Synthesis and binding affinity analysis of α1-2- and α1-6-O/S-linked dimannosides for the elucidation of sulfur in glycosidic bonds using quartz crystal microbalance sensors

Article abstract of DOI:10.1016/j.carres.2017.09.015

The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds have been developed, and the recognition properties assessed in competition binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors were produced using photoinitiated, nitrene-mediated immobilization methods, and the subsequent binding study was performed in an automated flow-through instrumentation, and correlated with data from isothermal titration calorimetry. The recorded K_d-values corresponded well with reported binding affinities for the O-linked dimannosides with affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas the α1-2-analog displayed very similar binding compared to the O-linked structure.

Full text of DOI:10.1016/j.carres.2017.09.015

Accepted Manuscript
Synthesis and binding affinity analysis of α1-2- and α1-6-O/S-linked dimannosides for
the elucidation of sulfur in glycosidic bonds using quartz crystal microbalance sensors
Oscar Norberg, Bin Wu, Niranjan Thota, Jiantao Ge, Germain Fauquet, Ann-Kathrin
Saur, Teodor Aastrup, Hai Dong, Mingdi Yan, Olof Ramström
PII:
S0008-6215(17)30445-7
10.1016/j.carres.2017.09.015
CAR 7454
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 23 June 2017
Revised Date: 26 September 2017
Accepted Date: 30 September 2017
Please cite this article as: O. Norberg, B. Wu, N. Thota, J. Ge, G. Fauquet, A.-K. Saur, T. Aastrup,
H. Dong, M. Yan, O. Ramström, Synthesis and binding affinity analysis of α1-2- and α1-6-O/S-linked
dimannosides for the elucidation of sulfur in glycosidic bonds using quartz crystal microbalance sensors,
Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.09.015.
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ACCEPTED MANUSCRIPT
Graphical Abstract
OH
O
OH
O
HO
HO
HO
HO
HO
HO
O
S
HO
HO
HO
HO
HO
HO
O
O
Measured by QCM
Similar affinity to Con A
OMe
Kd, QCM = 3.05
OMe
Kd, QCM = 3.02
M

ACCEPTED MANUSCRIPT
Synthesis and Binding Affinity Analysis of α1-2- and α1-6-O/S-linked
Dimannosides for the Elucidation of Sulfur in Glycosidic Bonds using
Quartz Crystal Microbalance Sensors
Oscar Norberg,a Bin Wu,b Niranjan Thota,a Jiantao Ge,b Germain Fauquet,a, Ann-Kathrin
Saur,a Teodor Aastrup,c Hai Dong,b* Mingdi Yan,a,d* and Olof Ramström a,d
*
aKTH - Royal Institute of Technology, Department of Chemistry, Teknikringen 36, S-10044
Stockholm, Sweden; Email: ramstrom@kth.se
b School of Chemistry & Chemical Engineering, Huazhong University of Science &
Technology, Luoyu Road 1037, Wuhan, 430074, P. R. China; Email:
hdong@mail.hust.edu.cn
c Attana AB, Björnnäsvägen 21, S-11419, Stockholm, Sweden
dDepartment of Chemistry, University of Massachusetts Lowell, 1 University Ave., MA
01854 Lowell, USA; Email: mingdi_yan@uml.edu
ABSTRACT
The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance
methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-
glycosidic bonds have been developed, and the recognition properties assessed in competition
binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors
were produced using photoinitiated, nitrene-mediated immobilization methods, and the
subsequent binding study was performed in an automated flow-through instrumentation, and
correlated with data from isothermal titration calorimetry. The recorded Kd-values
corresponded well with reported binding affinities for the O-linked dimannosides with
affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked
analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker
affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas
the α1-2-analog displayed very similar binding compared to the O-linked structure.
