemical synthesis requires stereo- and regioselective
control of glycosidic bond formation, thus multiple protection
aEcole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, 11 Allée de
Beaulieu, CS 50837, 35708 Rennes Cedex 7, France.
and deprotection schemes are needed to achieve the required
selectivity. Low yields of the desired products are also a result
of the difficulty in purifying the deprotected compound along
with product loss during each step of a multistep synthesis.13,22
Nature’s solution to the assembly of glycosidic bonds are
enzymes belonging to the families of glycosyl transferases and of
glycosidases. These enzymes have therefore enormous potential
for the synthesis of biologically relevant carbohydrate struc-
tures.23 These biocatalysts offer a significant advantage over their
chemical counterparts in their ability to form a specific glycosidic
linkage in the presence of other reactive functional groups.22
E-mail: vincent.ferrieres@ensc-rennes.fr; Tel: +33 223238058
bUniversité européenne de Bretagne, France
cDepartment of Biochemistry and Microbiology, Institute of Chemical Technology of
Prague, Techniká 3, 166 28 Prague 6, Czech Republic
dLaboratory of NMR spectroscopy, Institute of Chemical Technology of Prague,
Techniká 3, 166 28 Prague 6, Czech Republic
eLaboratory of Immunobiology, Institute of Molecular Genetics AS CR,
Videnska 1083, 142 20 Prague 4, Czech Republic
†Present address: Institut de Chimie Organique et Analytique (ICOA), UMR
CNRS 7311, Université d’Orléans, BP 6759, Rue de Chartres, 45067 Orléans
Cedex 2, France.
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In this context, only few groups have been interested in
the chemo-enzymatic synthesis of furanosyl-containing
Results
conjugates.3,24–27 Furanosyl transferases are however rare and In the first place, unavailability of the −1 subsite of Araf51 to
require both nucleotide-diphospho (NDP)-sugars as donors pyranosidic acceptors was confirmed spectrophotometrically
and suitable acceptors.28–30 Another approach, recently pro- (405 nm) by incubation of individual pNP pyranosides 2–8
posed by Thorson, is based on the glycosyl transferase reversi- with the enzyme for 2 h at 60 °C in a phosphate buffer at pH
bility, that uses simple glycosides as precursors of NDP-sugars 7.4. No release of p-nitrophenolate was recorded, indicating
for further transfer onto more complex natural products.31,32 that these compounds cannot act as donors, thus confirming
To the best of our knowledge, this strategy was not applied for our first hypothesis. Subsequently, to screen enzyme readiness
the synthesis of furanosides. Recently, our team developed an to catalyze transglycosylation, individual analytical-scale reac-
enzyme-based protocol for the preparation of di- and oligofura- tions were performed starting from donor 1 and each of pNP
nosides using the thermophilic Araf51 as biocatalyst.33 Consid- glycopyranoside, and in the presence of Araf51. Monitoring by
ering the structural similarity between L-arabino- and thin layer chromatography showed the formation of disacchar-
D-galactofuranosyl (D-Galf ) entities, and the wide presence of ides when the donor 1 was incubated with an equal molar
D-Galf residues in natural glycoconjugates and polysaccharides, quantity or with a 5-fold molar excess of pyranosidic acceptor,
we now propose to expand the chemo-enzymatic methodology after only short incubation periods (5–20 min). These com-
to transglycosylation reactions in order to transfer a galacto- pounds resulted from both self-condensation of 1 and desired
furanosyl entity to a variety of pyranosidic acceptors (Fig. 1). transglycosylation reactions.
These substrates were chosen so as to perform the synthesis of
As a model of biocatalyzed coupling, the progress of the
furanosyl-pyranoside sequences relevant to pathogenic micro- reaction of 1 with 2 (pNP α-D-Glcp) was monitored by HPLC
organisms3 as well as to estimate the specificity of this enzyme (Fig. 2). Between 5 and 20 minutes, digalactofuranosides con-
towards carbohydrate acceptors. As we expected that such pyra- stituted the main reaction products.33 At the same time, these
nosides are resistant to hydrolysis by the furanosidase Araf51, products of self-condensation were hydrolyzed more rapidly
the presence of the pNP group in the anomeric position was than furanosyl-pyranoside disaccharides, which resisted to
envisaged to enable simple UV detection of reaction products. hydrolysis for more than 3 h. This could be explained by the
Moreover, the D-gluco- (D-Glcp), D-galacto- (D-Galp) and D-man- structures of the resulting disaccharides which significantly
nopyranosidic (D-Manp) epimers, as well as their anomeric differ from those of natural substrates of the enzyme. In the
configurations, offer stereochemical variations that may influ- present model reaction, traces of trisaccharides were observed
ence the fate of the coupling process. Finally, we made a focus from the early stage of the reaction (5–10 min) and were con-
on the L-rhamnopyranoside (L-Rhap) 8 for structural and bio- firmed by mass spectrum analysis, but precise structures
logical reasons but also because it displays a 1C4 conformation could not be elucidated. Importantly, when the ratio donor/
instead a 4C1 one observed for other acceptors 2–7.
acceptor was increased to 1 : 10, no products of self-conden-
sation were detected. Moreover, synthesis of furano-pyrano-dis-
accharides was strongly favored over hydrolysis of 1. After
careful chromatographic purification, two main disaccharides
9 and 10 were isolated and their structures were elucidated by
NMR spectroscopy. The major product 9 was obtained in the
yield of 44%. It exhibited an intense three-bond coupling in
the 13C–1H (HMBC) spectrum between H-1′ (5.09 ppm) and C-2
(79.2 ppm) as well as C-1′ (109.4 ppm) and H-2 (3.69 ppm) and
was identified as pNP β-D-galactofuranosyl-(1→2)-α-D-glucopyr-
anoside 9 (Fig. 2). The second regioisomer, isolated in 34%
yield, presented a correlation between H-1′ (5.24 ppm) and C-3
(79.4 ppm) as well as between C-1′ (108.3 ppm) and H-3
(3.96 ppm). The compound was identified as (1→3)-linked dis-
accharide 10. The time-course analysis monitored by HPLC
revealed that the (1→3)-disaccharide 10 was kinetically syn-
thesized, followed by the formation of the (1→2)-isomer 9.
This monitoring also showed that these compounds dis-
appeared with time in favor of a third regioisomer. The yield of
the latter reached its maximum after 4 hours of reaction and it
was still present after 24 hours. The precise nature of the
corresponding glycosidic linkage could however not be clearly
elucidated. As a result, the overall yield of transglycosylation
reached a maximum, slightly greater than 80%, between 5 and
20 minutes. In the present model reaction, traces of trisacchar-
Fig. 1 Structure of glycosyl donor 1 and acceptors 2–8.
