Pseudoenantiomeric glycoclusters: Synthesis and testing of heterobivalency in carbohydrate-protein interactions

Article abstract of DOI:10.1039/c9ob00124g

Multivalent carbohydrate-protein interactions are key events in cell recognition processes and have been extensively studied by means of synthetic glycomimetics. To date, frequently the valency, i.e. the multiplicity of the ligand attached to a polyvalent

Full text of DOI:10.1039/c9ob00124g

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Article
Pseudoenantiomeric glycoclusters: Synthesis and testing of
heterobivalency in carbohydrate-protein interactions
Jasna Brekalo, Guillaume Despras* and Thisbe K. Lindhorst*
Received 00th January 20xx,
Accepted 00th January 20xx
Multivalent carbohydrate-protein interactions are key events in cell recognition processes and have been extensively
studied by the means of synthetic glycomimetics. To date, frequently the valency, i.e. the multiplicity of the ligand attached
to a polyvalent scaffold, has been considered in the design of multivalent structures but these studies have not led to a
conclusive understanding of glycan recognition at the molecular level. In this work, we add a new aspect to carbohydrate-
lectin recognition studies by designing the first heterobivalent diastereomeric glycoclusters in order to investigate the
influence of both, heteromultivalency and of relative ligand orientation. Two enantiomeric scaffolds, derived from D- and L-
serine, respectively, were glycosylated with two distinct carbohydrate ligands to obtain a library of pseudoenantiomeric
glycoclusters. They all have an α-D-mannosyl residue in common as a specific ligand for the lectins FimH and ConA, while
they differ in the second carbohydrate portion, consisting of a β-D-glucosyl, a β-D-galactosyl or a β-D-glucosaminyl residue
as unspecific ligands. The synthesised heteroclusters were tested in standard binding-inhibition assays investigating FimH-
mediated bacterial adhesion and ConA binding to mannosylated surfaces. A striking difference was observed between the
potencies of the two pseudoenantiomeric glucose-containing glycoclusters as inhibitors of FimH-mediated bacterial
adhesion. For the other diastereomeric glycocluster pairs smaller inhibitory potency differences were detected in the
bacterial adhesion assay. In contrast, the assays with ConA showed no significant variation for all tested clusters pairs. The
results obtained with the diastereomeric glucose-mannose glycocluster pair were rationalised by molecular docking. Binding
energies for the FimH carbohydrate recognition domain were calculated for both diastereomers and are in agreement with
experimental data obtained in the bacterial adhesion assays.
DOI: 10.1039/x0xx00000x
www.rsc.org/
affinity interactions in a cooperative fashion. These avidity or
5
cluster effects, respectively, have long been known and have
been exploited extensively, in particular employing synthetic
Introduction
Cell surface carbohydrates play an essential role in key cellular polyvalent glycomimetics. These studies have advanced our
communication processes, which are, i.a., mediated by understanding of carbohydrate-protein interactions and also
carbohydrate-lectin interactions. These include signalling, cell have opened the door to the application of carbohydrates as
1
6
trafficking and cell adhesion. In particular, the adhesion of antiadhesives to treat infectious diseases.
pathogens to tissues, an initial step in infection, is often
However, multivalency effects in carbohydrate recognition
mediated by the specific recognition of host cell are not conclusively understood as yet. While some of the
2
glycoconjugates by pathogen adhesins (lectins). For instance, synthetic multivalent glycomimetics were able to bind lectins
the adhesion of uropathogenic E. coli bacteria (UPEC) to the with high avidity (up to the picomolar range),
7
others
endothelial cells of the host organism occurs through binding of unexpectedly exhibited poor binding ability despite of a large
α-mannosyl residues by the bacterial lectin FimH, which is number of displayed ligands.3b,6d In light of these contrasting
3
located at the tips of so-called type 1 fimbriae. Fimbriae are results, it was important to investigate multivalency effects in
adhesive organelles, multiple projected from the bacterial cell an extended and advanced way than only taking into account
surface.4 Hence, carbohydrate-specific bacterial adhesion the multiplicity of the sugar epitope. For instance, proper
clearly relies on multivalent interactions between cell surfaces. distances between ligands, their spatial arrangement as well as
As manifold copies of a variety of carbohydrate epitopes are the flexibility of the linker moieties are important factors and
8
expressed on cell surfaces, multivalent interactions with lectins, have been investigated in several studies.
In order to test more
9
which often possess several recognition domains, leads to complex glycoconjugates which are structurally closer to the
strong binding as a result of a proper combination of single low- heterogeneous sugar coat of eukaryotic cells (glycocalyx),
García-Fernández and co-workers prepared heteroglycoclusters
and evaluated their glycobiology. Strikingly, these compounds
Christiana Albertina University of Kiel, Otto Diels Institute of Organic Chemistry,
Otto-Hahn-Platz 3-4, D-24118, Kiel, Germany.
E-mails: gdespras@oc.uni-kiel.de; tklind@oc.uni-kiel.de.
Electronic Supplementary Information (ESI) available: Synthetic procedures, NMR
spectra, biological testing with inhibition curves, and molecular modelling data.
See DOI: 10.1039/x0xx00000x
showed a remarkable "heterocluster effect” towards binding to
lectins such as Concanavalin A (ConA) and peanut butter
agglutinin (PNA), in comparison with their counterparts
†
1
0
displaying only the specific lectin ligand. With these findings,
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Org. Biomol. Chem, 2018, 00, 1-3 | 1
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a new thinking about multivalency effects in carbohydrate βGlcNAc-Man. In order to have a bivalent reference
View Article Online
1
1
DOI: 10.1039/C9OB00124G
recognition has begun.
Further, some reports showed that the presentation mode homobivalent mannose glycocluster (αMan)
of carbohydrate ligands on a surface is also critical for proper symmetrical diol 2-azido-propanediol (Scheme 1). As our
compound in hand, we also prepared the respective
2
from the
1
2
recognition by lectins. Among others, our group showed that synthetic approach is based on enantiomeric bivalent scaffold
the specific recognition of α-D-mannosyl residues by the lectin molecules from the chiral pool, it is highly flexible and allows for
FimH is clearly impacted by the orientation of the sugar the rapid preparation of libraries of "mirror image"
epitopes. This was demonstrated by using photoswitchable glycoclusters, without the need of separation of diastereomers
azobenzene mannosides attached onto gold surfaces during the synthesis. The employed carbohydrates, −mannose,
(
glycoSAMs) or the surface of human cells to systematically re- glucose, galactose, and GlcNAc−, were selected for their
1
2b,c
orient sugar ligands.
biological relevance as lectin ligands. All cluster glycosides
Based on the reported findings, we believe that prepared herein were thus tested in binding studies, on the one
1
4
multivalency effects in carbohydrate recognition strongly hand with the plant lectin ConA and on the other hand in
depend on the three-dimensional organization of carbohydrate bacterial adhesion studies with live type 1-fimbriated E. coli
1
3
ligands and glycoepitopes, respectively. In order to further bacteria, where carbohydrate binding is mediated by the
3
investigate how the relative positioning of ligands governs sugar adhesin FimH.
recognition, we approached a new design of heterobivalency in
which two different sugars are arranged on enantiomeric
scaffold molecules. This leads to a pair of diastereomeric
Results and Discussion
glycoclusters, which can be regarded as pseudoenantiomers
with respect to the configuration at the focal point of the
scaffold moiety (Figure 1A). Hence, the sugar ligands conjugated
in a particular bivalent heterocluster are displayed in different
relative orientation. We targeted three principal heterobivalent
pseudoenantiomeric glycocluster pairs, combining -D-
glucopyranosyl (Glc), -D-galactopyranosyl (Gal) and -D-
glucosaminyl (GlcNHR) moieties, respectively, with an α-D-
mannopyranoside (Figure 1B).
The diastereomeric pairs of heterobivalent glycoclusters are
based on scaffold molecules derived from D-and L-serine,
respectively. These enantiomeric α-amino acids were converted
into the respective mono-protected 2-azido-propanediol
derivatives (Figure 1C) and submitted to two sequential
glycosylation reactions, employing suitable glycosyl donors to
achieve the target glycoclusters βGlc-Man, βGal-Man, and
Synthesis
At first, we required the homobivalent mannoside cluster 6
(Scheme 1) as a reference compound for later testing of
heterobivalency effects. The synthesis of 6 was accomplished in
three steps starting from serinol (1), which was converted into
the respective azido-functionalised diol 3 with imidazole
1
5
1
6
sulfonyl azide (2) in 72 % yield. The bivalent scaffold 3 was
then glycosylated using the known thiomannoside 4 as glycosyl
1
7
donor. The mannosyl donor 4 was activated at -77 °C with N-
1
8
iodosuccinimide (NIS) and a catalytic amount of triflic acid.
Then, the reaction mixture was allowed to warm to room
temperature over three hours to provide the benzoyl-protected
bivalent cluster mannoside 5 in 60 % yield after work up and
purification. Deprotection of 5 with sodium methoxide in
methanol gave the pure target molecule 6.
Pseudoenantiomers
Diastereomeric pairs of heterodivalent cluster glycosides
A
B
OH
OH
OH
O
O
HO
HO
HO
HO
HO
HO
OH
O
OH
O
OH
O
O
O
OH
OH
Gal-Man

Glc-Man
GlcNHR-Man
HO
HO
O
O
O
O
O
*
*
N3 N3
O
O
O
O
HO
HO
HO
HO
O
O
* N3
* N3
*
N3
HO
OH
N3
PGO
OH
OH
NHR
C
D- and
L-serine
R = Ac or TFA
OH
N3
PGO
+
Figure 1 Pseudoenantiomeric cluster glycosides (A) were targeted, comprising two different carbohydrate moieties (black and white chairs), and differing in the configuration at
the focal point stereocenter (*) of the scaffold moiety. Four diastereomeric pairs of hetereobivalent glycoclusters were synthesised (B), Glc-Man, Gal-Man, GlcNHR-Man,
together with the analogous homobivalent mannoside cluster (Man)
2
for comparison (not shown, cf. Scheme 1). The diastereomeric glycoclusters were built on mono-protected
2
-azido-propanediol enantiomers (C), which were derived from D- and L-serine as enantiopure chiral pool scaffold molecules. TFA = trifluoroacetyl; PG = protecting group.
2
| J. Name., 2012, 00, 1-3
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ARTICLE
O
O
tried to reduce the methyl esters with lithiumViewa micleinium
A Online
lurt
DOI: 10.1039/C9OB00124G
OH
N
N
S
N3
OH
N3
hydride, but remarkably, this gave only poor yields. The primary
alcohols 9a and 9b were further used as acceptor molecules in
the first glycosylation step. However, we found that the N-Boc
protecting group was labile under the acidic conditions required
for the glycosylation reaction, whereas under the same
conditions the TBDPS group was stable. Therefore, compounds
9a and 9b were first treated with trifluoroacetic acid in order to
cleave the N-Boc group and the resulting free amines were
converted into the respective azides in a diazo transfer reaction
2
HO
HO
NH2
CuSO4, K2CO3
MeOH, CH2Cl2, H2O
2 %
1
3
OBz
OBz
7
O
BzO
BzO
NIS, TfOH
CH2Cl2
°
S
4
3
-
A MS
77 °C to rt
OR
RO
RO
OR
OR
O
16
employing 2 under similar conditions as described above. The
two enantiomeric scaffold molecules 10a and 10b were isolated
in 80 % and 79 %, respectively, over two steps. Notably, no
racemization was observed in any of the described steps.
With the enantiomeric building blocks 10a and 10b in hand,
the first glycosylation reaction was carried out to obtain the
pivotal mono-mannosylated acceptor molecules as precursors
for the synthesis of all bivalent heteroclusters. As expected, the
glycosylation of 10a and 10b proceeded much better than when
the carbamates 9a and 9b were employed. In first
mannosylation attempts, the well-known tetraacetylated α-D-
5
6
R = Bz (60 %)
R = H (70 %)
OR
O
NaOMe
MeOH
RO
RO
O
O
N3
Scheme 1 Synthesis of the homobivalent cluster mannoside 6.