Keywords: Photochemistry, Thiosaccharides, CuAAC, Carbohydrates, Lectins, Biosensor,
Quartz Crystal Microbalance
1. INTRODUCTION
With the increasing awareness of the role of glycans as key recognition entities in many
fundamental cellular processes, more carbohydrate structures are targeted for therapeutic
development, with several drugs already on the market [1–3]. Many glycans and
glycoconjugates have so far been associated with cancer, viruses, bacteria and parasites, and
have thus been targeted as vaccines. However, since carbohydrates are much less
immunogenic than peptides, glycans conjugated with adjuvants have recently received much
attention [1,4,5]. However, enzymatic degradation of the glycan-antigen remains a problem,
thereby reducing the immune response, and more stable glycan analogs are needed. In this
context, several types of glycans have been envisaged in which the glycosidic oxygen linkage
has been exchanged for a carbon, sulfur or nitrogen atom [6–10]. Although these analogs are
generally more stable, the structural modification may affect the resulting recognition effects.
Not only may the interactions with the glycosidic bond change, but the overall conformation
of the glycan is an important factor to consider [11].
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Studies have shown that various S-linked disaccharides, such as thiolactose [10], thiomaltose
[12], thiocellobiose [13], and glycosyldisulfides [14], display increased conformational
flexibility compared to their natural analogs, and varying degrees of inhibition towards
targeted enzymes. Contrary to these findings, Zhong et al. found that a series of α1-3- and α1-
6-linked thiooligomannosides, derived from the natural substrate, resulted in complete loss of
binding when tested against the enzyme Golgi α-mannosidase II [15]. Glycosidases have
proven unable to hydrolyze thioglycosides [16–20], hypothetically due to the poor hydrogen
bonding ability of the sulfur atom, thus hampering the acidic cleavage of S,O-acetals [21–23].
This effect is however not universal, and O- and S-linked N-acetylglucosamine (GlcNAc)
residues have been shown to undergo cleavage with comparable efficiencies in the presence
of a human O-GlcNAcase [24]. Although these effects are difficult to predict, thioglycosides
are progressively being identified as new therapeutics [25]. For instance, recent studies
explored S-linked glycan analogs as hydrolytically stable immunogens, used either by
themselves or coupled to tetanus toxoid or bovine serum albumin for increased
immunogenicity. The results showed that the glycans efficiently induced immune responses,
which detected not only the S-linked glycan analog but also the naturally occurring O-linked
glycan target [26–28]. Another example is morphine- and codeine-6-glucuronide (M6G and
C6G), two abundant analgesic metabolites of morphine which are both more potent than
morphine and display less side effects making them more attractive as analgesics. However,
the bioavailability is low due to hydrolysis of the glycosidic bond, an issue which was
addressed by MacDougall et al. by introducing an S-linkage [29]. The results showed that the
thioanalogs were full agonists with M6G and that the S-linkage is a viable strategy for
improving the pharmacological properties. Glycan thiols further present potential advantages
in the in vitro synthesis of glycoconjugates/-proteins where site-specific glycosylation is
problematic due to the many competing functional groups in natural glycans. Sulfhydryl
groups present increased nucleophilicity compared to hydroxyl groups, can be oxidized to the
corresponding disulfides, as well as react selectively in many other types of reactions [30–37].
Herein, the role of sulfur in specific glycosidic bonds, with respect to protein binding effects,
has been evaluated using quartz crystal microbalance (QCM) instrumentation where the chips
were functionalized with α-D-mannopyranoside monosaccharides [38–41]. We present the
synthesis of a series of O- and S-linked dimannosides, together with subsequent affinity
evaluation against lectins with known specificity towards oligomannoside glycans. The
binding studies were performed as competition assays where the binding of the tested lectins
towards mannose-functionalized surfaces was detected in a QCM flow-through
instrumentation, which also enabled comparison of the technique with isothermal titration
calorimetry (ITC). Recurring injections of solutions of lectin with the dimannoside inhibitors
of varying concentration generated inhibitory response curves, which fitted well to the
binding data. The resulting EC50-values correlated well Kd-values estimated by ITC for the O-
linked dimannosides.
2. RESULTS AND DISCUSSION
Two sets of disaccharides were chosen for the study (Figure 1), based on previous binding
affinity studies towards the target lectin Con A [42]. α1-6-Linked dimannoside 1 has been
shown to bind with relatively low affinity (similar to the affinity of methyl α-D-
mannopyranoside) whereas the corresponding α1-2-linked dimannoside 3 displayed a 17-fold
increase in affinity. Consequently, binding affinity analysis of the target disaccharides would
illustrate the effect of sulfur in glycosidic linkages, both in high and low affinity binding
events.
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Figure 1. Dimannoside structures studied.