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Fig. 2 Progress of the reaction of 1 with 2 or 3 catalyzed by Araf51.
ides were observed from the early stage of the reaction
On the other hand, the reaction with the α-mannopyrano-
(5–10 minutes) and confirmed by mass spectrum analysis, but side 6 (entry 3) was relatively rapid compared to other pyrano-
precise structures could not be elucidated. sidic acceptors and the degree of hydrolysis of formed
Under similar conditions, using the β-glucopyranoside 3 as disaccharides was slightly higher. The reaction in a preparative
an acceptor (Fig. 2), and after 20 minutes of reaction, three dis- scale was thus carried out for only 10 minutes. pNP β-D-Galf-
accharides were chromatographically separated: pNP β-D-Galf- (1→6)-α-D-Manp 19 was obtained in 15% yield. 2-D NMR ana-
(1→2)-β-D-Glcp 11, pNP β-D-Galf-(1→3)-β-D-Glcp 12, and pNP lyses confirmed that three other regioisomers were obtained as
β-D-Galf-(1→6)-β-D-Glcp 13 were isolated in 26%, 25% and 30% a mixture in an overall yield of 43%. The time-course analysis
yields, respectively. The time-course analysis revealed that the monitored by TLC revealed that these regioisomers are readily
(1→2)-linked regioisomer 11 was the major kinetic product formed from the first minutes of reaction with the maximum
and is present together with the (1→3)-linked regioisomer 12 yields reached between 5 and 10 minutes. The (1→6)-linked
from the first minutes of the reaction. From about 15 minutes, regioisomer 19 was formed from about 5 minutes of reaction.
the (1→6)-linked regioisomer 13 was formed and became the
The transglycosylation with the β-anomer 7 was apparently
prevalent one after 1 hour. It was still detected in 15% yield slower than that of the α-anomer and the conversion of pNP
after 24 hours. It is interesting to note that, when 12 was incu- β-D-Galf still did not reach a maximum after two hours of reac-
bated with the biocatalyst and an excess of 3, it was trans- tion. However, the degree of hydrolysis was quite low, similarly
formed into 13. This emphasizes the striking stability of this to the other pNP pyranosides. After 25 minutes of the prepara-
(1→6)-bond in the presence of the furanosyl hydrolase Araf51.
tive-scale reaction, two regioisomers were isolated (entry 4). pNP
To go further with this methodology, we applied the Araf- β-D-Galf-(1→4)-β-D-Manp 20 was obtained in a very high yield of
51-assisted synthesis of furanosyl-containing disaccharides to 49%. The second regioisomer 21, identified as the (1→6)-disac-
three other families of acceptors. The use of galactosidic charide, was isolated in 16% yield. The time-course analysis
acceptors 4 and 5 did not fundamentally change the obser- confirmed kinetic preferences for the (1→4)-linked disaccharide
vations previously exposed with the gluco series. Three disac- 20. In about 40 minutes of reaction, both regioisomers were
charides with (1→2)- (14), (1→3)- (15), and (1→4)-linkage (16) present equivalently. At the end of 2 hours incubation, the
were obtained from the α-anomer 4 and isolated in 73% favoured (1→6)-linked regioisomer 21 prevailed. Structures were
overall yield (Table 1, entry 1). Substrate 5, characterized by a unambiguously established according to NMR data.
β-configuration, afforded only the (1→2)- (17) and (1→6)-disac-
Finally, we also studied the L-rhamnopyranoside 8 since it
charides (18) in a near equimolecular ratio and in a 52% presents significantly different reactivity and because it is
overall yield (entry 2).
widely found in Nature. While three disaccharides were
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Table 1 Araf51-mediated synthesis of disaccharides 9–22 using pNP β-D-Galf 1 as donor and the linkages identified in individual regioisomers
together with the isolated yields after two hours of reaction
Product (yield, %)
Entry
1
Acceptor
Time (min)
20
(1→2)
(1→3)
(1→4)
(1→6)
Overall yield (%)
73
4 (α-D-Galp)
14 (41)
17 (27)
15
(32)
16
2
3
4
5
5 (β-D-Galp)
6 (α-D-Manp)
7 (β-D-Manp)
8 (α-L-Rhap)
20
15
25
20
18 (25)
19 (15)
21 (16)
52
58
65
38
Mixture (1→2/3/4) (43)
20 (49)
22 (38)
observed after 20 minutes of the biocatalytic process, exclusively
Starting from β-D-mannopyranoside and α-L-rhamnopyrano-
one regioisomer 22, characterized by a (1→4)-connection, was side, the kinetically preferred linkage was the (1→4). It results
indeed isolated and in a fairly good 38% yield (entry 5). Evi- from this observation that the adopted conformations within
dence of the precise structure was established on the basis of the active site of Manp and Rhap derivatives vary to some
NMR data. More specifically, the C-4 signal in the starting sub- extent compared to the glucosidic and galactosidic mono-
strate 8 (δC-4 = 71.4 ppm) was shifted to 77.8 ppm within the dis- saccharides. Thus the axial C-2 hydroxyl group in mannose, in
accharide 22, thus demonstrating the (1→4)-coupling, and this contrast to glucose, seems to impact the conformation within
result was corroborated by 2D NMR experiments.
the active site more markedly than the orientation of C-4 OH
group distinguishing glucose from galactose. Overall, in all the
three series where both anomers were tested (D-Glcp, D-Galp,
D-Manp), the reactions involving the α-anomer proceeded
markedly faster than those of their β-anomeric counterparts.
The time-course analyses also revealed the crucial effect of the
reaction time on the ratio of regioisomers formed in individual
reactions. Thus, in most of cases, it is possible to isolate either
kinetic or thermodynamic disaccharide with a very low quan-
tity of the other regioisomer with respect to the time-course of
the reaction.
In order to elucidate binding modes of acceptors 2–7 in
transglycosylation reactions, complexes of Araf51 with all puta-
tive transglycosylation products were subjected to molecular
dynamics simulations (24 × 10 ns). Each putative product was
docked into the active site by rigid fit of its Galf moiety onto
the Araf moiety in the experimental structure. The pyranosyl
and pNP-moieties were adjusted manually before the simu-
lation. The results of simulations for disaccharides 11, 14, 17
and 18 are shown in Fig. 3. These selected products showed
stable binding in the active site illustrated by low root-mean-
square deviation (RMSD). The results also demonstrated that
binding modes of (1→2) products are very similar. The pNP-
moiety is oriented perpendicularly to the access of the active
site. On the other hand, the pNP-moiety in 18 [β-D-Galf-(1→6)-
Discussion
All results obtained for the transfer of a galactofuranosyl
residue to pyranosidic acceptors mediated by the arabinofura-
nosidase Araf51 underline the ability of this enzyme to accept
within its +1 subsite all the seven tested pyranosidic acceptors.