The synthesis of the targeted diastereomeric pairs of
heterobivalent cluster glycosides (Figure 1B) was based on D-
and L-serine, 7a and 7b, respectively, as enantiopure chiral pool
compounds (Scheme 2). First of all, the -amino function of
each enantiomeric amino acid was Boc-protected under
standard conditions,19 and the resulting crude material directly
2
2
mannosyl trichloroacetimidate was used as the glycosyl
donor. However, the resulting glycosides were isolated in only
unsatisfying yields (36 to 56 %) which we could not further
optimise. This was due to acetyl migration resulting in
converted into the respective methyl ester in the presence of
iodomethane and potassium carbonate. The enantiomeric
serine derivatives 8a and 8b were thus received in 79 and 82 %
2
3
acetylation of the acceptor alcohol. Thus, we used the
1
7
benzoylated thiophenyl mannoside 4, as benzoyl groups are
2
4
2
0
less prone to acyl migration under acidic conditions. The
thioglycoside 4 was again activated under standard conditions
employing NIS and triflic acid to provide the mannosides 11a
and 11b in very satisfying yields around 80 %. Then, the
subsequent desilylation with TBAF (n-tetrabutylammonium
fluoride) afforded compounds 12a and 12b in good yields,
hence setting a further alcohol group available for the second
glycosylation step.
respective yield over two steps. In order to block the primary
alcohol of the serine scaffold, the tert-butyldiphenylsilyl
TBDPS) ether was used and introduced with tert-
butyldiphenylsilyl chloride and imidazole. The resulting crude
silyl ethers were carried on in the next reduction step with
lithium borohydride and after purification, the alcohols 9a and
(
2
1
9
b were both obtained in 90 % yield over two steps. We also
OH
OH
OTBDPS
* NHBoc
OTBDPS
N3
1
. K CO Boc O
dioxane
1. TBDPSCl, imidazole
CH Cl 0 °C to rt
2
3,
2
HO
H CO
2
2,
HO
1. TFA, CH Cl
2 2
HO
*
3
* NHBoc
*
NH2
2. K CO CH3I
2
3,
2
. LiBH , THF
2. 2, CuSO , K CO
4 2 3
MeOH, CH Cl , H O
2 2 2
O
O
4
DMF, 0 °C to rt
0
°C to rt
7
a (D-serine)
b (L-serine)
8a (R) (79 %)
8b (S) (82 %)
9a (S) (90 %)
10a (S) (80 %)
10b (R) (79 %)
7
9b (R) (90 %)
OBz
1
1a (S) R = TBDPS (85 %)
OBz
O
4
, NIS, TfOH, CH Cl
2
2
1
1b (R) R = TBDPS (80 %) BzO
TBAF, HOAc, THF
OR
N3
°
4
A MS, 0 °C to rt
BzO
0
°C to rt
1
2a (R) R = H (74 %)
2b (S) R = H (76 %)
O
*
1
Scheme 2 Synthesis of a diastereomeric pair of mannosides, derived from D- and L-serine as enantiopure scaffold molecules from the chiral pool. The obtained mannosides 12a and
1
2b were needed as acceptor alcohols for all following glycosylation reactions (cf. Scheme 3).
We targeted three principal diastereomeric pairs of pairs βGlc-αMan and βGal-αMan were obtained employing the
1
7
heterobivalent cluster glycosides (Figure 1B). For this, we used benzoyl-protected thioglucoside (13) and the respective
glucose, galactose and GlcNAc donors for the glycosylation of thiogalactoside donor (16) (Scheme 3). The glycosylation
the mannosides 12a and 12b. The protected diastereomeric reactions proceeded efficiently, furnishing 14a/b and 17a/b in
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yields from 61 to 71 %. Deprotection under Zemplén Zemplén deprotection delivered the diastereomeric βGlcNTFA-
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conditions25 provided the final diastereomeric cluster pairs αMan pair 21a/21b. In order to achieve the respective βGlcNAc-
DOI: 10.1039/C9OB00124G
1
5a/b and 18a/b, respectively, in very good to excellent yields αMan pair, the N-trifluoroacetyl (TFA) derivatives were cleaved
2
8
after purification by size exclusion chromatography.
with aqueous lithium hydroxide and subsequent N-acetylation
For the synthesis of the βGlcNAc-αMan glycocluster, the in methanol under basic conditions afforded compounds 22a
2
6
acetylated trifluoromethyl oxazoline 19, derived from D- and 22b (Scheme 3). Both heterobivalent diastereomeric pairs
glucosamine, was used as the donor in a glycosylation reaction involving glucosamine, 21a/21b (βGlcNTFA-αMan) and 22a/22b
promoted by
a
catalytic amount of trimethylsilyl (βGlcNAc-αMan), were used in lectin binding studies (see
2
7
trifluoromethanesulfonate at room temperature. The - below).
configured glycosides 20a and 20b were isolated in good yields
and no α-anomer was obtained. The following standard
OR
OR
RO
RO
OBz
O
OBz
O
RO
RO
OR
O
BzO
BzO
OR
O
BzO
BzO
S
S
OR
OR
OBz
OBz
RO
O
O
1
6
13
1
2a
2b
O
RO
RO
O
O
1
O
* N3
RO
* N
TfOH, NIS, CH Cl
2
2
TfOH, NIS, CH Cl
2
2
3
°
°
OR
OR
3
A MS, 0 °C to rt
3 A MS, 0 °C to rt
1
7a (r) R = Bz (64 %)
7b (s) R = Bz (61 %)
OAc
O
14a (r) R = Bz (73 %)
14b (s) R = Bz (71 %)
1
NaOMe
MeOH
AcO
AcO
TMSOTf, CH Cl
NaOMe
MeOH
2
2
°
3A MS, rt
1
8a (r) R = H (97 %)
8b (s) R = H (85 %)
15a (r) R = H (95 %)
15b (s) R = H (89 %)
N
O
1
F3C
1
9
0a (R) R1 = Bz, R2 = Ac, R = TFA (72 %)
3
2
OR1
OR1
1
20b (S) R1 = Bz, R2 = H, R = TFA (75 %)
3
R O
1
NaOMe
MeOH
R O
O
2
2
1a (R) R1 = R2 = H, R3 = TFA (98 %)
1b (S) R1 = R2 = H, R3 = TFA (100 %)
OR2
O
O
(i) aq. LiOH
(ii) Ac2O,
NaOMe,
MeOH
2
R O
2
O
* N
3
R O
NHR3
22a (R) R1 = R2 = H, R3 = Ac (60 %)
2b (S) R1 = R2 = H, R3 = Ac (58 %)
2
Scheme 3 Scheme 3 Synthesis of four diastereomeric pairs of heterobivalent glycoclusters. In the diastereomeric pairs Glc-Man and Gal-Man, the focal point represents a
pseudoasymmetric centre as the two attached sugar moieties only differ in their configuration (Glc, Man, and Gal have the same constitution). Thus, the configuration at the focal
point is assigned by using small letters, r and s. Otherwise, the two diastereomeric pairs GlcNHAc-Man and GlcNHTFA-Man comprise sugars of different mass and hence
capitalised descriptors R and S are used to assign the configuration at the focal point. (Note, that according to IUPAC, the diastereomeric pairs comprising mannosyl and glucosaminyl
residues cannot be called “pseudoenantiomeric“.) Please note further that for assigning the CIP priorities of the sugar moieties, their anomeric configuration is decisive, with a sugar
with anomeric (R)-configuration having a higher priority than its isomer with anomeric (S)-configuration. CIP = Cahn-Ingold-Prelog.
Biological Testing
compound reflects the concentration at which bacterial or ConA
binding is reduced by 50% and thus correlates with its inhibitory
potency. Bacterial binding (adhesion) and ConA binding to the
surface were measured by fluorescence read-out. For this, we
used the E. coli strain PKL1162, expressing the green fluorescent
The synthesised bivalent glycoclusters were tested in solution
as inhibitors of the adhesion of type 1 fimbriae-mediated E. coli
bacteria and of binding of ConA, respectively, to a mannan-
coated microtiter plate surface. The type 1-fimbrial lectin is
FimH, exhibiting a pronounced specificity for α-D-mannosyl
3
0
protein (GFP), and fluorescein-labelled ConA. In all assays,
methyl α-D-mannopyranoside (MeMan) was tested as reference
inhibitor on the same plate in order to allow the quantitative
comparison of all tested cluster glycosides, even when tested in
different experiments. Hence, we report relative inhibition
potency (RIP) values which are all related to the inhibitory
potency of MeMan, tested in the same experiment. We also
compared the various heteroclusters with the homobivalent
mannoside 6, tested on the same microplate, to observe
3
residues. ConA on the other hand, recognises α-D-mannosides
2
9
as well as α-D-glucosides. Depending on the pH, ConA exists in
two forms, as a dimer (at pH ≤ 5.6) or as a tetramer (at pH 5.8
1
4
to 7). Accordingly, in the assay performed here, ConA is tested
as a tetramer.
In the employed assays, a soluble inhibitor has to compete
with the bacterial lectin FimH or ConA, respectively, for binding
to a mannosylated (mannan-coated) surface. Using 96-well
plates, serial dilutions of each tested inhibitor were applied in
order to measure dose-response inhibition curves (cf. ESI) from
which IC50 values can be determined. The IC50 value of a specific
1
0
whether a heteromultivalency effect occurs. In this respect,
we also report valency-corrected RIP values (RIPvc), where the
determined RIP value is divided by the number of clustered
mannose ligands (two in the case of 6).
4
| J. Name., 2012, 00, 1-3
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The so determined IC50 values are summarised in Table 1 for observed for the galactose-containing clusters 1V8ieaw Aartnicdle O1n8libne,
the inhibition of both, adhesion of type 1-fimbriated E. coli and although their RIP values differ only by aDOfaI:c1t0o.r10o3f9/aCp9pOrBo0x0.1224iGn
ConA binding. The corresponding RIP values are compared in favour of 18b (RIP = 7.97). The N-TFA-β-GlcNAc-containing
Figure 2. Overall, the homobivalent cluster mannoside 6 and glycoclusters 21a and 21b, exhibited a small RIP difference, in
every tested heterobivalent glycocluster show more or less this case in favour of the D-serine-derived cluster 21a (RIP =
pronounced multivalency effects with RIP values higher than 1, 14.89). The GlcNAc-containing clusters 22a/b only displayed
with the exception of the N-GlcNAc-containing glycoclusters weak inhibitory potencies with almost no difference between
2
2a/b as inhibitors of ConA binding (Fig. 2, top chart). As the diastereomers. It might be concluded at this point that the
inhibitors of FimH-mediated bacterial adhesion (Fig. 2, bottom N-trifluoroacetyl group seems to have a beneficial effect on
chart), some of the heterobivalent glycoclusters, 15b, 18b, 21a inhibition of bacterial adhesion.
and 21b, also exhibit a heterocluster effect with RIP values 2- to
6
-fold higher than RIPvc of 6 (always tested in parallel). On the
other hand, in the ConA assay, no significant heterocluster
effects were observed. This is not surprising according to the
results reported by García Fernández et al. with ConA, that
evidenced the heteromultivalency effect occurring only at a
1
0
high density of ligands on the scaffold.
Table 1. IC50 values of synthetic inhibitors of type 1 fimbriae-mediated adhesion of E. coli
cells and of ConA binding, respectively, both to mannan-coated microtiter plates,
employing the E. coli strain GFP-PKL1162 and FITC-labelled ConA.
FITC-ConA
GFP-PKL1162
a
a
Inhibitor
MeMan
IC50 (SD) [mmol]
IC50 (SD) [mmol]
5.55 (±0.48)
4.37 (±0.33)
2.71 (±0.76)
1.62 (±0.12)
4.95 (±1.29)
3.43 (±0.97)
3.88 (±0.92)
1.27 (±0.33)
5.78 (±1.06)
3.60 (±0.21)
3.60 (±0.18)
1.78 (±0.17)
4.76 (±0.36)
6.48 (±0.15)
6.56 (±0.41)
1.77 (±0.10)
11.87 (±0.96)
8.17 (±0.9)


Glc-Man 15a
Glc-Man 15b
0.43 (±0.06)
0.60 (±0.10)
13.31 (±4.00)
3.63 (±0.10)
1.68 (±0.19)
1.54 (±0.33)
6.47 (±1.13)
0.44 (±0.24)
0.57 (±0.19)
1.00 (±0.09)
6.67 (±0.67)
2.31 (±0.88)
2.66 (±0.29)
0.37 (±0.04)
Man)
2
6
MeMan


Gal-Man 18a
Gal-Man 18b
Man)
2
6
MeMan
Figure 2. Relative inhibitory potencies (RIP values) of the tested compounds deduced
from the measured IC50 values as listed in Table 1. Top chart: Inhibition of ConA (FITC-
labelled) binding to mannan; bottom chart: Inhibition of E. coli (GFP-PKL1162) adhesion
to mannan. RIP values are based on the inhibitory potency of methyl α-D-
mannopyranoside (MeMan) tested on the same microplate (MeMan, IP ≡ 1); RIP =


GlcTFA-Man 21a
GlcTFA-Man 21b
Man)
2
6
MeMan
IC50(MeMan)/IC50(tested compound). For the homobivalent cluster mannoside (αMan)
6) also the valency-corrected value RIPvc (6vc) is depicted for comparison.