2.1. Synthetic design
The synthetic routes towards the target disaccharides were initially designed based on
glycosylation with suitable selectively protected glycosyl acceptors and thioglycosyl donors
[43], where the donors were chosen based on their reported versatility in synthetic
transformations and relatively mild reaction conditions needed for activation [44]. Glycosyl
acceptors with one free hydroxyl or sulfhydryl group were thus designed to react with fully
protected thioglycosyl donors, under N-iodosuccinimide-mediated and triflic acid-catalyzed
conditions, to produce the corresponding fully protected disaccharides. Although
glycosylation with sulfhydryl acceptors and thioglycosyl donors may potentially fail due to
cross-selectivity between the acceptor sulfur entity and the donor thioether, no reports thereof
could be found.
This strategy proved generally viable, but alternative strategies were also applied. Thus,
glycosyl donors possessing an anomeric acetyl group were additionally designed as
alternative donors for the S-linked disaccharide synthesis, and the synthetic strategy was
subsequently further complemented with a 1-thiopyranose donor and a thioglycosyl acceptor
pair.
2.2. Synthesis of glycosyl acceptors
The syntheses of selectively protected glycosyl acceptors 9 and 12 originated from
commercially available methyl α-D-mannopyranoside 5 and were carried out in
straightforward four/six-steps routes Scheme 1. Methyl α-D-mannopyranoside 5 was first
fully protected with benzylidene groups using benzaldehyde dimethyl acetal in the presence
of catalytic amounts of molecular iodine [45]. The use of iodine is an attractive alternative to
the commonly used hazardous and moisture sensitive acid catalysts and the product was
obtained after 3 h with a yield of 92%. The 2,3-O-benzylidene acetal was then selectively
opened using diisobutylaluminum hydride (DIBAL-H) in hexane, leaving the more stable six-
membered cyclic 4,6-O-acetal intact and producing a mixture of isomers 7a and 7b [46]. The
main product was in this case isomer 7b with the benzyl ether in the axial position. Both
isomers were however used in the subsequent benzylation step; producing fully protected
mannoside 8 in 93% yield over two steps. Reductive opening of the 4,6-O-benzylidene acetal
was then achieved under copper(II) triflate (Cu(OTf)2)-catalysis, successfully leaving the
primary hydroxyl group unprotected and producing hydroxyl glycosyl acceptor 9 with a yield
of 97% [47]. Sulfhydryl glycosyl acceptor 12 was then produced through a protection-
substitution-deprotection route from glycosyl acceptor 9. Initially, triflic anhydride was
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evaluated for the leaving group transformation but discarded due to stability issues of the
triflate product. Tosyl chloride (TsCl) was therefore employed giving mannoside tosylate 10
in 98% yield. The reaction was highly moisture sensitive requiring dry conditions for
completion. Subsequently, the tosylate was replaced by thioacetate in an SN2-reaction using
potassium thioacetate in dimethylformamide (DMF), giving mannoside 11 in 92% yield.
Finally, deacetylation produced sulfhydryl glycosyl acceptor 12 in 76% yield. The reaction
proved very fast and the resulting product was highly prone to oxidation thus reducing the
yield. Limiting the reaction time to 5-10 min sufficiently prevented the formation of the
disulfide.
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Scheme 1. Synthesis of glycosyl acceptors 9, 12, 17, 19 and 21 (Cf. supporting information
for abbreviations and details). a) PhC(OMe)2, I2, MeCN, rt, 3 h (92%); b) DIBAL-H (hexane),
DCM, 0 °C, 2 h (7a + 7b: 93%); c) BnBr, NaH, DMF, 0 °C-rt, 2.5 h (99%); d) BH3·THF,
Cu(OTf)2, THF, rt, 2 h (87%); e) pyridine, MS 4 Å, TsCl, rt, 4 h (98%); f) KSAc, DMF, 60
°C, 4 h (92 %); g) NaOMe, MeOH, rt, 5-10 min, (76 %); h) TMSCl, DIPEA, DCM, 0 °C, 25
h (quant.); i) i) PhCHO, TMSOTf, -78 °C, 2 h, ii) PhCHO, Et3SiH, -78 °C, 3 h (94%); j)
pyridine, Tf2O, DCM, -15 °C, 2.5 h (99%); k) TBANO2, toluene, 70 °C, 24 h (70%); l) KSAc,
5

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DMF, 40 °C, 16 h (89%); m) NaOMe, MeOH, rt, 5-10 min (77%); n) PhC(OMe)2, TsOH,
DMF, rt, 6 h (85%); o) Pyridine, Tf2O, DCM, -20 °C, 2 h (85%).