Importantly, the anomeric configuration of the latter modu-
lated the behavior of the furanosyl transfer. Usually, the (1→2)-
disaccharides were the most common in the gluco and galacto
series, and readily formed and stable. The (1→3) linkage was
formed subsequently and was also frequently presented in
these series. Interestingly, none of these α-anomers displayed
the (1→6)-connection although for the β-anomers, it rep-
resents the major thermodynamically formed linkage. In
Nature, exo-acting α-L-arabinofuranosidases release the arabi-
nosyl decorations at C-2 and C-3 position of arabinogalactans
and arabinoxylans from a range of plant structural polysac-
charides, where both the galactose and xylose moieties are pre-
sented in
a β-D-pyranose form. Thus, considering the
structural similarity of xylose and glucose, the transglycosyla-
tion preferences correspond to the hydrolytic activity on
natural substrates.
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Fig. 3 Predicted binding modes of 11, 14, 17 and 18 by 10 ns molecular dynamics simulations. The figure shows predicted binding modes at the
end of each simulation (left) and corresponding RMSD profiles (right). These were obtained by fitting protein Cα atoms and calculating RMSD for the
product.
β-D-Galp], as an example of (1→6) product, is placed along the large excess of acceptor allowed overcoming the self-conden-
axis of the active site. The fact that the pNP-moiety may orient sation side reaction. Secondly, transglycosylation products
in different angles explains relatively broad acceptor specificity. were obtained in the early stage of the biocatalyzed process
Most other putative products have shown high RMSD which and increased reaction times did not affect the target furano-
indicate either inability of the product to bind into the active syl-pyranoside disaccharides since the degree of hydrolysis of
site or incorrect docking. Unfortunately, attempts to quantita- the latter remained very low. These factors together were fruit-
tively predict acceptor preferences from molecular simulations ful from a synthetic point of view. Consequently, and with
or free-energy methods were not successful (not shown).
regard to the very good yields obtained with the assistance of
Among the disaccharides prepared throughout this part, the hydrolytic furanosidase Araf51, this chemo-enzymatic
several display a configuration reported from uncommon cell approach constitutes a very interesting alternative to multi-step
wall glycoconjugates of some pathogenic species. Galactofura- chemical synthesis of various mimetics of biologically signifi-
nosyl residue connected to D-Glcp moiety through the (1→3)- cant structures. The +1 subsite of Araf51 was able to recognize
linkage has been identified e.g. in Streptococcus species,34 the simple glycopyranosides as acceptors, even a Rhap derivative
1
one with the (1→6)-linkage is reported from Escherichia coli and its C4 conformation. Among targets were prepared disac-
K-12 strain.35 The (1→3)-linkage to D-Galp was identified in charides biosynthesized by Mycobacteria, Leishmania, Trypano-
Fibrobacter succinogenes36 or in the O-antigen repeating unit of soma or Paracoccidioides microorganisms.
Klebsiella pneumoniae.36 In Mycobacterium species37–40 and
Plesiomonas shigelloides,41 β-D-Galf is connected to L-Rhap
through the (1→4)-linkage. The sequence β-D-Galf-(1→2)-
Experimental section
General remarks
D-Manp is present in many microorganisms, among the patho-
gens notably Cryphonectria parasitica42 or Trichoderma.43
β-D-Galf-(1→3)-D-Manp is frequent in Aspergillus,44 Trypano-
Prior to NMR analysis, fractions were exchanged in D2O
(99.9% purity) at room temperature with intermediate freeze-
soma cruzi12,45 and Leishmania,11 β-D-Galf-(1→6)-D-Manp e.g. in
Aspergillus,44 Leishmania11 or Paracoccidioides brasiliensis.46
drying, and then dissolved in 400 μL of D2O. 1H, 13C, COSY,
HSQC, HMBC, TOCSY and NOESY NMR spectra were recorded
at the Laboratory of NMR spectroscopy (ICT Prague, Czech
Republic) on a Bruker 600 Avance spectrometer equipped with
Conclusions
1
a cryoprobe at 600 MHz for H and 125 MHz for 13C, and on a
A chemo-enzymatic synthesis of galactofuranosyl-containing Bruker ARX 400 at 400 MHz for 1H, 100 MHz for 13C at ENSCR
disaccharides was proposed mediated by the thermophilic ara- (France). Chemical shifts are given in δ-units (ppm). Coupling
binofuranosidase Araf51. We have first demonstrated that a constants J are given in Hz.
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The HRMS were measured at the Centre Régional de pNP β-D-galactofuranoside (20 mg, 66 μmol; or 10 mg,
Mesures Physiques de l’Ouest (CRMPO, Université de Rennes 33 μmol) with individual pNP pyranosides (200 mg, 660 μmol
1, France) with a MS/MS ZabSpec TOF Macromass using for pNP D-Glcp and pNP D-Galp; 100 mg, 330 μmol for pNP
m-nitrobenzylic alcohol as the matrix and accelerated caesium D-Manp and pNP L-Rhap) and 4800 U of Araf51 (for reactions
ions for ionization and at the Laboratory of Mass Spectrometry with pNP D-Glcp and pNP D-Galp) or 2400 U of Araf51 (for reac-
(ICT Prague, Czech Republic) using a Q-TOF Micro (Waters, tions with pNP D-Manp and pNP L-Rhap) in 50 mM PBS pH 7.4
USA), where electrospray-ionisation mass spectra (ESI-MS) in a total volume with regard to the solubility of substrates:
were recorded on samples dissolved in MeOH injected in a 5 mL (reactions with α- and β-D-Galp and β-D-Glcp) or 10 mL
volume of 2–5 μL into a flow (100 μL min−1) of MeOH. Sample (α-D-Glcp and α- and β-D-Manp) or 15 mL (α-L-Rhap).
cone voltage was 42 V and the source temperature was 150 °C.
Based on the results from analytical-scale reactions, individ-
Measurements were performed in positive ([M + Na]+ ion ual reactions proceeded for 20 min with the exception of
detection) mode in the range of 100–1000 Da. Finally, at the α-D-Manp (15 min) and β-D-Manp (25 min) to obtain the
Mass Spectrometry Group (Institute of Organic Chemistry and maximum ratio of transglycosylation/hydrolysis. The reactions
Biochemistry, Academy of Sciences of the Czech Republic) on were stopped by enzyme denaturation at 100 °C for 10 min.