2


GlcNAc-Man 22a
GlcNAc-Man 22b
(
Man)
2
6
To rationalise the remarkable difference in inhibitory power,
which was seen in the bacterial adhesion assay with the
pseudoenantiomeric heterobivalent glycoclusters 15a and 15b,
we studied their interactions with the bacterial adhesin FimH by
molecular modelling. At this point we concentrated on 15a/b
and did not consider the molecular interactions of the other
pseudoenantiomeric pairs which showed less pronounced
effects.
a
IC50 values are averaged from the mean values obtained in at least two
independent adhesion experiments (cf. ESI). Note, that the IC50 values can vary
significantly in independent experiments as live bacteria are investigated. SD:
standard deviation; GFP: green fluorescent protein; FITC: fluorescein
isothiocyanate.
We were especially excited to test, if the pseudoenantiomeric
pairs of heterobivalent glycoclusters would show any significant
difference in their inhibitory potencies. In the ConA-based
assay, the pseudoenantiomeric glycoclusters performed very
similar or equal. Only some RIP difference was observed with
the βGlc-αMan pair (1.27 and 2.05, respectively, for 15a and
Molecular modelling
We figured that the spatial orientation of the glucosyl residue
in 15a versus 15b might have a significant influence on the
recognition of the bivalent structure by the bacterial adhesin
FimH. To support this assumption, we examined the interaction
of both cluster glycosides with the lectin by molecular modelling
based on force-field methods. We first performed a molecular
docking study involving 15a and 15b and the homobivalent
cluster mannoside 6 for comparison. For docking, Glide was
used, a specific software implemented in the Schrödinger
1
5b). However, a striking difference was indeed observed for
the same βGlc-αMan pair 15a and 15b when tested as inhibitors
of bacterial adhesion. Hence, 15b, based on the L-serine-derived
scaffold, is nearly 20-fold more potent (RIP = 27.44), than its
isomer 15a (RIP = 1.45). We confirmed this exciting result in
3
1
several independent assays (cf. ESI). A similar trend was
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J. Name., 2013, 00, 1-3 | 5
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 6 of 14
ARTICLE
Journal Name
suite.32 FimH has been crystallised in two conformations, a comparing the position of the different ligands bound to FimH
View Article Online
closed" form33 and an "open" one, depending on the (Figure 3). While the α-D-mannosyl residue of each cluster
34
DOI: 10.1039/C9OB00124G
"
orientation of two tyrosine residues (Tyr 48 and Tyr 137) which equally fits into the binding pocket (Figure 3A), there is a clear
flank the carbohydrate recognition domain (CRD) and form the difference in the orientation of the glucoside moiety. In case of
so-called "tyrosine gate". Thus, we used both, the closed and 15b, a stacking of the glucosyl residue with a polar domain of
the open crystal structures of the lectin (pdb codes 1UWF and the CRD is visible and the unpolar azido group points towards
1
KLF, respectively) for docking. Because docking scores do not the tyrosine gate. In case of 15a on the other hand, the azido
generally reflect the affinity of ligands, each docking output was function is further away from the tyrosine gate while the
next submitted to a MM-GBSA calculation (molecular glucoside portion is shifted closer to a hydrophobic domain of
mechanics energies combined with the generalised Born and the CRD.
3
5
surface area continuum solvation) to provide binding
energies.36 These values are more reliable than docking scores
a
b
Table 2. Docking scores and binding energies of the clusters 6, 15a, and 15b in complex
with the open and closed gate conformation of FimH.
3
6
to estimate and compare protein-ligand interactions.
Table 2 depicts the docking score for the clusters 6, 15a and
5b along with the corresponding lowest binding energy for
both the open and the closed gate conformation of the lectin
FimH. The obtained ranking of the glycoclusters is consistent
with the RIP values deduced from the adhesion inhibition
assays, 15b forming a more stable complex with FimH than 6
and 15a.
Ligand
Docking
score
Docking
score
Binding energy
(kJ mol )
Binding energy
(kJ mol )
c
c
-1
-1
1
Open gate
Closed gate
Open gate
-74.892
-72.526
-78.104
Closed gate
6
-9.911
-8.231
-68.407
15a
15b
-9.561
-8.552
-60.940
-9.340
-8.858
-70.465
a
b
Calculated with Glide; calculated using the MM-GBSA method based on the
docking output; the lower the docking score, the better the predicted binding.
c
As the difference in energy between 15b and 15a is more
important in the closed gate conformation (about 9.5 kJ·mol ),
we selected the corresponding docking conformations for
-1
2
Figure 3. Interaction of FimH (closed gate conformation, pdb code 1UWF) with clusters 15a, 15b (diastereomeric Glc-Man pair) and 6 (αMan) . A: Partial charge coloured Connolly
description38 (negative charges in red, positive in blue); left image: overlay of 15a (grey) and 15b (blue) in the carbohydrate recognition domain (CRD) of FimH; right image: overlay
of 6 (purple) and 15b (blue) in the FimH CRD. B: H-bond network between 15a (left), 15b (middle) and 6 (right) and the CRD of FimH: the amino acid residues interacting with the
cluster outside of the binding pocket are highlighted in green, suggesting that 15b and 6 are better FimH ligands that 15a.
The hydrogen bond network was also examined (Figure 3B). with the same asparagine residue Asn 138 is observed for 15a.
Hence, the mannoside ligand in both diastereomers, 15a and Hence, the results of our modelling study provide a good basis
1
5b, forms exactly the same H-bond pattern with the amino for a possible explanation of the differences between the
acids of the FimH binding pocket. The difference between 15a heterobivalent diastereomeric glycoclusters 15a and 15b (the
and 15b resides in the non-covalent interactions of the pseudoenantiomeric Glc-Man pair) which was observed in
glucoside portion. In 15b, two hydrogen bonds are provided by the bacterial adhesion-inhibition assay. It might also give hints
the two protein residues forming the aforementioned polar for the interpretation of data obtained earlier and likewise for
domain (Asn 138 and Asp 140). In contrast, only a single H-bond
6
| J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
1
1
1
13
1
13
1
1
the future design of inhibitors and the understanding of techniques ( H- H COSY, H- C HSQC, H- C HMBC and H- H
carbohydrate recognition in a complex environment.
View Article Online
NOESY). ESI mass spectra were recorded on a Mariner 5280
DOI: 10.1039/C9OB00124G
instrument (Applied Biosystems).
For diastereomeric compounds, the one based on D-serine is
specified as "a", while the other one, based on L-serine, is called "b".
Conclusions
In conclusion, we introduced the first pseudoenantiomeric Molecule names are according to IUPAC nomenclature. To facilitate
cluster glycosides as a new tool for investigating the influence assignment of NMR peaks, the glycocluster skeleton was numbered
of the relative epitope orientation on lectin binding. A small as shown in Fig. 4. The nature of the carbohydrate portion is
library of heterobivalent glycoclusters was readily prepared indicated by “Gal” (D-galactose), “Glc” (D-glucose), “GlcNAc” (N-
from two enantiomeric scaffolds derived from L- and D-serine,
leading to pairs of diastereomers according to the configuration
at the focal point of the molecules. All synthesised
heterobivalent glycoclusters contain one α-mannosyl residue
and vary in the nature of the second sugar epitope. They were
tested as inhibitors of bacterial adhesion, mediated by the lectin
FimH, and as ligands of the lectin ConA. Both lectins, FimH and
ConA, specifically recognise α-mannosyl epitopes. The results
reveal a sharp difference in the inhibitor potency of the
diastereomeric Glc-Man pair 15a/b when tested in bacterial
acetyl-D-glucosamine), “GlcNTFA” (N-trifluoroacetyl-D-glucosamine),
and “Man” (D-mannose) subscripts, respectively.
BzO
BzO
BzO
BzO
O
BzO
4
6
BzO
O
BzO
BzO
5
O
9
1
2
3
8
O
N3
7
adhesion. Much smaller potency gaps were detected with the Figure 4. Numbering of the synthesized molecules for assignment of NMR data.
other pseudoenantiomeric glycocluster pairs. In the ConA-
based assays on the other hand, almost no significant variations General procedure for glycosylation (general procedure A).
were seen. In addition to the effect of the focal point To a round bottom flask containing the acceptor, the donor and 3 Å
stereochemistry, a marked heterocluster effect was observed molecular sieves, dry dichloromethane (c = 0.1 M) was added. After
with several derivatives, but again only in the context of
inhibition of bacterial adhesion.
stirring at room temperature for 15 min, the mixture was cooled to
°C then N-iodosuccinimide (1.5 eq.) and trifluoromethanesulfonic
acid (0.15 eq.) were sequentially added. After stirring at 0 ˚C for
0 min, the mixture was allowed to warm to room temperature and
stirred until completion then diluted with dichloromethane and
filtered over celite. After washing with satd. aq. sodium bicarbonate
and satd. aq. sodium thiosulfate, the aqueous layer was extracted
with dichloromethane then the combined organic layers were dried
over magnesium sulfate, filtered and concentrated. The crude
residue was purified by flash chromatography to afford the expected
compound.
General procedure for glycosylation (general procedure B).
To a round bottom flask containing the acceptor, the donor and 3 Å
molecular sieves, dry dichloromethane (c = 0.1 M) was added. After
stirring at room temperature for 15 min, trimethylsilyl
trifluoromethanesulfonate (0.1 eq.) was added and the mixture was
stirred until completion. The reaction mixture was neutralised with
triethylamine then diluted with dichloromethane, filtered over celite
and concentrated. The crude residue was purified by flash
chromatography to afford the expected compound.
0
Molecular docking with FimH provided means for
rationalizing the experimental data found with the βGlc-αMan
clusters. In fact, the models of the cluster-lectin complexes
showed a clear difference in the orientation of the glucosyl
residue and the resulting stabilization of the sugar-FimH
complex by H-bonds in the periphery of the CRD depending on
sugar scaffolding. We believe that our study opens new
prospects in the design of multivalent glycomimetics and the
understanding of carbohydrate recognition. Further analytical
methods, more complex glycoconjugates as well as other lectins
shall be employed to deepen the approach we have introduced
herein.
3
Experimental Section
General information
Air- or moisture-sensitive reactions were carried out under nitrogen
in dry glassware unless otherwise stated. All reactions were
monitored by thin layer chromatography (TLC) on silica gel plates (F
General procedure for ester cleavage (general procedure C).
To a solution of the ester-protected glycocluster in dry methanol (c
2
54, Merck). Detection of spots was effected by UV light and/or
subsequent charring with 10% sulphuric acid in ethanol, vanillin, or
ninhydrin followed by heat treatment at ~150 °C. Flash
chromatography was performed on silica gel 60 (0.040-0.063 mm)
using distilled solvents. Optical rotations were measured with a
Perkin-Elmer 241 polarimetry (sodium D-line: 589 nm) in the solvents
indicated. H and 13C NMR spectra were recorded on a Bruker DRX-
=
0.03 M), sodium methoxide (c = 5.4 M in methanol, two drops) was
added and the mixture was stirred at room temperature until
+
completion then neutralised with Amberlite IR120-H , diluted with
methanol, filtered and concentrated. The residue was taken up into
a 1:1 mixture of water and methanol then washed with diethyl ether
and the aqueous layer was concentrated to dryness. The residue was
purified by size exclusion chromatography on Sephadex G10 gel and
eluting with deionised water.
1
5
00 spectrometer at 300 K. Chemical shifts (in ppm) are relative to
residual non-deuterated solvent as an internal reference. Full
assignment of the peaks was achieved with the aid of 2D NMR
General procedure for silyl cleavage (general procedure D).