The syntheses of glycosyl acceptors 17 and 19 started from methyl α-D-glucopyranoside 13
and were based on the key intermediate triflate 16 Scheme 1. Full protection using
trimethylsilyl chloride (TMSCl) efficiently produced protected glucose derivative 14 in
quantitative yield. Then, a one-pot two-step deprotection/protection reaction readily gave
glucoside 15 selectively deprotected in the 2-O-position [48]. A 4,6-benzylidene acetal was
first obtained using benzaldehyde and the Lewis acid trimethylsilyl triflate (TMSOTf),
followed by reductive etherification using benzaldehyde and triethylsilane. Interestingly,
Wang et al. reported the use of tetrabutylammonium fluoride (TBAF) to cleave off the final
trimethylsilyl protecting-group in the 2-O-position [48]. However, this was deemed
unnecessary since the deprotected glucoside 15 was already observed. Subsequent triflation of
the hydroxyl group in the C2-position and nitrite/thioacetate inversion would then produce the
correct mannose-configuration. Triflation was easily performed using triflic anhydride in
pyridine, producing compound 16 in 99% yield without the need for column purification.
Inversion was then performed either with tetrabutylammonium nitrite (TBANO2) producing
hydroxyl glycosyl acceptor 17 in 70% yield, or with tetrabutylammonium
thioacetate/potassium thioacetate (TBASAc/KSAc) producing mannoside 18 in 89% yield,
according to a modified procedure by Pei et al [49]. Higher yields were generally obtained
with KSAc in DMF compared to TBASAc in toluene, likely because of the feasible lower
reaction temperature (40 °C instead of 70 °C) and less demanding purification. Thioacetate 18
was then treated with sodium methoxide in methanol to give sulfhydryl glycosyl acceptor 19
in 77% yield.
Following unsuccessful glycosylations using 2-thio-acceptors and different donors, the
reverse strategy was instead pursued with 1-thio-α-D-mannopyranosides and corresponding 2-
O-triflate glucoside acceptors. The best results were in this case obtained with thioglycoside
acceptor 21, prepared in two efficient steps from methyl α-D-glucopyranoside 13 according to
Knapp et al [50]. Thus, the 4,6-benzylidene acetal was first produced using benzaldehyde
dimethyl acetal under acidic conditions, and compound 21 obtained following pyridine-
assisted triflate formation in dichloromethane at -20 °C.
2.3. Synthesis of glycosyl donors
Peracetylated and perbenzylated glycosyl donors 23 and 25 were initially envisioned for the
glycosylation reactions, primarily for their facile synthesis Scheme 2. Lewis acid catalyzed
coupling of ethanethiol gave glycosyl donor 23 in moderate yield, and, following Zemplén
deprotection and perbenzylation, produced glycosyl donor 25 in 40% yield over two steps. As
reported for several types of glycosyl donors, the electronic properties of the neighboring
protecting groups significantly affect the reactivity of the activatable species, in this case the
thioethyl-moeity [51–55]. For this reason, “superarmed” glycosyl donor 27 was synthesized
in a straightforward five step route through key intermediate 26 Scheme 2. Orthoester 26 was
synthesized according to Beignet et al [56]. starting from peracetylated D-mannose 22. Lewis
acid-promoted ring-opening under dry conditions then produced glycosyl donor 27 in 68%
yield. In addition to the 1-thioethyl glycosyl donors 23, 25 and 27, 1-acetyl-glycosyl donor 29
was synthesized from key intermediate 26 in a two-step deprotection-protection reaction
according to Ponpipom [57]. Orthoester 26 was thus hydrolyzed in aqueous acetic acid and
acetylated using acetic anhydride, giving glycosyl donor 29 in 77% yield over two steps.