LTQ-Orbitrap XL (THERMO), where ESI+ spectra were recorded The reaction products were repeatedly separated by silica gel
on samples dissolved in MeOH–H2O (1 : 1), sample cone flash chromatography using EtOAc–AcOH–H2O in ratios from
voltage was 40 V.
15 : 1 : 1 to 40 : 1 : 1. Separations were monitored by TLC, frac-
Thin layer chromatography (TLC) analyses were conducted tions corresponding to individual regioisomers were collected,
on Kieselgel 60 F254 (Merck) plates with 0.2 mm layer thick- evaporated and lyophilized several times with intermediate
ness. Spots were visualized by UV (254 nm) and by exposure to dissolving in H2O, finally in D2O and subjected to structural
0.2% w/v orcinol in H2SO4 (20% v/v) in ethanol. For column analyses.
chromatography, either Si 60 (40–63 μm) Silica gel or pre-
packed Chromabond®Flash RS 15 SiOH columns (Macherey- side (9). This compound was obtained according to the
Nagel) were used. described general procedure by incubation of 20 mg (66 μmol)
p-Nitrophenyl β-D-galactofuranosyl-(1→2)-α-D-glucopyrano-
Gel permeation chromatography was performed on P-2 Bio- of 1 with 200 mg (660 μmol) of pNP α-D-glucopyranoside 2 in
Gel (Bio-Rad) using FPLC system consisting of a solvent deliv- the presence of 4800 U of Araf51 and was isolated in 44% yield
ery system Biologic F40 DuoFlow, Biologic QuadTec UV-Vis (13.3 mg) after purification. TLC: Rf = 0.45 (AcOEt–AcOH–H2O,
Detector and Biologic BioFrac Fraction Collector (all Bio-Rad). 7/2/2). 1H NMR (400 MHz, D2O): δ = 8.19 (d, 2H, J = 9.2 Hz,
Deionized filtrated (0.22 μm PVDF membrane, Millipore) water Hm, C6H4), 7.23 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.87 (d, 1H, J =
was used as a mobile phase with a flow rate of 0.15 mL min−1
.
3.6 Hz, 1a-H), 5.08 (d, 1H, J = 2.0 Hz, 1b-H), 4.07 (dd, 1H, J =
Separation was monitored by UV absorbance at 280 and 2.0, 4.2 Hz, 2b-H), 3.93 (dd, 1H, J = 4.2, 6.8 Hz, 3b-H), 3.92 (dd,
405 nm by operating software Biologic DuoFlow. Collected frac- 1H, J = 9.0, 9.8 Hz, 3a-H), 3.70 (dd, 1H, J = 3.6, 9.8 Hz, 2a-H),
tions were lyophilized (FreeZone Freeze Dry System, 3.60–3.50 (m, 3H, 6a-H, 5a-H), 3.56 (dd, 1H, J = 3.1, 6.8 Hz, 4b-
Labconco).
H), 3.53–3.47 (m, 1H, 5b-H), 3.50 (dd, 1H, J = 9.0, 9.8 Hz,
Recombinant arabinofuranosidase Araf51 was produced in 4a-H), 3.10 (dd, 1H, J = 8.1, 11.5 Hz, 6b-H), 2.86 (dd, 1H, J =
E. coli and purified as already described.47 The hydrolytic 4.0, 11.5 Hz, 6′b-H) ppm. 13C NMR (100 MHz, D2O): δ = 161.6
activity toward p-nitrophenyl pyranosides was tested by incu- (Cipso C6H4), 142.7 (Cp C6H4), 126.1 (Cm C6H4), 116.7
bation of the solution of enzyme (obtained after affinity Ni- (Co C6H4), 109.5 (1b-C), 96.3 (1a-C), 82.6 (4b-C), 81.1 (2b-C), 79.3
NTA chromatography and exchanged to 50 mM PBS pH 7.4 by (2a-C), 76.1 (3b-C), 72.4 (5a-C), 71.5 (3a-C), 69.7 (5b-C), 69.0
PD10 gel chromatography) with individual glycopyranosides (4a-C), 62.7 (6b-C), 60.1 (6a-C) ppm. HRMS (ESI): m/z calcd for
(5 mM) in 50 mM potassium phosphate buffer pH 7.4 at 60 °C. C18H25O13NNa [M + Na]+ 486.12181; found 486.12173.
The release of p-nitrophenolate was continuously measured at
p-Nitrophenyl β-D-galactofuranosyl-(1→3)-α-D-glucopyrano-
405 nm (Microplate Spectrophotomerer PowerWave XS/XS2, side (10). This compound was obtained according to the
BioTek) and data evaluated with Gen5 Data Analysis Software described general procedure by incubation of 1 (20 mg,
(BioTek). pNP α-D-Galp, pNP β-D-Galp, pNP α-D-Glcp, pNP 66 μmol) with pNP α-D-glucopyranoside 2 (200 mg, 660 μmol)
β-D-Glcp, pNP α-D-Manp, pNP β-D-Manp and pNP α-L-Rhap are in the presence of 4800 U of Araf51. It was isolated in 34%
commercially available.
yield (10.5 mg). TLC: Rf = 0.5 (AcOEt–AcOH–H2O, 7/2/2). 1H
NMR (400 MHz, D2O): δ = 8.12 (d, 2H, J = 9.2 Hz, Hm, C6H4),
7.18 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.70 (d, 1H, J = 3.5 Hz, 1a-
H), 5.24 (d, 1H, J = 1.5 Hz, 1b-H), 4.10 (dd, 1H, J = 1.5, 3.5 Hz,
General procedure for the transglycosylation reactions
Prior to preparative-scale reactions, the time-course analyses 2b-H), 4.03–3.99 (m, 2H, 3b-H, 4b-H), 3.96 (dd, 1H, J = 9.2,
were performed in the analytical scale (5 μmol of individual 9.6 Hz, 3a-H), 3.80 (dd, 1H, J = 3.5, 9.6 Hz, 2a-H), 3.76 (ddd,
pNP pyranosides, 0.5 μmol of pNP β-D-galactofuranoside and 1H, J = 3.2, 4.4, 7.6 Hz, 5b-H), 3.67–3.57 (m, 5H, 6a-H, 6′a-H,
36 U of Araf51 in 100 μL of 50 mM PBS pH 7.4). The reaction 6b-H, 6′b-H, 5a-H), 3.48 (dd, 1H, J = 9.2, 9.6 Hz, 4a-H) ppm.