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 8 of 14
ARTICLE
Journal Name
D
1
The silylated starting material was dissolved in anhydrous [α]20 = +53.5 (c 0.7, water); H NMR (500 MHz, D
2
O) δ 4.89-4.86 (m,
View Article Online
tetrahydrofuran (c = 0.1 M) then the mixture was buffered with acetic 2H, 2 H-1), 3.98-3.87 (m, 6H, 2 H-2, 2 H-4, 2DHO-6I:a1)0,.130.8395/-C39.8O0B(0m01,234HG,
acid (6 eq.) before dropwise adding 1 M n-tetrabutylammonium 2 H-3, H-8), 3.76-3.70 (m, 3H, H-9a, H-9b, H-6b), 3.66-3.59 (m, 5H, H-
fluoride in tetrahydrofuran (3 eq.). The mixture was stirred until 7a, H-7b, 2 H-5, H-6b) ppm; 13C NMR (126 MHz, D
O) δ 100.3, 99.8
2
completion then diluted with ethyl acetate. After washing with satd. (2C, 2 C-1), 73.1, 73.1 (2C, 2 C-4), 70.5 (2C, 2 C-3), 69.9 (2C, 2 C-2),
aq. sodium bicarbonate then 1 N hydrochloric acid, the aqueous 67.2 (C-6), 66.7 (2C, 2 C-5), 66.7 (C-6), 61.0 (2C, C-7, C-9), 60.1 (C-8)
phases were extracted with ethyl acetate then the combined organic ppm; IR (ATR) vmax/cm-1 3332, 2927, 2097, 1048; ESI-HRMS: m/z
+
+
layers were dried over magnesium sulfate, filtered and concentrated. calcd. for C15
The crude residue was purified by flash chromatography to afford the (S)–2–Azido–1–O–(tert–butyldiphenylsilyl)–3–O–(2,3,4,6–tetra–O–
expected compound.
benzoyl––D–mannopyranosyl)–1,3–propanediol (11a). General
General procedure for cleavage of the N-trifluoroacetyl group and procedure A was applied to acceptor 10a (169 mg, 476 µmol) and
subsequent N-acetylation (general procedure E).
donor 4 (434 mg, 572 µmol, 1.2 eq.). Reagents and conditions: N-
To a solution of the N-trifuoroacetyl derivative in methanol (c iodosuccinimide (160.7 mg, 715 µmol, 1.5 eq.), trifluoromethane
0.03 M), 2 M aq. lithium hydroxide (40 eq.) was added at room sulfonic acid (4.8 l, 47.0 µmol, 0.1 eq.), dichloromethane (c = 0.1 M,
27 3
H N O12 + Na : 464.14869; [M+Na] ; found 464.14822.
=
temperature. The solution was sonicated at 40 °C until completion 4.76 mL). Flash chromatography with ethyl acetate/cyclohexane 1/9
+
then neutralised with Amberlite IR120-H , diluted with methanol, yielded compound 11a (374 mg, 85 %) as a white foam; R = 0.3 (ethyl
f
filtered and concentrated to dryness. The residue was dissolved in acetate/cyclohexane 1/9); [α]20D = -26.9 (c 0.4, dichloromethane); 1H
dry methanol (c = 0.03 M) then sodium methoxide (c = 5.4 M in NMR (500 MHz, CDCl ) δ 8.11-8.09 (m, 2H, 2 H-Ar), 8.06-8.04 (m, 2H,
3
methanol, two drops) and acetic anhydride (5 eq.) were sequentially 2 H-Ar), 7.96-7.94 (m, 2H, 2 H-Ar), 7.85-7.84 (m, 2H, 2 H-Ar), 7.70-
added under nitrogen. After stirring overnight at room temperature, 7.67 (m, 4H, 4 H-Ar), 7.62-7.48 (m, 3H, 3 H-Ar), 7.46-7.33 (m, 15H, 15
sodium methoxide (c = 5.4 M in methanol, two drops) was added and H-Ar), 6.14 (dd, 3J3,4 = 3J4,5 = 9.9 Hz, 1H, H-4), 5.91 (dd, J = 10.1 Hz,
3
3,4
the mixture was stirred for a further 30 min then neutralised with 3J2,3 = 3.3 Hz, 1H, H-3), 5.71 (dd, 3J2,3 = 3.3 Hz, 3J1,2 = 1.8 Hz, 1H, H-2),
Amberlite IR120-H+, diluted with methanol, filtered and 5.10 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 4.70 (dd, 2J6a, 6b = 13.4 Hz, 3J5,6a
=
=
3.6 Hz, 1H, H-6a), 4.50-4.43 (m, 2H, H-5, H-6b), 3.93 (dd, 2J7a,7b
concentrated.
–Azido–1,3–di–O–(2,3,4,6–tetra–O–benzoyl––D–
mannopyranosyl)–1,3–propanediol (5). General procedure A was 7b), 1.08 (s, 9H, Si-C(CH ) ) ppm; C NMR (126 MHz, CDCl ) δ 166.1,
9.9 Hz, 3J7a,8 = 7.0 Hz, 1H, H-7a), 3.84-3.73 (m, 4H, H-8, H-9a, H-9b, H-
2
1
3
3
3
3
applied to acceptor 3 (27.0 mg, 227 µmol) and donor 4 (207 mg, 165.4, 165.3(4C, 4 PhC=O) , 135.54, 133.4, 133.2, 133.0, 132.7, 130.0,
73 µmol, 1.2 eq.). Reagents and conditions: N- iodosuccinimide 129.9, 129.8, 129.7, 129.2, 129.0, 128.9, 128.6, 128.4, 128.3, 127.9
76.8 mg, 340 µmol, 1.5 eq.), trifluoromethanesulfonic acid (2.3 l, (36C, 36 C-Ar) 97.7 (C-1), 70.1 (C-2), 69.9 (C-3), 69.2 (C-5), 67.7 (C-7),
2
(
2
3 3
0.0 µmol, 0.15 eq.), dichloromethane (c = 0.1 M, 2.8 mL), T = -77 ˚C 66.6 (C-4), 63.5 (C-9), 62.7 (C-6), 62.0 (C-8), 26.6 (Si-C(CH ) ), 19.2 (Si-
-1
to room temperature after three hours. Flash chromatography with C(CH
ethyl acetate/cyclohexane 2.5/7.5 afforded compound 5 as a white 503; ESI-HRMS: m/z calcd. for C53
foam (174 mg, 60 %); R
=
8
3
)
3
) ppm; IR (ATR) vmax/cm 2103, 1724, 1451, 1260, 1093, 705,
+
51
H N
3
O
11Si + Na : 956.31851
D
+
f
= 0.3 (ethyl acetate/cyclohexane 3/7); [α]20
[M+Na] ; found 956.31719.
1
-46.1 (c 0.7, dichloromethane); H NMR (500 MHz, CDCl
3
) δ 8.13- (R)–2–Azido–1–O–(tert–butyldiphenylsilyl)–3–O–(2,3,4,6–tetra–O–
.10 (m, 4H, 4 H-Ar), 8.06-8.04 (m, 4H, 4 H-Ar), 7.98-7.96 (m, 4H, 4 H- benzoyl––D–mannopyranosyl)–1,3–propanediol (11b). General
Ar), 7.84-7.82 (m, 4H, 4 H-Ar),7.61-7.55 (m, 4H, 4 H-Ar), 7.50-7.45 (m, procedure A was applied to acceptor 10b (300 mg, 843 µmol) and
3
7
2
2
H, 3 H-Ar), 7.44-7.37 (m, 10H, 10 H-Ar), 7.35-7.31 (m, 4H, 4 H-Ar) donor 4 (768 mg, 1.01 mmol, 1.2 eq.). Reagents and conditions: N-
.28-7.26 (m, 1H, H-Ar), 7.25-7.23 (m, 2H, 2 H-Ar), 6.20-6.14 (m, 2H, iodosuccinimide (284 mg, 1.26 mmol, 1.5 eq.), trifluoromethane
H-4), 5.97-5.92 (m, 2H, 2 H-3), 5.77 (dd, 3J2,3 = 3.3 Hz, 3J1,2 = 1.8 Hz, sulfonic acid (8.4 l, 50.0 µmol, 0.1 eq.), dichloromethane (c = 0.1 M,
H, 2 H-2), 5.21 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 5.19 (d, 3J1, 2 = 1.7 Hz, 1H, 8.4 mL). Flash chromatography with ethyl acetate/cyclohexane 1/9
H-1), 4.79-4.75 (m, 2H, 2 H-6a), 4.58-4.47 (m, 4H, 2 H-6b, 2 H-5), 4.07- afforded compound 11b (665 mg, 85 %) as a white foam; R = 0.3
f
4
.03 (m, 3H, H-8, H-7a, H-9a), 3.84-3.74 (m, 2H, H-7b,H-9b) ppm; 13
NMR (126 MHz, CDCl
C
(ethyl acetate/cyclohexane 1/9); [α]20D
=
-33.8 (c 0.6,
1
3
) δ 166.1, 165.5, 165.4, 165.3(8C, 8 PhC=O), dichloromethane); H NMR (500 MHz, CDCl ) δ 8.12-8.02 (m, 4H, 4 H-
3
1
1
6
6
33.5, 133.4, 133.1, 129.9, 129.8, 129.2, 129.0, 128.9, 128.6, 128.5, Ar), 7.91-7.89 (m, 2H, 2 H-Ar), 7.85-7.83 (m, 2H, 2 H-Ar), 7.65 (m, 4H,
28.4, 128.3 (48C, 48 C-Ar), 98.2, 97.8 (2C, 2 C-1), 70.2 (2C, 2 C-2), 4 H-Ar), 7.64-7.47 (m, 4H, 4 H-Ar), 7.47-7.33 (m, 14H, 14 H-Ar), 6.18-
9.9, 69.8 (2C, 2 C-3), 69.4, 69.4 (2C, 2 C-5), 67.6, 67.5 (2C, C-7, C-9), 6.06 (dd, 3J3,4 = 3J4,5 = 10.0 Hz, 1H, H-4), 5.89 (dd, 3J3,4 = 10.1 Hz, 3J2,3
6.7, 66.6 (2C, 2 C-4), 62.7 (2C, 2 C-6), 59.9 (C-8) ppm; IR 3.3 Hz, 1H, H-3), 5.71 (dd, 3J2,3 = 3.3 Hz, 3J1,2 = 1.8 Hz, 1H, H-2), 5.12
=
(
ATR) vmax/cm 2097, 1721, 1258, 1092, 705; ESI-HRMS: m/z calcd. (d, 3J1,2 = 1.7 Hz, 1H, H-1), 4.71-4.61 (dd, 2J6a,6b = 11.8 Hz, 3J5,6a
-1
=
+
+
2.1 Hz, 1H, H-6a), 4.43 (m, 2H, H-5, H-6b), 3.99 (dd, 2J7a,7b = 8.4 Hz,
59 3
for C71H N O20 + Na : 1296.35590 [M+Na] ; found 1296.35841.
2
–Azido–1,3–di–O–(–D–mannopyranosyl)–1,3–propanediol (6). 3J7a,8 = 2.4 Hz, 1H, H-7a), 3.87-3.82 (m, 2H, H-9a, H-9b), 3.77-3.70 (m,
General procedure C was applied to compound 5 (500 mg, 2H, H-8, H-7b), 1.09 (s, 9H, Si-C(CH ) ) ppm; 13C NMR (126 MHz,
3
3
3
92 µmol). Reagents and conditions: sodium methoxide (c = 5.4 M in CDCl ) δ 166.2, 165.5, 165.4, 165.3 (4C, 4 PhC=O), 135.6, 133.5,
3
methanol, two drops), methanol (c = 0.03 M, 7.84 mL). Compound 6 133.2, 133.1, 132.8, 132.7, 130.0, 129.8, 129.9, 129.8, 129.7, 129.8,
100 mg, 70 %) was obtained as a white foam after lyophilisation; 129.7, 129.2, 129.0, 128.9, 128.6, 128.5, 128.3, 127.9, (36C, 36 C-Ar),
(
8
| J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
9
8.2 (C-1), 70.2 (C-2), 69.8 (C-3), 69.2 (C-5), 67.7 (C-7), 66.7 (C-4), 63.5 (r)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
View Article Online
(
C-9), 62.6 (C-6), 62.0 (C-8), 26.9 (Si-C(CH
3
)
3
), 19.2 (Si-C(CH
3 3
)
); IR mannopyranosyl)–3–O–(2,3,4,6–tetra–O–bDeOnzI:o1y0l.–103– 9D /–C9OB00124G
-1
(ATR) vmax/cm 2101, 1724, 1451, 1259, 1067, 704, 504; ESI-HRMS: glucopyranosyl)–1,3–propanediol (14a). General procedure A was
+
+
m/z calcd. for C19
9
25
H N
3
O
2
Si + Na : 956.31851 [M+Na] ; found applied to acceptor 12a (325 mg, 468 µmol) and donor 13 (426 mg,
561 µmol, 1.5 eq.). Reagents and conditions: N-iodosuccinimide
56.31800.