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Scheme 2. Synthesis of glycosyl donors 23, 25, 27, and 29 (Cf. supporting information for
abbreviations and details); a) EtSH, BF3·Et2O, DCM, 0 °C-rt, 24 h (51%); b) NaOMe, MeOH,
rt, 3 h (quant.); c) BnBr, NaH, DMF, 0 °C-rt, 4 h (40%); d) EtSH, HgBr2, MeCN, 60 °C, 24 h
(68%); e) AcOH:H2O (1:2), 100 °C, 4 h; f) Ac2O, pyridine, DMAP, rt, 1 h (77% over two
steps).
2.4. Glycosylation reactions
Initially, the hydroxyl glycosyl acceptor 9 was allowed to react with the peracetylated
mannosyl donor 23 under iodonium promotion, as described by Veeneman et al [43]. It was
thus reported that glycosylation with a β-galactose derivative resembling mannosyl donor 23
(benzoyl instead of acetyl) underwent quick and selective formation of the β-anomer in up to
98% yield, whereas the same reaction with a β-glucose derivative resembling mannosyl donor
25 resulted in a mixture of α/β-anomers. Surprisingly, in the present case, no reaction could
be observed using peracetylated mannosyl donor 23, presumably due to the deactivating
acetyl protecting groups. On the other hand, glycosylation using perbenzylated mannosyl
donor 25 proceeded quickly, resulting in a complex mixture. However, electrospray ionization
mass spectrometry (ESI-MS) and 1H-NMR analysis confirmed the presence of the
disaccharide formation as an anomeric mixture, albeit in low yield.
Glycosyl donor 27 was subsequently applied to the same reaction conditions with glycosyl
acceptors 9 and 17, successfully yielding α1-6- and α1-2-linked disaccharides 30 and 33
Scheme 3. Here, the combination of lower deactivation from the less electron withdrawing
benzyl groups and the activating and directing acetyl group efficiently produced the α-anomer
in short time [53,54]. Deprotection was performed in a two-step one-pot reaction, starting
with deacetylation using sodium methoxide in methanol. After completion of the starting
material, as determined by TLC, acetic acid was added to quench the base and the reaction
mixture was stirred in the presence of palladium under hydrogen atmosphere, forming
unprotected disaccharides 1 and 3 in 77% and 85% yield, respectively.
7

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Scheme 3. Glycosylation reactions leading to dimannosides 1-4. (Cf. supporting information
for abbreviations and details); a) 27, NIS, TfOH, DCM, MS 4 Å, -15 °C, 15 min (30, 80%,
33, 39%); b) i: NaOMe, MeOH, rt, 6-24 h, ii: H2, Pd/C, AcOH, MeOH, 24-72 h (1, 77%, 3,
85%); c) 29, BF3·Et2O, DCM, MS 3 Å, 0 °C-rt, 13 h (61%); d) i: Li, NH3 (l), THF, -78 °C, 2
min, ii: pyridine, Ac2O 17 h (85%); e) Li, MeOH, 2.5 h (94%); f) 1-thio-α-D-mannopyranose
sodium salt, DMF, 50 °C, 18 h, (25%); g) DCM/MeOH/AcOH 1:1:2 (v/v), 50 °C, (76%).
The sulfhydryl acceptors proved more challenging. Reactions under the aforementioned
conditions with glycosyl acceptor 12 and glycosyl donor 27 yielded no disaccharide but
instead the 1-methoxy mannoside side product, as determined by NMR-analysis. Attempts
were made to pre-activate the glycosyl donor 27 by mixing it with N-iodosuccinimide and
triflic acid prior to addition of the glycosyl acceptor, however resulting in the same product
mixture. The glycosyl acceptor 12 was instead allowed to react with orthoester 26 and HgBr2,
8

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since these conditions proved successful in the formation of glycosyl donor 27.
Unfortunately, even after 24 h, no disaccharide was formed and only the corresponding
disulfide as well as the hydrolyzed orthoester could be observed. The glycosyl donor 29 was
subsequently evaluated according to a modified procedure by Abronina et al. Scheme 3 [58].