was monitored by TLC (7 : 2 : 2 EtOAc–AcOH–H2O). Preparative- 13C NMR (100 MHz, D2O): δ = 161.1 (Cipso C6H4), 141.9 (Cp
scale reactions were performed at 60 °C by incubation of the C6H4), 125.7 (Cm C6H4), 116.4 (Co C6H4), 108.3 (1b-C), 96.6
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(1a-C), 82.9 (4b-C), 81.2 (2b-C), 79.4 (3a-C), 76.7 (3b-C), 72.7 142.7 (Cp C6H4), 126.1 (Cm C6H4), 116.5 (Co C6H4), 107.9 (1b-
(5a-C), 70.8 (2a-C), 70.6 (5b-C), 67.5 (4a-C), 62.8 (6b-C), 60.1 C), 99.3 (1a-C), 83.0 (4b-C), 81.0 (2b-C), 76.8 (3b-C), 75.3 (3a-C),
(6a-C) ppm. HRMS (ESI): m/z calcd for C18H25O13NNa 75.2 (5a-C), 72.7 (2a-C), 70.8 (5b-C), 69.4 (4a-C), 66.3 (6a-C),
[M + Na]+ 486.12181; found 486.12160.
62.7 (6b-C) ppm. HRMS (ESI): m/z calcd for C18H25O13NNa
p-Nitrophenyl β-D-galactofuranosyl-(1→2)-β-D-glucopyrano- [M + Na]+ 486.12181; found 486.12172.
side (11). This compound was obtained according to the
p-Nitrophenyl β-D-galactofuranosyl-(1→2)-α-D-galactopyrano-
described general procedure by incubating 1 (20 mg, 66 μmol) side (14). This compound was obtained according to the
with pNP β-D-glucopyranoside 3 (200 mg, 660 μmol) in the general procedure starting from donor 1 (20 mg, 66 μmol),
presence of 4800 U of Araf51, and was isolated in 26% yield acceptor 4 (200 mg, 660 μmol) and 4800 U of Araf51. Com-
(7.9 mg). TLC: Rf = 0.4 (AcOEt–AcOH–H2O, 7/2/2). 1H NMR pound 14 was isolated in 41% yield (12.6 mg). TLC: Rf = 0.4
1
(400 MHz, D2O): δ = 8.18 (d, 2H, J = 9.2 Hz, Hm, C6H4), 7.17 (d, (AcOEt–AcOH–H2O, 7/2/2). H NMR (400 MHz, D2O): δ = 8.19
2H, J = 9.2 Hz, Ho, C6H4), 5.28 (d, 1H, J = 7.2 Hz, 1a-H), 5.26 (d, 2H, J = 9.2 Hz, Hm, C6H4), 7.23 (d, 2H, J = 9.2 Hz, Ho,
(d, 1H, J = 1.4 Hz, 1b-H), 4.03 (dd, 1H, J = 1.4, 2.5 Hz, 2b-H), C6H4), 5.89 (d, 1H, J = 3.2 Hz, 1a-H), 5.08 (s, 1H, 1b-H), 4.10
3.94–3.90 (m, 2H, 3b-H, 4b-H), 3.81 (dd, 1H, J = 5.7, 12.4 Hz, (dd, 1H, J = 3.3, 10.4 Hz, 3a-H), 4.07 (dd, 1H, J = 2.1, 4.3 Hz,
6a-H), 3.70–3.65 (m, 2H, 5b-H, 2a-H), 3.65 (dd, 1H, J = 9.5, 9.5 2b-H), 3.99 (dd, 1H, J = 2.0, 3.3 Hz, 4a-H), 3.93 (ddd, 1H, J =
Hz, 3a-H), 3.64 (dd, 1H, J = 2.2, 12.4 Hz, 6′a-H), 3.59–3.55 (m, 0.7, 4.3, 6.9 Hz, 3b-H), 3.91 (dd, 1H, J = 3.7, 10.3 Hz, 2a-H),
1H, 5a-H), 3.44 (dd, 1H, J = 4.3, 11.6 Hz, 6b-H), 3.43 (t, 1H, J = 3.89 (ddd, 1H, J = 1.9, 4.7, 7.7 Hz, 5a-H), 3.67–3.57 (m, 2H,
9.5 Hz, 4a-H), 3.39 (dd, 1H, J = 7.3, 11.6 Hz, 6′b-H) ppm. 13C 6a-H, 6′a-H), 3.56 (dd, 1H, J = 2.9, 6.9 Hz, 4b-H), 3.52 (ddd, 1H,
NMR (100 MHz, D2O): δ = 161.6 (Cipso C6H4), 142.7 (Cp C6H4), J = 2.9, 4.2, 8.2 Hz, 5b-H), 3.42 (dd, 1H, J = 4.0, 6.4 Hz, 6b-H),
126.1 (Cm C6H4), 116.5 (Co C6H4), 108.3 (1b-C), 98.1 (1a-C), 3.11 (dd, 1H, J = 8.0, 11.2 Hz, 6′b-H) ppm. 13C NMR (100 MHz,
83.7 (4b-C), 81.0 (2b-C), 78.2 (2a-C), 76.7 (3b-C), 76.1 (5a-C), D2O):
δ = 161.6 (Cipso C6H4), 142.7 (Cp C6H4), 126.2
75.6 (3a-C), 70.8 (5b-C), 69.0 (4a-C), 62.7 (6b-C), 60.3 (6a-C) (Cm C6H4), 116.8 (Co C6H4), 109.7 (1b-C), 96.6 (1a-C), 82.5
ppm. HRMS (ESI): m/z calcd for C18H25O13NNa [M + Na]+ (4b-C), 81.2 (2b-C), 76.2 (2a-C and 3b-C), 71.8 (5a-C), 69.6
486.12181; found 486.12173.
(5b-C), 69.0 (4a-C), 68.0 (3a-C), 62.6 (6b-C), 60.8 (6a-C) ppm.
p-Nitrophenyl β-D-galactofuranosyl-(1→3)-β-D-glucopyrano- HRMS (ESI): m/z calcd for C18H25O13NNa [M + Na]+ 486.12181;
side (12). This disaccharide was prepared as described in the found 486.12163.