(
R)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
(157 mg, 702 µmol, 1.5 eq.), trifluoromethane sulfonic acid (4.7 l,
mannopyranosyl)–1,3–propanediol (12a). General procedure D was 50.0 µmol, 0.1 eq.), dichloromethane (c = 0.1 M, 4.68 mL). Flash
applied to compound 11a (593 mg, 634 µmol). Reagents and chromatography with ethyl acetate/cyclohexane 1/4 yielded
conditions: tetrabutylammonium fluoride (c = 1 M, 1.91 mL, compound 14a (434 mg, 73 %) as a white foam; R = 0.3 (ethyl
f
1
.91 mmol, 3 eq.), acetic acid (0.22 mL, 3.81 mmol, 6 eq.), acetate/cyclohexane 3/7); [α]20D = -15.8 (c 1.0, dichloromethane); 1H
tetrahydrofuran (c = 0.1 M, 6.35 mL). Flash chromatography with NMR (600 MHz, CDCl ) δ 8.11-8.07 (m, 2H, 2 H-Ar), 8.06-8.01 (m, 4H,
3
ethyl acetate/cyclohexane 1/9 to 2/8 afforded compound 12a 4 H-Ar), 7.98-7.96 (m, 4H, 4 H-Ar), 7.92-7.90 (m, 2H, 2 H-Ar), 7.86-
(
348 mg, 78 %) as a white foam; R
f
= 0.3 (ethyl acetate/cyclohexane 7.81 (m, 4H, 4 H-Ar), 7.60-7.58 (m, 2H, 2 H-Ar), 7.53-7.44 (m, 4H, 4 H-
D
1
2
/3); [α]20 = -57.8 (c 0.5, dichloromethane); H NMR (500 MHz, Ar) 7.45-7.33 (m, 15H, 15 H-Ar), 7.29-7.27 (m, 3H, 3 H-Ar), 6.10 (dd,
CDCl
) δ 8.09-8.07 (m, 2H, 2 H-Ar), 8.05-8.03 (m, 2H, 2 H-Ar), 7.96- 3J4,5 = 3J3,4 = 10.1 Hz, 1H, H-4Man), 5.94 (dd, 3J3,4 = 3J3,2 = 9.7 Hz, 1H, H-
3
7
.94 (m, 2H, 2 H-Ar), 7.84-7.82 (m, 2H, 2 H-Ar), 7.63-7.48 (m, 3H, 3 H- 3Glc), 5.85 (dd, 3J3,4 = 10.1 Hz, 3J3,2 = 3.3 Hz, 1H, H-3Man), 5.72 (dd, 3J3,4
Ar), 7.45-7.34 (m, 8H, 8 H-Ar), 7.29-7.26 (m, 1H, H-Ar), 6.12 (dd, J3,4 = 3J4,5 = 9.9 Hz,1H, H-4Glc), 5.70-5.68 (dd, J = 3.3 Hz, 3J1,2 = 1.8 Hz,,
3
3
2,3
=
3
1
4
1
4
3
J
4,5 = 10.0 Hz, 1H, H-4), 5.92 (dd, 3J3,4 = 10.1 Hz, 3J3,2 = 3.4 Hz, 1H, H- 1H, H-2Man), 5.57 (dd, 3J2,3 = 9.8 Hz, 3J1,2 = 7.8 Hz, 1H, H-2Glc), 5.00 (d,
), 5.73 (dd, 3J2, 3 = 3.3 Hz, 3J1,2 = 1.8 Hz, 1H, H-2), 5.15 (d, J1,2 = 1.8 Hz,
3
3
J1,2 = 1.6 Hz, 1H, H-1Man), 4.98 (d, 3J1,2 = 7.9 Hz, 1H, H-1Glc), 4.70 (dd,
H, H-1), 4.74-4.69 (dd, 2J6a,6b = 11.9 Hz, 3J5,6a = 2.3 Hz, 1H, H-6a), 2J6a,6b = 12.2 Hz, 3J5,6a = 3.1 Hz, 1H, H-6aGlc), 4.65 (dd, 2J6a,6b = 12.2 Hz,
.54-4.45 (m, 2H, H-5, 6b), 4.02 (dd, 2J7a,7b = 10.0 Hz, J7a,8 = 7.5 Hz, 3J5,6a = 2.5 Hz, 1H, H-6aMan), 4.54 (dd, 2J6a,6b = 12.2 Hz, 3J5,6a = 4.9 Hz,
3
H, H-7a), 3.94-3.88 (m, 1H, H-8), 3.83 (dd, J9a,9b = 11.5 Hz, 3J8,9a
2
=
1H, H-6bGlc), 4.46 (dd, 2J6a,6b = 12.2 Hz, 3J5,6a = 4.2 Hz, 1H, H-6bMan),
.4 Hz, 1H, H-9a), 3.78 (dd, 2J7a,7b = 10.0 Hz, 3J7b,8 = 4.3 Hz, 1H, H-7b), 4.43-4.38 (m, 1H, H-5Man), 4.25-4.20 (m, 1H, H-5Glc), 4.11 (dd, 2J9a,9b
=
=
.71 (dd, 2J9a,9b = 11.5 Hz, 3J8,9b = 5.9 Hz, 1H, H-9b) ppm; 13C NMR 10.1 Hz, 3J8,9a = 3.9 Hz, 1H, H-9a), 3.90 (dd, 2J7a,7b = 10.4 Hz, 3J
7a,8
2
(126 MHz, CDCl
3
) δ 166.2 165.5, 165.4, (4C, 4 PhC=O), 129.9, 129.8, 3.6 Hz, 1H, H-7a), 3.88-3.80 (m, 2H, H-8, H-9b), 3.59-3.57 (dd, J
7
a,7b
29.7, 128.6, 128.5, 128.3 (24C, 24 C-Ar), 97.7 (C-1), 70.2 (C-2), 69.8 = 10.5 Hz, 3J7a,8 = 5.6 Hz, 1H, H-7b) ppm; C NMR (151 MHz, CDCl ) δ
13
1
3
(
9
5
C-3), 69.3 (C-5), 67.9 (C-7), 66.7 (C-4), 62.4 (C-6), 62.1 (C-8), 62.0 (C- 166.1, 165.8, 165.5, 165.3, 165.2, 165.1 (8C, 8 PhC=O), 133.5, 133.3,
-1
) ppm; IR (ATR) vmax/cm 2933, 2095, 1723, 1451, 1259, 1093, 705, 133.2, 133.1, 130.0, 129.9, 129.8, 129.8, 129.7, 129.5, 129.2, 129.0,
+
+
03; ESI-HRMS: m/z calcd. for C37
33
H N
3
O
11+ Na : 718.20073 [M+Na] ; 128.9, 128.8, 128.6, 128.5, 128.4, 128.3, (48C, 48 C-Ar) 101.5 (C-1Glc),
found 718.19969.
97.9 (C-1Man), 72.8 (C-3Glc), 72.4 (C-5Glc), 71.7 (C-2Glc), 70.1 (C-2Man),
(
S)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
69.9 (C-3Man), 69.5 (C-4Glc), 69.4 (C-9), 69.2 (C-5Man), 67.9 (C-7), 66.6
mannopyranosyl)–1,3–propanediol (12b). General procedure D was (C-4Man), 62.8 (C-6Glc), 62.7 (C-6Man), 60.2 (C-8) ppm; IR (ATR) vmax/cm-
1
applied to compound 11b (393 mg, 427 µmol). Reagents and
conditions: tetrabutylammonium fluoride (c = 1 M, 1.28 mL, 3eq.), C H N O +H : 1274.37647 [M+H] ; found 1274.37451.
2933, 2095, 1722, 1451, 1258, 1093, 704; ESI-HRMS; m/z calcd. for
+
+
7
1 59 3 20
acetic acid (150 µl, 2.56 mmol, 6eq.), tetrahydrofuran (c = 0.1 M, (s)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
.3 mL). Flash chromatography with ethyl acetate/cyclohexane 1/9 mannopyranosyl)–3–O–(2,3,4,6–tetra–O–benzoyl––D–
4
to 2/8 afforded compound 12b (586 mg, 76 %) as a white foam; R
f
=
glucopyranosyl)–1,3–propanediol (14b). General procedure A was
D
0
.3 (ethyl acetate/cyclohexane 2/3); [α]20 = -45.6 (c 0.42, applied to acceptor 12b (102 mg, 147 µmol) and donor 13 (134 mg,
1
dichloromethane); H NMR (500 MHz, CDCl
Ar), 8.06-8.04 (m, 2H, 2 H-Ar), 7.98-7.96 (m, 2H, 2 H-Ar), 7.85-7.83
m, 2H, 2 H-Ar), 7.64-7.34 (m, 10H, 10 H-Ar), 7.29-7.27 (m, 2H, 2 H-
3
) δ 8.10-8.08 (m, 2H, 2 H- 176 µmol, 1.5 eq.). Reagents and conditions: N-iodosuccinimide
49.6 mg, 220 µmol, 1.5 eq.), trifluoromethane sulfonic acid (1.5 l,
5.0 µmol, 0.1 eq.), dichloromethane (c = 0.1 M, 1.47 mL). Flash
chromatography with ethyl acetate/cyclohexane 2.5/7.5 afforded
compound 14b (210 mg, 71 %) as a white foam; R = 0.3 (ethyl
acetate/cyclohexane 3/7); [α]20 = -11.9 (c 1.0, dichloromethane); 1H
NMR (500 MHz, CDCl ) δ 8.13-8.11 (m, 2H, 2 H-Ar), 8.07-8.01 (m, 4H,
H-Ar), 7.99-7.89 (m, 6H, 6 H-Ar), 7.86-7.78 (m, 4H, 4 H-Ar), 7.59 (m,
H, 2 H-Ar), 7.54-7.47 (m, 3H, 3 H-Ar), 7.46-7.22 (m, 19H, 19 H-Ar),
(
1
(
Ar), 6.13 (dd, 3J4,5 = J3,4 = 10.0 Hz, 1H, H-4), 5.90 (dd, 3J3,4 = 10.1 Hz,
3J2,3 = 3.3 Hz, 1H, H-3), 5.73 (dd, 3J2,3 = 3.3 Hz,3J1,2 = 1.8 Hz, 1H, H-2),
f
5
2
1
.16 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 4.74-4.69 (dd, 2J6a,6b = 11.9 Hz, 3J5,6a
.3 Hz, 1H, H-6a), 4.53-4.45 (m, 2H, H-5, H-6b), 4.07 (dd, 2J7a,7b
0.1 Hz, 3J7a,8 = 4.1 Hz, 1H, H-7a), 3.89-3.72 (m, 4H, H-8, H-9a, H-9b,
) δ 166.2,
65.5, 165.4 (4C, 4 PhC=O), 133.6, 133.5, 133.3, 133.2, 129.9, 129.8,
=
=
D
3
4
2
6
9
5
1
1
2
6
H-7b), 1.88 (br s, 1H, OH) ppm; 13C NMR (126 MHz, CDCl
3
1
1
7
.11 (dd, 3J4,5 = 3J3,4 = 10.1 Hz, 1H, H-4Man), 5.93 (dd, 3J2,3 = 3J3,4
=
29.2, 129.0, 128.9, 128.6, 128.5, 128.4 (24C, 24 C-Ar), 98.0 (C-1),
0.2 (C-2), 69.8 (C-3), 69.2 (C-5), 67.8 (C-7), 66.7 (C-4), 62.8 (C-6), 62.1
.7 Hz, 1H, H-3Glc), 5.87 (dd, 2J3,4 = 10.1 Hz, 3J2,3 = 3.3 Hz, 1H, H-3Man),
.73-5.66 (m, 2H, H-2Man, H-4Glc), 5.54 (dd, 2J2,3 = 9.8 Hz, 3J1,2 = 7.8 Hz,
-1
(2C, C-8, C-9) ppm IR (ATR) vmax/cm 2932, 2094, 1722, 1451, 1258,
H, H-2Glc), 4.96-4.90 (m, 2H, H-1Man, H-1Glc), 4.69 (dd, 2
J
6a,6b
=
=
1
7
093, 705, 504; ESI-HRMS: m/z calcd. for C37
33
H N
3
O
11 + Na+:
2.2 Hz, 3J5,6a =3.1 Hz, 1H, H-6aGlc), 4.63 (dd, 2J6a,6b = 12.2 Hz, 3J5,6a
+
18.20073 [M+Na] ; found 718.19968.