general procedure starting from donor 1 (20 mg, 66 μmol) and
p-Nitrophenyl β-D-galactofuranosyl-(1→3)-α-D-galactopyrano-
pNP β-D-glucopyranoside 3 (200 mg, 660 μmol) in the presence side (15). The disaccharide 15 was obtained according to the
of 4800 U of Araf51. The target compound 12 was isolated in described general procedure by incubation of donor 1 (20 mg,
25% yield (7.6 mg) after purification. TLC: Rf = 0.48 (AcOEt– 66 μmol) with acceptor 4 (200 mg, 660 μmol) in the presence
1
AcOH–H2O, 7/2/2). H NMR (400 MHz, D2O): δ = 8.18 (d, 2H, of 4800 U of Araf51, and was isolated in regioisomeric mixture
J = 9.2 Hz, Hm, C6H4), 7.17 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.26 with 16 in 32% yield (9.9 mg). According to the 1H NMR signal
(d, 1H, J = 1.7 Hz, 1b-H), 5.21 (d, 1H, J = 7.6 Hz, 1a-H), 4.10 integration, it was obtained in 18% yield. TLC: Rf = 0.48
1
(dd, 1H, J = 1.7, 3.3 Hz, 2b-H), 4.02 (dd, 1H, J = 3.8, 6.4 Hz, 4b- (AcOEt–AcOH–H2O, 7/2/2). H NMR (400 MHz, D2O): δ = 8.19
H), 4.01 (ddd, 1H, J = 0.4, 3.3, 6.4 Hz, 3b-H), 3.87–3.84 (m, 1H, (d, 2H, J = 9.2 Hz, Hm, C6H4), 7.23 (d, 2H, J = 9.2 Hz, Ho,
6a-H), 3.77–3.72 (m, 1H, 5b-H), 3.72–3.68 (m, 1H, 3a-H), C6H4), 5.74 (d, 1H, J = 3.8 Hz, 1a-H), 5.12 (d, 1H, J = 2.1 Hz,
3.70–3.66 (m, 2H, 2a-H, 6′a-H), 3.66–3.57 (m, 2H, 5a-H, 6b-H), 1b-H), 4.10 (dd, 1H, J = 3.7, 10.2 Hz, 3a-H), 4.07 (dd, 1H, J =
3.61–3.55 (m, 1H, 6′b-H), 3.49 (dd, 1H, 4a-H) ppm. 13C NMR 2.2, 4.7 Hz, 2b-H), 4.02 (dd, 1H, J = 1.3, 3.0 Hz, 4a-H), 3.97 (dd,
(100 MHz, D2O): δ = 161.6 (Cipso C6H4), 142.7 (Cp C6H4), 126.1 1H, J = 3.7, 10.4 Hz, 2a-H), 3.95 (dd, 1H, J = 4.4, 7.1 Hz, 3b-H),
(Cm C6H4), 116.5 (Co C6H4), 108.1 (1b-C), 99.2 (1a-C), 83.0 (4b- 3.92–3.87 (m, 1H, 5a-H), 3.85 (dd, 1H, J = 3.7, 7.1 Hz, 4b-H),
C), 81.5 (2a-C), 81.1 (2b-C), 76.6 (3b-C), 76.0 (5a-C), 72.8 (3a-C), 3.72–3.67 (m, 1H, 5b-H), 3.57–3.50 (m, 4H, 6a-H, 6′a-H, 6b-H,
70.6 (5b-C), 67.7 (4a-C), 62.8 (6b-C), 60.5 (6a-C) ppm. HRMS 6′b-H) ppm; 13C NMR (100 MHz, D2O): δ = 161.6 (Cipso C6H4),
(ESI): m/z calcd for C18H25O13NNa [M + Na]+ 486.12181; found 142.7 (Cp C6H4), 126.2 (Cm C6H4), 116.8 (Co C6H4), 108.9 (1b-
486.12171.
C), 96.6 (1a-C), 82.2 (4b-C), 81.4 (2b-C), 76.1 (3b-C), 75.6 (4a-C),
p-Nitrophenyl β-D-galactofuranosyl-(1→6)-β-D-glucopyrano- 71.9 (5a-C), 70.1 (5b-C), 69.6 (3a-C), 67.8 (2a-C), 62.7 (6b-C),
side (13). This compound was synthesized as described in the 61.3 (6a-C) ppm. HRMS (ESI): m/z calcd for C18H25O13NNa
general procedure using 1 (20 mg, 66 μmol), pNP β-D-gluco- [M + Na]+ 486.12181; found 486.12161.
pyranoside 3 (200 mg, 660 μmol), and 4800 U of Araf51. The
p-Nitrophenyl β-D-galactofuranosyl-(1→4)-α-D-galactopyrano-
desired disaccharide 13 was isolated in 30% yield (9.2 mg). side (16). This compound was synthesized according to the
TLC: Rf = 0.4 (AcOEt–AcOH–H2O, 7/2/2). 1H NMR (600 MHz, described general procedure starting from 1 (20 mg, 66 μmol),
D2O): δ = 8.22 (d, 2H, J = 9.1 Hz, Hm, C6H4), 7.21 (d, 2H, J = 9.1 pNP α-D-galactopyranoside 4 (200 mg, 660 μmol), 4800 U of
Hz, Ho, C6H4), 5.22 (d, 1H, J = 2.7 Hz, 1a-H), 4.94 (d, 1H, J = Araf51, and was isolated in regioisomeric mixture with 15 in
1.5 Hz, 1b-H), 4.04–3.97 (m, 2H, 6a-H, 2b-H), 4.98 (dd, 1H, J = 32% yield (9.9 mg). According to the 1H NMR signal inte-
3.4, 6.2 Hz, 3b-H), 3.90–3.86 (m, 1H, 4b-H), 3.80–3.71 (m, 3H, gration, this compound was obtained in 14% yield. TLC: Rf =
5a-H, 5b-H, 6′a-H), 3.57–3.48 (m, 5H, 3a-H, 2a-H, 6b-H, 6′b-H, 0.5 (AcOEt–AcOH–H2O, 7/2/2). 1H NMR (400 MHz, D2O): δ =
4a-H) ppm; 13C NMR (125 MHz, D2O): δ = 161.6 (Cipso C6H4), 8.19 (d, 2H, J = 9.2 Hz, Hm, C6H4), 7.23 (d, 2H, J = 9.2 Hz, Ho,
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C6H4), 5.75 (d, 1H, J = 3.2 Hz, 1a-H), 5.15 (d, 1H, J = 1.8 Hz, 1b- described general procedure by incubation of 1 (20 mg,
H), 4.10 (dd, 1H, J = 1.8, 3.6 Hz, 2b-H), 4.09–4.02 (m, 2H, 4a-H, 66 μmol) with of pNP α-D-mannopyranoside 6 (200 mg,
3a-H), 4.03 (dd, 1H, J = 3.3, 10.4 Hz, 2a-H), 3.96 (ddd, 1H, J = 660 μmol) in the presence of 4800 U of Araf51, and was iso-
1
0.9, 3.4, 6.7 Hz, 3b-H), 3.94 (dd, 1H, J = 4.0, 6.6, 4a-H), lated in 15% yield (4.6 mg). H NMR (600 MHz, D2O): δ = 8.22
3.90–3.86 (m, 1H, 5a-H), 3.74–3.68 (m, 1H, 5b-H), 3.58–3.52 (d, 2H, J = 9.3 Hz, Hm, C6H4), 7.23 (d, 2H, J = 9.3 Hz, Ho,
(m, 4H, 6a-H, 6′a-H, 6b-H, 6′b-H) ppm; 13C NMR (100 MHz, C6H4), 5.71 (d, 1H, J = 1.7 Hz, 1a-H), 4.91 (d, 1H, J = 1.7 Hz, 1b-
D2O): δ = 161.6 (Cipso C6H4), 142.7 (Cp C6H4), 126.2 (Cm H), 4.14 (dd, 1H, J = 1.7, 3.4 Hz, 2a-H), 3.99 (dd, 1H, J = 3.4, 9.1
C6H4), 116.8 (Co C6H4), 109.2 (1b-C), 96.6 (1a-C), 82.7 (4b-C), Hz, 3a-H), 3.91–3.82 (m, 3H, 5a-H, 2b-H, 3b-H), 3.72–3.62 (m,
81.4 (2b-C), 76.7 (3b-C), 72.0 (3a-C), 71.9 (5a-C), 71.9 (5a-C), 3H, 4a-H, 6a-H, 6a′-H), 3.62–3.56 (m, 1H, 5b-H), 3.52–3.44 (m,
70.6 (5b-C), 66.9 (2a-C), 62.6 (6b-C), 60.8 (6a-C) ppm. HRMS 3H, 4b-H, 6b-H, 6a′-H) ppm. 13C NMR (125 MHz, D2O): δ =
(ESI): m/z calcd for C18H25O13NNa [M + Na]+ 486.12181; found 160.7 (Cipso C6H4), 142.0 (Cp C6H4), 126.0 (Cm C6H4), 116.8
486.12162.