.5 Hz, 1H, H-6aMan), 4.53 (dd, 2J6a,6b = 12.2 Hz, 3J5,6b =5.1 Hz, 1H, H-
b
Glc), 4.44 (dd, 2J6a,6b = 12.3 Hz, 3J5,6b = 4.0 Hz, 1H, H-6bMan), 4.37-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 10 of 14
ARTICLE
Journal Name
4
.31 (m, 1H, H-5Man), 4.21 (m, 1H, H-5Glc), 4.04 (dd, 2J9a,9b = 10.6 Hz, 48.0 µmol, 0.1 eq.), dichloromethane (c = 0.1 M, 2.V0ie5wmArLtic).le OFlnalisnhe
3J8,9a = 5.0 Hz, 1H, H-9a), 3.96-3.89 (m, 1H, H-8), 3.86 (dd, 2J7a,7b
chromatography with ethyl acetate/cyclohexane 2.5/7.5 afforded
DOI: 10.1039/C9OB00124G
=
1
0.1 Hz, 3J7a,8 = 7.7 Hz, 1H, H-7a), 3.75-3.65 (m, 2H, H-9b, H-7b) ppm; compound 17a (196 mg, 64 %) as a white foam; R
f
= 0.3 (ethyl
) δ 166.1, 165.8, 165.4, 165.2, 165.0 (8C, 8 acetate/cyclohexane 3/7); [α]20 = -16.3 (c 1, dichloromethane);1H
) δ 8.13 – 8.07 (m, 4H, 4 H-Ar), 8.03-8.01 (m,
29.5, 129.3, 129.1, 129.0, 128.8, 128.60, 128.5, 128.4, 128.3, (48C, 4H, 4 H-Ar), 7.98-7.92 (m, 4H, 4 H-Ar), 7.83-7.77 (m, 4H,4 H-Ar), 7.62-
13C NMR (126 MHz, CDCl
D
3
PhC=O), 133.5, 133.4, 133.3, 133.2, 133.1, 130.0, 129.9, 129.8, 129.7, NMR (600 MHz, CDCl
3
1
4
8 C-Ar) 101.5 (C-1Glc), 97.7 (C-1Man), 72.7 (C-3Glc), 72.5 (C-5Glc), 71.7 7.28 (m, 22H, 22 H-Ar), 7.24-7.22 (m, 2H, 2 H-Ar) 6.11 (dd, J4,5 = 3J3,4
3
(
(
C-2Glc), 70.1, 69.8, 69.6 (3C, C-4Glc, C-2Man, C-3Man), 69.2 (C-5Man), 68.6 = 10.1 Hz, 1H, H-4Man), 6.01 (dd, 3J3,4 = 3.4 Hz, J4,5 = 1.0 Hz, 1H, H-4Gal),
C-9), 67.9 (C-7), 66.6 (C-4Man), 62.9 (C-6Glc), 62.6 (C-6Man), 60.4 (C-8); 5.86 (dd, 3J3,4 = 10.1 Hz, 3J2,3 = 3.3 Hz, 1H, H-3Man), 5.80 (dd, 3J2,3
=
IR (ATR) vmax/cm-1 2933, 2095, 1722, 1451, 1258, 1092, 705; m/z 10.4 Hz, 3J1,2 = 7.9 Hz, 1H, H-2Gal), 5.67 (dd, J2,3 = 3.3 Hz, 3J2,1 = 1.8 Hz,
3
+
+
1H, H-2Man), 5.63 (dd, 3J2,3 = 10.4 Hz, 3J3,4 = 3.5 Hz, 1H, H-3Gal), 4.90-
4.87 (m, 2H, H-1Man, H-1Gal), 4.71 (dd, 2J6a,6b = 11.4 Hz, 3J5,6a = 6.5 Hz,
calcd. for C71
59 3
H N O20 +H : 1274.37647 [M+H] ; found 1274.37450.
(
r)–2–Azido–1–O–(–D–mannopyranosyl)–3–O–(–D–
glucopyranosyl)–1,3–propanediol (15a). General procedure C was 1H, H-6aMan), 4.63 (dd, 2J6a,6b = 12.2 Hz, 3J5.6a =2.5 Hz, 1H, H-6aGal),
applied to compound 14a (410 mg, 322 µmol). Reagents and 4.48-4.41 (m, 2H, H-6bMan, H-6bGal), 4.40-4.35 (1H, H-5Gal), 4.35-4.30
conditions: sodium methoxide (c = 5.4 M in methanol, two drops), (m, 1H, H-5Man), 4.11 (dd, J9a,9b = 10.6 Hz, J8,9a = 5.1 Hz, 1H, H-9a),
methanol (c = 0.03 M, 10.7 mL). Compound 15a (135 mg, 96 %) was 3.99-3.91 (m, 1H, H-8), 3.85 (dd, J7a,7b = 10.2 Hz, J7a,8 = 7.9 Hz, 1H, H-
obtained as a white foam after lyophilisation; [α]20 = +27.0 (c 0.8, 7a), 3.74-3.67 (m, 2H, H-7b, H-9b) ppm; 13C NMR (151 MHz, CDCl
D
) δ
3
1
O) δ 4.87 (d, 3J1,2 = 1.6 Hz, 1H, H-1Man), 166.1, 166.0, 165.6, 165.54, 165.4, 165.4, 165.3, 165.2 (8C, 8 PhC=O),
water); H NMR (600 MHz, D
2
4
4
.47 (d, 3J1,2= 8.0 Hz, 1H, H-1Glc), 4.05 (dd, 2J9a,9b= 11.1 Hz, 3J8,9a
.0 Hz, 1H, H-9a), 4.00-3.85 (m, 5H, H-2Man, H-5Man, H-6aMan, H-6aGlc
=
,
133.6, 133.5, 133.5, 133.4, 133.3, 133.2, 133.1, 130.0, 129.9, 129.8,
129.8, 129.8, 129.8, 129.7, 129.7, 129.4, 129.2, 129.2, 129.0, 128.9,
H-7a), 3.82 (dd, 3J2,3 = 9.1 Hz, 3J3,4= 3.4 Hz, 1H, H-3Man), 3.79-3.58 (m, 128.7, 128.6, 128.5, 128.3, 128.2 (48C, 48 C-Ar), 101.9 (C-1Gal), 97.6
6
3
8
1
7
6
H, H-8, H-4Man, H-6bGlc, H-9b, H-7b, H-6bMan), 3.52-3.41 (m, 2H, H- (C-1Man), 71.6 (C-3Gal or C-5Gal), 71.5 (C-3Gal or C-5Gal), 70.0 (C-2Man),
Glc, H-5Glc), 3.40-3.34 (m, 1H, H-4Glc), 3.28 (dd, 3J2,3 = 9.4 Hz, 3J2,1
69.8 (C-3Man), 69.6 (C-2Gal), 69.2 (C-5Man), 68.7 (C-9), 68.1 (C-4Gal or C-
.0 Hz, 1H, H-2Glc) ppm; 13C NMR (151 MHz, D
O) δ 102.8 (C-1Glc), 7), 68.0 (C-4Gal or C-7), 66.5 (C-4Man), 62.5 (C-6Gal), 61.9 (C-6Man), 59.9
00.3 (C-1Man), 75.9 (C-5Glc), 75.7 (C-3Glc), 73.1 (C-2Glc), 73.0 (C-4Man), (C-8) ppm; IR (ATR) vmax/cm 2096, 1722, 1451, 1257, 1091, 705; ESI-
=
2
-1
+
+
59 3
0.4 (C-3Man), 69.9 (C-5Man), 69.6 (C-4Glc), 69.0, 67.2 (C-6Man or C-6Glc), HRMS: m/z calcd. for C71H N O20 + Na : 1296.35841 [M+Na] ; found
6.7 (C-7), 61.0 (C-6Glc or C-6Man), 60.7 (C-9), 60.7 (C-8) ppm; IR 1296.35802.
-1
(
ATR) vmax/cm 3325, 2932, 2127, 1672, 1021; ESI-HRMS: m/z calcd. (s)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
+
+
27
for C15H N
3
O
12 + Na = 464.14869 [M+Na] ; found 464.14853.
mannopyranosyl)–3–O–(2,3,4,6–tetra–O–benzoyl––D–
(
s)–2–Azido–1–O–(–D–mannopyranosyl)–3–O–(–D–
galactopyranosyl)–1,3–propanediol (17b). General procedure A was
glucopyranosyl)–1,3–propanediol (15b). General procedure C was applied to acceptor 12a (226 mg, 324 µmol) and donor 16 (369 mg,
applied to compound 14b (123 mg, 96.0 µmol). Reagents and 486 µmol, 1.5 eq.). Reagents and conditions: N-iodosuccinimide
conditions: sodium methoxide (c = 5.4 M in methanol, two drops), (195 mg, 648 µmol, 2 eq.), trifluoromethane sulfonic acid (6.5 l,
methanol (c = 0.03 M, 3.2 mL). Compound 15b (37.2 mg, 89 %) was 64.0 µmol, 0.1 eq.) dichloromethane (c = 0.1 M, 3.24 mL). Flash
D
obtained as a white foam after lyophilisation; [α]20 = +51.9 (c 0.8, chromatography with ethyl acetate/cyclohexane 2.5/7.5 afforded
1
O) δ 4.89 (d, 3J1,2 = 1.5 Hz, 1H, H-1Man), compound 17b (251 mg, 61 %) as a white foam; R = 0.3 (ethyl
water); H NMR (600 MHz, D
4
9
2
f
.47 (d, 3J1,2 = 8.0 Hz, 1H, H-1Glc), 4.00-3.95 (m, 3H, H-2Man, H-3Man, H- acetate/cyclohexane 3/7); [α]20D = -21.1 (c 1, dichloromethane); 1H
a), 3.92-3.79 (m, 5H, H-4Man, H-9b, H-6aMan, H-6aGlc, H-7a), 3.77-3.68 NMR (600 MHz, CDCl ) δ 8.11-8.08 (m, 4H, 4 H-Ar), 8.03-8.01 (m, 6H,
3
(
(
(
m, 3H, H-7b, H-6bMan, H-6bGlc), 3.66-3.62 (m, 2H, H-5Man, H-8), 3.47 6 H-Ar), 7.96-7.95 (m, 2H, 2 H-Ar), 7.84-7.78 (m, 4H, 4 H-Ar), 7.63-
dd, 3J2,3 = J3,4 = 9.2 Hz, 1H, H-3Glc)3.45-3.41 (m, 1H, H-5Glc), 3.39-3.34 7.33 (m, 22H, 22 H-Ar), 7.27-7.23 (m, 2H, 2 H-Ar), 6.11 (dd, 3J4,5 = 3J3,4
3
dd, 3J3,4 = 3J4,5 = 9.4 Hz,, 1H, H-4Glc), 3.28 (dd, 3J2,3 = 9.3 Hz, 3J1,2
=
=10.1 Hz, 1H, H-4Man), 6.02 (dd, J3,4 = 3.4 Hz, J =0.7 Hz, 1H, H-4Gal),
4,5
3
8
.0 Hz, 1H, H-2Glc) ppm; 13C NMR (151 MHz, D O) δ 102.4 (C-1Glc), 99.7 5.87-5.82 (m, 2H, H-3Man, H-2Gal), 5.68 (dd, J = 3.3 Hz, 3J1,2 = 1.8 Hz,
2
2,3
(
(
C-1Man), 76.0 (C-3Glc), 75.7 (C-5Glc), 73.1 (C-4Man), 73.0 (C-2Glc), 70.4 1H, H-2Man), 5.64 (dd, 3J3,4 = 10.4 Hz, 3J2,3 = 3.5 Hz, 1H, H-3Gal), 4.99 (d,
C-2Man), 69.9 (C-3Man), 69.72 (C-4Glc), 69.6 (C-5Man), 68.7 (C-6Man or C- 3J1,2 = 1.6 Hz, 1H, H-1Man), 4.96 (d, 3J1,2 = 7.9 Hz, 1H, H-1Gal), 4.73 (dd,
6
Glc), 66.9 (C-6Man or C-6Glc), 61.0 (C-7), 60.7 (C-9), 60.2 (C-8) ppm; IR 2J6a,6b = 11.2 Hz, 3J5,6a = 6.3 Hz, 1H, H-6aMan), 4.65 (dd, J6a,6b = 12.2 Hz,
(
[
ATR) vmax/cm-1 3338, 2932, 2115, 1259, 1033; ESI-HRMS: m/z J5.6a =2.5 Hz, 1H, H-6aGal), 4.48-4.43 (m, 2H, H-6bGal, H-6bMan), 4.42-
+
27 3
H N O
12 + Na = 464.14869 [M+Na] ; found 4.38 (m, 2H, H-5Man, H-5Gal), 4.17 (dd, 2J9a,9b = 10.4 Hz, J = 3.8 Hz,
+
+
M+Na] calcd. for C15
64.14847.
r)–2–Azido–1–O–(2,3,4,6–tetra–O–benzoyl––D–
mannopyranosyl)–3–O–(2,3,4,6–tetra–O–benzoyl––D–
8,9a
1H, H-9a), 3.92-3.87 (m, 2H, H-8, H-7a), 3.85 (dd, 2J9a,9b = 10.3 Hz,
4
(
3J8,9b = 7.7 Hz, 1H, H-9b), 3.61 (dd, 2J7a,7b = 11.6 Hz, 3J7b,8 = 6.7 Hz, 1H,
H-7b) ppm; 13C NMR (151 MHz, CDCl
3
) δ 166.1, 166.0, 165.6, 165.5,
galactopyranosyl)–1,3–propanediol (17a). General procedure A was 165.3, (8C, 8 PhC=O), 133.6, 133.5, 133.3, 133.2, 133.1, 132.9, 130.1,
applied to acceptor 12a (167 mg, 241 µmol) and donor 16 (274 mg, 129.9, 129.8, 129.8, 129.6, 129.5, 129.3, 129.2, 129.0, 129.0, 128.9,
3
61 µmol, 1.5 eq.). Reagents and conditions: N-iodosuccinimide 128.7, 128.7, 128.6, 128.5, 128.5, 128.4, 128.3, (48C, 48 C-Ar), 102.0
(
109 mg, 482 µmol, 2 eq.), trifluoromethane sulfonic acid (4.8 l, (C-1Gal), 97.9 (C-1Man), 71.6 (C-3Gal or C-5Gal), 71.5 (C-3Gal or C-5Gal),
1
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 11 of 14
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
3
7
6
(
0.1 (C-2Man), 69.8 (C-3Man or C-2Gal), 69.7 (C-9), 69.6 (C-3Man or C-2Gal), 10.2 Hz, 3J2,3 = 3.3 Hz, 1H, H-3Man), 5.72 (dd, 3J2,3 = 3.2 Hz, J1,2
=
View Article Online
9.2 (C-5Man), 68.0 (C-7), 67.9 (C-4Gal), 62.6 (C-6Gal), 61.9 (C-6Man), 60.3 1.9 Hz, 1H, H-2Man), 5.43 (dd, 3J3,4 = 10.7 HDzO, I 3: J 12 0, 3. 10=399/.3C9HOzB, 010H1,24HG-
C-8) ppm; IR (ATR) vmax/cm-1 2933, 2094, 1721, 1451, 1258, 1066,
3
GlcNTFA), 5.16-5.10 (m, 2H, H-1Man, H-4GlcNTFA), 4.84 (d, 3J1,2 = 8.3 Hz,
+
7
05; ESI-HRMS: m/z calcd. for C71
59
H N
3
O
20 + Na : 1296.35841 1H, H-1GlcNTFA), 4.79 (dd, 2J6a,6b = 12.2 Hz, 3J5,6a = 2.9 Hz, 1H, H-6aMan),
4.54-4.41 (m, 2H, H-5Man, H-6bMan), 4.31 (dd, 2J6a,6b = 12.4 Hz, 3J5,6a
4.7 Hz, 1H, H-6aGlcNTFA), 4.19 (dd, 2J6a,6b = 12.4 Hz, J5,6b = 2.3 Hz, 1H, H-
+
[
M+Na] ; found 1296.35793.
=
(
r)–2–Azido–1–O–(–D–mannopyranosyl)–3–O–(–D–
galactopyranosyl)–1,3–propanediol (18a). General procedure C was 6bGlcNTFA), 4.15-3.98 (m, 3H, H-2GlcNTFA, H-7a, H-9a), 3.87-3.78 (m, 3H,
applied to compound 17a (196 mg, 153 µmol). Reagents and H-8, H-5GlcNTFA, H-7b ), 3.75 (dd, 2J9a,9b = 10.4 Hz, 3J8.9a = 5.7 Hz, 1H, H-
conditions: sodium methoxide (c = 5.4 M in methanol, two drops), 9b), 2.08, 2.05, 2.03 (each s, each 3H, 3 CH
3
C=O) ppm; 13C NMR
) δ 170.8, 170.7, 169.3, 166.4, 165.8, 165.5, 165.4
C=O), 157.6, (q, J = 37.4 Hz, CF C=O) 133.6, 133.5,
methanol (c = 0.03 M, 4.02 mL). Compound 18a (71.3 mg, 98 %) was (126 MHz, CDCl
obtained as a white foam after lyophilisation; [α]20 = +37.1 (c 0.8, (7C, 4 PhC=O, 3 CH
3
D
3
3
1
O) δ 4.89 (d, 3J1,2 = 1.6 Hz, 1H, H-1Man), 133.4, 133.2, 129.8, 129.8, 129.7, 129.1, 128.9, 128.6, 128.5, 128.4
water); H NMR (600 MHz, D
4
9
2
.40 (d, 3J1,2 = 7.9 Hz, 1H, H-1Gal), 4.01-3.95 (m, 3H, H-2Man, H-3Man, H- (24C, 24 C-Ar), 100.7 (C-1GlcTFA), 98.0 (C-1Man), 72.2 (C-5GlcNTFA), 71.5
a), 3.92-3.70 (m, 9H, H-4Man, H-5Man, H-6Man, H-6Gal, H-9b, H-7), 3.67 (C-3GlcNTFA), 70.1 (C-2Man), 70.0 (C-3Man), 69.2 (C-5Man), 68.8 (C-9), 68.2
dd, 3J3,4 = 8.0 Hz, 3J4,5 = 4.1 Hz, 1H, H-4Gal), 3.65-3.61 (m, 3H, H-3Gal
H-8, H-5Gal), 3.51 (dd, 3J2,3 = 9.9 Hz, 3J2, 1 = 7.9 Hz, 1H, H-2Gal) ppm; 13
NMR (151 MHz, D
C-3Gal or C-5Gal), 72.7 (C-3Gal or C-5Gal), 70.7 (C-2Gal), 70.4 (C-4Man or HRMS: m/z calcd. for C51
C-5Man), 69.9 (C-3Man), 68.6 (C-4Man or C-5Man), 68.6 (C-9), 66.9 (2C, C- 1079.29950.
(
,
C
(C-4GlcNTFA), 67.7 (C-7), 66.6 (C-4Man), 62.7 (C-6Man), 61.7 (C-6GlcNTFA),
59.8 (C-8), 55.0 (C-2GlcNTFA), 20.7, 21.0, 20.4 (3C, 3 CH C=O) ppm; IR
O) δ 102.9 (C-1Gal), 99.7 (C-1Man), 75.2 (C-4Gal), 73.0 (ATR) vmax/cm 3327, 2933, 2308, 2103, 1723, 1219, 1027, 708; ESI-
2
3
-1
+
+
(
49 3 4
H F N O19 +H : 1079.30159 [M+H] ; found
, C-8), 61.0 (2C, C-6Man, C-6Gal), 60.2 (C-2Man) ppm; IR (ATR) vmax/cm- (S)–2–Azido–1–O–(2–deoxy–2–trifluoroacetamido–3,4,6–tri–O–
338, 2933, 2124, 1640, 1032; ESI-HRMS: m/z calcd. for C15
7
1
3
27
H N
3
O
12
acetyl–β–(D)–glucopyranosyl)–3–O–(2,3,4,6–tetra–O–benzoyl–α–
D–mannopyranosyl)–1,3–propanediol (20b). General procedure B
was applied to acceptor 12b (150 mg, 216 µmol) and donor 19
+
+
+
Na = 464.14869 [M+Na] ; found 464.14847.
(
s)–2–Azido–1–O–(–D–mannopyranosyl)–3–O–(–D–
galactopyranosyl)–1,3–propanediol (18b). General procedure C was (99.2 mg, 258 µmol, 1.2 eq.). Reagents and conditions: trimethylsilyl
applied to compound 17b (210 mg, 165 µmol). Reagents and trifluoromethanesulfonate (3.7 µl, 22.0 µmol, 0.1 eq.),
conditions: sodium methoxide (c = 5.4 M in methanol, two drops), dichloromethane (c = 0.1 M, 2.87 mL). Flash chromatography with
methanol (c = 0.03 M, 5.5 mL). Compound 18b (61.8 mg, 85 %) was ethyl acetate/cyclohexane 2/3 afforded compound 20b (175 mg, 75
D
D
obtained as a white foam after lyophilisation; [α]20 = +22.9 (c 0.8, %) as a white foam; R
f
= 0.3 (ethyl acetate/cyclohexane 2/3); [α]20
=
1
O) δ 4.88 (d, 3J1,2 = 1.6 Hz, 1H, H-1Man), -14.4 (c 0.8, dichloromethane); H NMR (600 MHz, CDCl
1
water); H NMR (600 MHz, D
2
3
) δ 8.11-8.09
4
4
.40 (d, 3J1,2 = 7.9 Hz, 1H, H-1Gal), 4.06 (dd, 2J9a,9b = 11.0 Hz, 3J8,9a
.0 Hz, 1H, H-9a), 3.99-3.86 (m, 5H, H-2Man, H-5Gal, H-6aMan, H-3Man
=
,
(m, 2H, 2 H-Ar), 8.03-8.01 (m, 2H, 2 H-Ar), 7.98-7.96 (m, 2H, 2 H-Ar),
7.85-7.83 (m, 2H, 2 H-Ar), 7.61-7.56 (m, 2H, 2 H-Ar), 7.53-7.51 (m,
H-7a), 3.82 (dd, 3J3,4 = 9.0 Hz, 3J4,5 = 3.4 Hz, 1H, H-4Gal), 3.79-3.71 (m, 1H, H-Ar), 7.47-7.36 (m, 7H, 7 H-Ar), 7.29-7.27 (m, 2H, 2 H-Ar), 6.94
4
5
H, H-6aGal, H-7b, H-9b, H-6bMan), 3.70-3.61 (m, 5H, H-8, H-3Gal, H- (d, J = 8.9 Hz, 1H, NHCOCF
Man, H-4Man, H-6bGal), 3.52 (dd, 3J2,3 = 9.9 Hz, 3J1,2 = 7.9 Hz, 1H, H-2Gal
5.86 (dd, 3J3,4 = 10.2 Hz, 3J2,3 = 3.3 Hz, 1H, H-3Man), 5.70 (dd, 3J2,3
O) δ 103.4 (C-1Gal), 100.4 (C-1Man), 75.2 3.2 Hz, 3J2, 1 = 1.8 Hz, 1H, H-2Man), 5.38-5.32 (m, 1H, H-3GlcNTFA), 5.16
C-5Man), 73.0 (C-4Man or C-3Gal), 72.7 (C-4Man or C-3Gal), 70.7 (C-2Gal), (dd, J4,5 = J3,4 = 9.6 Hz, 1H, H-4GlcNTFA), 5.09 (d, 3J1,2 = 1.6 Hz, 1H, H-
3
), 6.14 (dd, J4,5 = J3,4 = 10.0 Hz, 1H, H-4Man),
)
=
ppm; 13C NMR (151 MHz, D
2
(
7
(
3
+
0.4 (C-4Gal), 69.9 (C-5Gal), 69.0 (C-7), 68.7 (C-3Man), 67.2 (C-9), 66.7
1
J
Man), 4.83 (d, 3J1,2 = 8.3 Hz, 1H, H-1GlcNTFA), 4.75 (dd, 2J6a,6b = 11.8 Hz,
5,6a = 2.3 Hz, 1H, H-6aMan), 4.48 (m, 2H, H-5Man, H-6bMan), 4.32 (dd,
2J6a,6b = 12.4 Hz, 3J5,6a = 4.5 Hz, 1H, H-6aGlcNTFA), 4.22 (dd, 2J6a,6b
12.4 Hz, 3J5,6a = 2.3 Hz, 1H, H-6bGlcNTFA), 4.15 (dd, 2J9a,9b = 10.0 Hz, 3J8,9a
= 3.9 Hz, 1H, H-9a), 4.12-4.06 (m, 1H, H-2GlcNTFA), 3.98 (dd, 2J7a,7b
10.5 Hz, 3J7a,8 = 4.2 Hz, 1H, H-7a), 3.86-3.78 (m, 3H, H-5GlcNTFA, H-8, H-
mannopyranosyl)–1,3–propanediol (20a). General procedure B was 9b), 3.70 (dd, 2J7a,7b = 10.5 Hz, 3J7b,8 = 4.8 Hz, 1H, H-7b), 2.09, 2.06,
applied to acceptor 12a (188 mg, 271 µmol) and donor 19 (125 mg, 2.05 (each s, each 3H, 3 CH ) δ
C=O) ppm; 13C NMR (151 MHz, CDCl
25 µmol, 1.2 eq.). Reagents and conditions: trimethylsilyl 170.9, 170.7, 169.3, 166.3, 165.8, 165.5, 165.4 (7C, 4 PhC=O, 3
trifluoromethanesulfonate (5.0 µl, 27.0 µmol,
0.1 eq.), CH C=O), 133.6, 133.4, 133.2, 129.9, 129.8, 129.8, 129.7, 129.1,
dichloromethane (c = 0.1 M, 2.7 mL). Flash chromatography with 128.9, 128.6, 128.5, 128.4 (24C, 24 C-Ar), 100.5 (C-1GlcNTFA), 97.8 (C-
C-2Man), 61.0 (2C, C-6Gal, C-6Man), 60.7 (C-8) ppm; IR (ATR) vmax/cm-1
337, 2933, 2103, 1639, 1038; ESI-HRMS: m/z calcd. for C15
27
H N
3
O
12
=
+
+
Na = 464.14869 [M+Na] ; found 464.14846.