(Co C6H4), 108.1 (1b-C), 97.6 (1a-C), 82.5 (4b-C), 81.1 (2b-C),
p-Nitrophenyl β-D-galactofuranosyl-(1→2)-β-D-galactopyrano- 76.5 (3b-C), 73.2 (4a-C), 70.5 (5b-C), 70.2 (3a-C), 69.6 (2a-C),
side (17). The disaccharide was synthesized as described in 67.3(6a-CH2), 66.7 (5a-C), 62.8 (6b-CH2) ppm.
the general procedure incubating furanosyl donor 1 (20 mg,
p-Nitrophenyl β-D-galactofuranosyl-(1→4)-β-D-mannopyrano-
66 μmol) with pNP β-D-galactopyranoside 5 (200 mg, 660 μmol) side (20). This compound was obtained according to the
in the presence of 4800 U of Araf51, and was isolated in 27% general procedure by incubation of 1 (10 mg, 33 μmol) with
yield (8.1 mg). TLC: Rf = 0.4 (AcOEt–AcOH–H2O, 7/2/2). 1H mannopyranosidic acceptor 7 (100 mg, 330 μmol) in the pres-
NMR (400 MHz, D2O): δ = 8.18 (d, 2H, J = 9.2 Hz, Hm, C6H4), ence of 2400 U of Araf51, and was isolated in 49% yield
7.17 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.25 (d, 1H, J = 1.5 Hz, (7.5 mg). TLC: Rf = 0.49 (AcOEt–AcOH–H2O, 7/2/2). 1H NMR
1b-H), 5.24 (d, 1H, J = 7.5 Hz, 1a-H), 4.05 (dd, 1H, J = 2.9, 1.5 (400 MHz, D2O): δ = 8.16 (d, 2H, J = 9.2 Hz, Hm, C6H4), 7.13 (d,
Hz, 2b-H), 3.95 (dd, 1H, J = 2.9, 5.8 Hz, 3b-H), 3.96–3.91 (m, 2H, J = 9.2 Hz, Ho, C6H4), 5.43 (s, 1H, 1a-H), 5.02 (d, 1H, J =
1H, 5a-H), 3.91 (dd, 1H, J = 4.1, 5.8 Hz, 4b-H), 3.88 (dd, 1H, J = 1.80 Hz, 1b-H), 4.18 (d, 1H, J = 2.6 Hz, 2a-H), 4.06–4.03 (m, 2H,
7.5, 9.7 Hz, 2a-H), 3.84–3.78 (m, 2H, 3a-H, 4a-H), 3.73–3.67 (m, 4b-H, 3b-H), 4.02 (d, 1H, 2b-H), 3.87 (dd, 1H, J = 1.9, 12.2 Hz,
3H, 5b-H, 6a-H, 6′a-H), 3.45 (dd, 1H, J = 4.4, 11.7 Hz, 6b-H), 6a-H), 3.79 (dd, 1H, J = 2.6, 9.2 Hz, 3a-H), 3.80–3.76 (m, 1H,
3.39 (dd, 1H, J = 7.3, 11.7 Hz, 6′b-H) ppm. 13C NMR (100 MHz, 4a-H), 3.75–3.73 (m, 1H, 5b-H), 3.72 (dd, 1H, J = 4.5, 12.2 Hz,
D2O): δ = 161.9 (Cipso C6H4), 142.6 (Cp C6H4), 125.9 (Cm 6′a-H), 3.63 (ddd, 1H, J = 1.9, 5.4, 9.5 Hz, 5a-H), 3.60 (d, 1H, J =
C6H4), 116.4 (Co C6H4), 108.4 (1a-C), 98.5 (1b-C), 83.5 (4a-C), 6.7, 11.9 Hz, 6b-H), 3.58 (dd, 1H, J = 4.1, 11.9 Hz, 6′b-H) ppm.
81.0 (2a-C), 76.7 (3a-C), 76.4 (2b-C), 75.5 (4b-C), 72.6 3b-C), 13C NMR (100 MHz, D2O): δ = 161.5 (Cipso C6H4), 142.4 (Cp
70.8 (5a-C), 68.4 (5b-C), 62.6 (6a-C), 60.6 (6b-C) ppm. HRMS C6H4), 126.1 (Cm C6H4), 116.2 (Co C6H4), 108.0 (1b-C), 97.0
(ESI): m/z calcd for C18H25O13NNa [M + Na]+ 486.12181; found (1a-C), 82.8 (3b-C), 81.0 (4b-C), 76.0 (2b-C), 74.6 (4a-C), 71.2
486.12190.