(
R)–2–Azido–1–O–(2–deoxy–2–trifluoroacetamido–3,4,6–tri–O–
=
acetyl–β–D–glucopyranosyl)–3–O–(2,3,4,6–tetra–O–benzoyl–α–D–
3
3
3
3
ethyl acetate/cyclohexane 2/3 afforded compound 20a (215 mg, 73
1
Man), 72.3 (C-5GlcNTFA), 71.6 (C-3GlcNTFA), 70.0 (2C, C-2Man, C-3Man), 69.5
D
%
) as a white foam; R
f
= 0.3 (ethyl acetate/cyclohexane 2/3); [α]20
=
(C-9), 69.3 (C-5Man), 68.0 (C-4GlcNTFA), 67.7 (C-7), 66.6 (C-4Man), 62.7 (C-
6Man), 61.7 (C-6GlcNTFA), 59.7 (C-8), 55.0 (C-2GlcNTFA), 21.8, 20.6, 20.4 (3
1
-37.10 (c 0.8, dichloromethane); H NMR (500 MHz, CDCl
3
) δ 8.09-
.07 (m, 2H, 2 H-Ar), 8.03-8.01 (m, 2H, 2 H-Ar), 7.97-7.95 (m, 2H, 2 H- CH
Ar), 7.84-7.81 (m, 2H, 2 H-Ar), 7.61-7.56 (m, 2H, 2 H-Ar), 7.53-7.49 1067, 708; ESI-HRMS: m/z calcd. for C51
m, 1H, H-Ar), 7.47-7.35 (m, 7H, 7 H-Ar), 7.32-7.27 (m, 3H, 2 H-Ar, [M+H] ; found 1079.29953.
3
C=O) ppm; IR (ATR) vmax/cm-1 2933, 2308, 2105, 1724, 1260,
8
+
49 3
H F N
4
O
19 +H : 1079.30159
+
(
NHCOCF
3
), 6.17 (dd, 3J4,5 = J3,4 = 10.1 Hz, 1H, H-4Man), 5.89 (dd, 3J3,4
=
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 12 of 14
ARTICLE
Journal Name
(
R)–2–Azido–1–O–(2–deoxy–2–trifluoroacetamido–β–D–
5
2
6
GlcNAc), 75.7 (C-4Man), 73.2 (C-3Man), 72.7 (C-2Man), 72.6V(iCew-4 AG rl tc i Nc lAe cOonrliCne-
GlcNAc), 72.3 (C-9), 69.8 (C-6Man), 69.4 (C-3GlcNAc), 63.7 (C-7 or C-
DOI: 10.1039/C9OB00124G
glucopyranosyl)–3–O–(–D–mannopyranosyl)–1,3–propanediol
(
1
21a). General procedure C was applied to compound 20a (169 mg,
57 µmol). Reagents and conditions: sodium methoxide (c = 5.4 M in ppm IR (ATR) vmax/cm 3279, 2103, 1557, 1410, 1054; ESI-HRMS: m/z
3
GlcNAc), 63.5 (C-7 or C-6GlcNAc), 63.4 (C-5Man), 58.3 (C-8), 25.0 (CH C=O)
-1
+
+
30 4
methanol, two drops), methanol (c = 0.03 M, 5.23 mL). Compound calcd. for C17H N O12 + Na : 505.17524 [M+Na] ; found 505.17514.
2
1a (74.2 mg, 98 %) was obtained as a white foam after (S)–2–Azido–1–O–(2–deoxy–2–acetamido–β–D–glucopyranosyl)–
D
1
lyophilisation; [α]20 = +14.6 (c 0.8, water); H NMR (600 MHz, D
2
O) 3–O–(––D–mannopyranosyl)–1,3–propanediol (22b). General
δ 4.86 (d, 3J1,2 = 1.5 Hz, 1H, H-1Man), 4.65 (d, 3J1,2 = 8.5 Hz, 1H, H- procedure E was applied to compound 21b (74.0 mg, 137 µmol).
1
6
GlcNTFA), 4.00-3.84 (m, 5H, H-2Man, H-5GlcNTFA, H-9a, H-6aGlcNTFA, H- Reagents and conditions: (i) aq. lithium hydroxide (c = 2 M, 2.8 mL,
Man), 3.82-3.73 (m, 6H, H-6bMan, H-6bGlcNTFA, H-9b, H-7a, H-2GlcNTFA 5.51 mmol, 40 eq.), methanol (c = 0.03 M, 4.59 mL);(ii) sodium
a
,
H-3Man), 3.69-3.58 (m, 4H, H-7b, H-4Man, H-3GlcNTFA, H-4GlcNTFA), 3.48- methoxide (c = 5.4 M in methanol, two drops.), acetic anhydride
3
1
.45 (m, 2H, H-8, H-5Man) ppm; 13C NMR (151 MHz, D
GlcNTFA), 99.9 (C-1Man), 76.1 (C-8), 73.0 (2C, C-3GlcNTFA, C-4GlcNTFA), 70.4 22b (23.0 mg, 58 %) was obtained as a white foam after
2
O) δ 100.6 (C- (34 µl, 363 µmol, 5 eq.), methanol (c = 0.03 M, 2.4 mL). Compound
D
1
2
(C-3Man), 69.9 (C-2Man or C-5GlcNTFA), 69.8 (C-2Man or C-5GlcNTFA), 68.9 (C- lyophilisation; [α]20 = +3.4 (c 0.8, water); H NMR (500 MHz, D O) δ
9
5
1
), 66.8 (C-7), 66.6 (C-4Man), 60.8 (C-6GlcNTFA), 60.6 (C-6Man), 59.9 (C- 4.88 (d, 3J1,2 = 1.7 Hz, 1H, H-1Man), 4.56 (d, 3J1,2 = 8.4 Hz, 1H, H-1GlcNAc),
Man), 56.0 (C-2GlcNTFA) ppm; IR (ATR) vmax/cm-1 3306, 2933, 2102, 3.99-3.95 (m, 2H, H-2Man, H-6aMan), 3.94-3.85 (m, 3H, H-5Man, H-
+
707, 1023; ESI-HRMS: m/z calcd. for C17
27 3
H F N
4
O
12 + Na : 559.14776
5
GlcNAc, H-6bMan), 3.83-3.60 (m, 9H, H-3Man, H-2GlcNAc, H-7a, H-7b, H-
9a, H-9b, H-4Man, H-6GlcNAc), 3.57-3.50 (m, 1H, H-3GlcNAc), 3.46-3.40 (m,
2H, H-4GlcNAc, H-8), 2.04 (s, 3H, CH
C=O) δ ppm; 13C NMR (151 MHz,
O) δ 174.7 (CH C=O), 101.2 (C-1GlcNAc), 99.8 (C-1Man), 76.0 (C-
GlcNAc), 73.8 (C-3GlcNAc), 73.0 (C-4Man), 70.4 (C-3Man or C-5Man), 68.5 (C-
47 µmol). Reagents and conditions: sodium methoxide (c = 5.4 M in 8), 69.9 (C-5GlcNAc), 68.7 (C-3Man or C-5Man), 66.7 (C-2Man), 60.9 (C-6Man
methanol, two drops), methanol (c = 0.03 M, 5.0 mL). Compound 21b or C-6GlcNAC), 60.7 (C-6Man or C-6GlcNAC), 59.9 (C-2GlcNAc), 55.5 (C-7 or C-
74.3 mg, quantitative) was obtained as a white foam after 9), 48.9 (C-7 or C-9), 22.3 (CH
C=O) ppm; IR (ATR) vmax/cm-1 3266,
+
[
M+Na] ; found 559.14681.
(
S)–2–Azido–1–O–(2–deoxy–2–trifluoroacetamido–β–D–
3
glucopyranosyl)–3–O–(–D–mannopyranosyl)–1,3–propanediol
21b). General procedure C was applied to compound 20b (159 mg,
D
2
3
(
1
4
(
3
D
1
12 + Na+:
lyophilisation; [α]20 = +11.8 (c 0.8, water); H NMR (600 MHz, D
δ 4.83 (d, 3J1,2 = 1.7 Hz, 1H, H-1Man), 4.64 (d, 3J1,2 = 8.5 Hz, 1H, H- 505.17524 [M+Na] ; found 505.17505.
2
O) 2114, 1557, 1410, 1054; ESI-HRMS: m/z calcd. for C17
30
H N
4
O
+
1
GlcNTFA), 4.10 (dd, 2J9a,9b = 10.9 Hz, 3J8,9a = 3.5 Hz, 1H, H-9a), 3.95 (dd,
3J2,3 = 3.3 Hz, 3J1,2 = 1.6 Hz, 1H, H-2GlcNTFA), 3.94-3.91 (m, 1H, H-6aMan),
Conflicts of interest
Man, H-2GlcNTFA, H-5GlcNTFA, H6bMan), 3.69-3.60 (m, 4H, H-6bGlcNTFA, H- There are no conflicts to declare.
b, H-4Man, H-3GlcNTFA), 3.57-3.52 (m, 1H, H-7b), 3.50-3.45 (m, 2H, H-
3
3
9
5
1
2
6
.90-3.85 (m, 3H, H-7a, H-4GlcNTFA, H-6aGlcNTFA), 3.82-3.72 (m, 4H, H-
Man, H-8) ppm; 13C NMR (151 MHz, D
Man), 76.0 (C-8), 73.0 (C-3GlcNTFA or C-4Man), 70.4 (C-3Man), 69.9 (C-
2
O) δ 100.9 (C-1GlcTFA), 100.3 (C-
Acknowledgements
Man), 69.4 (C-9), 67.0 (C-7), 66.7 (C-3GlcNTFA or C-4Man), 60.6 (C- The authors are grateful for funding by the DFG (SFB 677) and
GlcNTFA), 60.5 (2C, C-5Man, C-6Man), 56.1 (C-2GlcNTFA) ppm; IR by the Fonds of the Chemical Industry (FCI). We are also very
ATR) vmax/cm 3288, 2933, 2108, 1707, 1022; ESI-HRMS: m/z calcd. thankful to Dr. Claudia Fessele for teaching J. B. the biological
-1
(
+
+
for C17
27 3
H F N
4
O
12 + Na : 559.14776 [M+Na] ; found 559.14623.
assays.
(
R)–2–Azido–1–O–(2–deoxy–2–acetamido–β–D–glucopyranosyl)–
3
–O–(–D–mannopyranosyl)–1,3–propanediol (22a). General
Notes and references
procedure E was applied to compound 21a (76.8 mg, 141 µmol).
Reagents and conditions: (i) aq. lithium hydroxide (c = 2 M, 2.80 mL,
1
(a) B. Alberts, A. Johnson, J. Lewis, D. Morgan, M. Raff, K.
Roberts and P. Walter, in Molecular Biology of the Cell, ed. B.
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Chem. Biol., 2004, 8, 617-625; (c) L. L. Kiessling and R. A.
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Crocker, Current Opinion in Structural Biology, 2002, 12, 609-
5
.66 mmol, 40 eq.), methanol (c = 0.03 M, 4.70 mL); (ii) sodium
methoxide (c = 5.4 M in methanol, two drops), acetic anhydride
44 µl, 465 µmol, 5 eq.), methanol (c = 0.03 M, 3.1 mL). Compound
2a (28.0 mg, 60 %) was obtained as a white foam after
(
2
D
1
lyophilisation; [α]20 = +3.1 (c 0.8, water); H NMR (500 MHz, D
2
O) δ
.84 (d, 3J1,2 = 1.7 Hz, 1H, H-1Man), 4.53 (d, 3J1,2 = 8.5 Hz, 1H, H-1GlcNAc),
.09 (dd, 2J9a,9b = 10.9 Hz, 3J8,9a = 3.5 Hz, 1H, H-9a), 3.96 (dd, 3J2,3
.4 Hz, 3J2,1 = 1.8 Hz, 1H, H-2Man), 3.93-3.83 (m, 4H, H-6aGlcNAc, H-7a,
6
15.
4
4
3
2
(a) I. Ofek, D. Mirelman and N. Sharon, Nature, 1977, 265,
623-625; (b) N. Sharon and H. Lis, Glycobiology, 2004, 14,
53R-62R; (c) K. Ohlsen, T. A. Oelschlaeger, J. Hacker and A. S.
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=
H-5GlcNAc, H-6aMan), 3.81 dd, 3J3,4 = 9.4 Hz, 3J2,3 = 3.4 Hz, 1H, H-3Man),
3
4
(a) S. D. Knight and J. Bouckaert, Top. Curr. Chem., 2009, 288,
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3
3
.78-3.62 (m, 6H, H-6bGlcNAc, H-4Man, H-8, H-3GlcNAc, H-7b, H-9b), 3.58-
.50 (m, 2H, H-5Man, H-6bMan), 3.44 (m,2H, H-4GlcNAc, H-2GlcNAc), 2.04
C=O) ppm; 13C NMR (126 MHz, D
(s, 3H, CH
3
O) δ 177.4 (CH C=O),
2 3
1
04.4 (C-1GlcNAc), 103.1 (C-1Man), 78.7 (C-4GlcNAc or C-2GlcNAc), 76.5 (C-
1
2 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB00124G