(3a-C), 70.5 (5b-C), 70.1 (2a-C), 62.6 (6b-C), 61.1 (5a-C), 60.3
p-Nitrophenyl β-D-galactofuranosyl-(1→6)-β-D-galactopyrano- (6a-C) ppm. HRMS (ESI): m/z calcd for C18H25O13NNa [M +
side (18). This compound was prepared according to the Na]+ 486.12181; found 486.12204.
described general procedure by incubation of 1 (20 mg,
p-Nitrophenyl β-D-galactofuranosyl-(1→6)-β-D-mannopyrano-
66 μmol) with of pNP β-D-galactopyranoside 5 (200 mg, side (21). This compound was prepared according to the
660 μmol) in the presence of 4800 U of Araf51, and was iso- described general procedure by incubation of 1 (20 mg,
lated in 25% yield (7.6 mg). TLC: Rf = 0.4 (AcOEt–AcOH–H2O, 66 μmol) with of pNP α-D-mannopyranoside 6 (200 mg,
7/2/2). 1H NMR (400 MHz, D2O): δ = 8.18 (d, 2H, J = 9.2 Hz, 660 μmol) in the presence of 4800 U of Araf51, and was iso-
1
Hm, C6H4), 7.17 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.12 (d, 1H, J = lated in 15% yield (4.9 mg). H NMR (600 MHz, D2O): δ = 8.20
7.6 Hz, 1a-H), 4.90 (dd, 1H, J = 1.2, 0.6 Hz, 1b-H), 4.00 (ddd, (d, 2H, J = 9.3 Hz, Hm, C6H4), 7.17 (d, 2H, J = 9.3 Hz, Ho,
1H, J = 0.9, 3.8, 8.4 Hz, 4a-H), 3.97 (d, 1H, J = 1.2 Hz, 2b-H), C6H4), 5.46 (br s, 1H, 1a-H), 4.95 (d, J = 4.4 Hz, 1H, 1b-H), 4.18
3.96 (ddd, 1H, J = 3.3, 0.6 Hz, 3b-H), 3.93 (dd, 1H, J = 0.9, 3.4 (d, 1H, J = 3.0 Hz, 2a-H), 4.00–3.00 (m, 2H, 5a-H, 2b-H), 3.97
Hz, 4a-H), 3.91 (dd, 1H, J = 1.5, 4.4 Hz, 4b-H), 3.83 (dd, 1H, J = (dd, 1H, J = 3.5, 6.0 Hz, 3b-H), 3.91 (dd, 1H, J = 4.3, 6.0 Hz, 4b-
3.8, 11.6 Hz, 6a-H), 3.76 (dd, 1H, J = 7.6, 9.9 Hz, 2a-H), H), 3.76–3.63 (m, 5H, 3a-H, 4a-H, 5b-H, 6a-H, 6a′-H), 3.60–3.53
3.73–3.72 (m, 1H, 5b-H), 3.71 (dd, 1H, J = 8.4, 11.6 Hz, 6′a-H), (m, 2H, 6b-H, 6b′-H) ppm. 13C NMR (125 MHz, D2O): δ = 161.5
3.70 (dd, 1H, J = 3.4, 9.9 Hz, 3a-H), 3.63–3.57 (m, 1H, 6b-H), (Cipso C6H4), 142.5 (Cp C6H4), 126.1 (Cm C6H4), 116.3
3.56–3.51 (m, 1H, 6′b-H) ppm. 13C NMR (100 MHz, D2O): δ = (Co C6H4), 107.9(1b-C), 97.0 (1a-C), 83.1 (4b-C), 80.9 (2b-C),
161.6 (Cipso C6H4), 142.7 (Cp C6H4), 126.1 (Cm C6H4), 116.5 76.8 (3b-C), 75.5 (4a-C), 72.5 (3a-C), 70.8 (5b-C), 70.2 (2a-C),
(Co C6H4), 107.8 (1b-C), 99.8 (1a-C), 83.1 (4b-C), 82.0 (2b-C), 66.6 (5a-C), 66.6 (6a-CH2), 62.7 (6b-CH2) ppm.
76.7 (3b-C), 74.5 (5a-C), 72.3 (3a-C), 70.7 (5b-C), 70.2 (2a-C),
p-Nitrophenyl β-D-galactofuranosyl-(1→4)-α-L-rhamnopyra-
68.5 (4a-C), 66.9 (6a-C), 62.5 (6b-C) ppm. HRMS (ESI): m/z noside (22). This compound was obtained according to the
calcd for C18H25O13NNa [M
486.12172.
+
Na]+ 486.12181; found described general procedure by incubation of pNP β-D-galacto-
furanoside 1 (10 mg, 33 μmol) with pNP α-L-rhamnopyranoside
p-Nitrophenyl β-D-galactofuranosyl-(1→6)-α-D-mannopyrano- 8 (100 mg, 330 μmol) in the presence of 2400 U of Araf51, and
side (19). This compound was prepared according to the was isolated in 38% yield (5.5 mg). TLC: Rf = 0.5 (AcOEt–
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1
AcOH–H2O, 7/2/2). H NMR (400 MHz, D2O): δ = 8.15 (d, 2H,
J = 9.2 Hz, Hm, C6H4), 7.13 (d, 2H, J = 9.2 Hz, Ho, C6H4), 5.55
(d, 1H, J = 1.2 Hz, 1a-H), 5.21 (d, 1H, J = 1.7 Hz, 1b-H), 4.06
(dd, 1H, J = 1.2, 3.4 Hz, 2a-H), 4.04 (dd, 1H, J = 1.8, 3.9 Hz, 2b-
H), 4.04–4.00 (m, 1H, 3a-H), 3.97 (dd, 1H, J = 3.9, 6.3 Hz,
3b-H), 3.87 (dd, J = 3.8, 6.3 Hz, 1H, 4b-H), 3.72 (ddd, J = 4.4,
4.5, 7.2 Hz, 1H, 5b-H), 3.58 (dd, 1H, J = 4.4, 11.6 Hz, 6b-H),
3.59–3.55 (m, 1H, 5a-H), 3.57–3.53 (m, 1H, 4a-H), 3.54 (dd, 1H,
J = 7.2, 11.6 Hz, 6′b-H), 1.11 (d, 1H, J = 5.7 Hz, CH3) ppm. 13C
NMR (100 MHz, D2O): δ = 161.3 (Cipso C6H4), 142.0 (Cp C6H4),
125.8 (Cm C6H4), 116.2 (Co C6H4), 108.4 (1b-C), 97.5 (1a-C),
82.7 (4b-C), 81.5 (2b-C), 77.8 (4a-C), 70.4 (5b-C), 70.3 (3a-C),
69.9 (2a-C), 68.2 (5a-C), 62.7 (6b-C), 17.1 (6a-C) ppm. HRMS
(ESI): m/z calcd for C18H25O12NNa [M + Na]+ 470.12690; found
470.12665.
Notes and references
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