Using the strong Lewis acid BF3·Et2O, the desired S-linked disaccharide 31 was thus
produced in 61% yield. Deprotection of the S-linked disaccharide 31 was initially evaluated
using FeCl3, however quickly abandoned due to purification issues. Deprotection using the
same procedure as for the O-linked disaccharides 30 and 33 was instead attempted, and
although this route initially looked promising, the resulting monosaccharide species indicated
that the glycosidic linkage was too labile under these conditions. Finally, efficient
deprotection was successfully achieved using Birch reduction and an acetylation-
deacetylation protocol according to Cumpstey et al [32]. The protected disaccharide 31 was
thus subjected to liquid ammonia containing dissolved lithium for 2 min before quenching
using ammonium chloride. To simplify purification, the resulting salt mixture was subjected
to acetic anhydride and pyridine overnight, giving the peracetylated disaccharide 32 in 85%
yield. Subsequent deacetylation using lithium in methanol provided the desired S-linked
disaccharide 2 in 94% yield.
The synthesis of the α1-2-S-linked disaccharide was initially evaluated by the same route as
for the α1-6-linked analog using glycosyl donor 29 and glycosyl acceptor 19. Unfortunately,
no disaccharide was formed although both starting materials were quickly consumed. The 1-
methoxy mannoside side product was instead formed together with a highly polar
carbohydrate residue and benzaldehyde, indicating that the 4,6-benzylidene acetal in glycosyl
acceptor 19 had been removed. Varying the reaction temperature did not result in better
selectivity. Attempts to replace the 4,6-benzylidene acetal by more robust benzyl protecting
groups proved unsuccessful, and an alternative route was instead devised using the reversed
reactivities of the two carbohydrate moieties. The synthesis of α1-2-S-linked dimannoside 4
was thus performed in a 2-step route from the unprotected 1-thio-α-D-mannopyranose sodium
salt [59], and triflate derivative 21. Nucleophilic substitution of the triflate group in
dimethylformamide at 50 °C for 18 h afforded dimannoside 34 in relatively low yield,
showing the difficulties in efficiently forming α1-2-S-linked dimmanosides. Deprotection of
the benzylidene moiety under mildly acidic conditions subsequently led to compound 4 in
76% yield.
2.5. Binding affinity study
The relative binding affinities of the synthesized mannosides towards the target lectin Con A
were determined by competition assays using a QCM flow-through instrumentation and
mannose-functionalized sensor chips, produced by methodology reported previously [39,40].
The surfaces were thus prepared using a three-step method from polystyrene-coated, gold-
plated QCM crystals (Scheme 4). This involved conjugation of NHS-activated 4-azido-
2,3,5,6-tetrafluorobenzoic acid to the polymer surfaces using UV-irradiation, followed by
amide formation with an alkyne-containing, amine-terminated diethyleneglycol linker
creating an alkyne-functionalized surface. Finally, an α-D-mannopyranosyl azide structure
was coupled to the surfaces using copper-catalyzed alkyne-azide cycloaddition (CuAAC)
chemistry.
9

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Scheme 4. Functionalization of QCM sensor surfaces using photoinitiated, nitrene-mediated
coupling and copper-catalyzed alkyne-azide cycloaddition.
The mannose-functionalized surfaces were subjected to injections of solutions containing Con
A (2 µM) and either of the mannoside inhibitors in different concentrations. The observed
reduction in measured frequency, compared to injections with pure Con A, was taken as a
measure of the competitive effect of the tested mannosides. The quantitative binding was then
subjected to nonlinear regression analysis using a dose-response inhibition model (equation 1,
cf. Supporting Information).
ꢇ
ꢋꢇ
ꢈꢌꢍ
ꢈꢉꢊ
ꢀ ꢁ ꢂꢃꢄꢅ
ꢆ
(1)
ꢞ
ꢠ
ꢞ
ꢠ
ꢧꢨ
ꢥꢦ
ꢑꢒꢓꢔꢔ ꢕꢖꢗꢘꢘ ꢙ ꢚꢛꢜꢝ ꢟ ꢡꢔꢖꢢ ꢣꢤ
ꢎꢏꢎꢐ
During the binding studies, an unexpected binding behavior was noticed during the injections
with high inhibitor concentrations Figure 2. Control injections in the absence of lectin
however showed the same behavior, indicating that the resulting frequency change emanated
from an instrumental artifact due to the change of buffer composition. Subtraction of the
lectin response curves with the corresponding inhibitor response curves thus resulted in the
corrected binding Figure 2. Control experiments using α/β-D-galactose at varying
concentrations confirmed the trend, simultaneously validating the methodology.
10

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Figure 2. Example of binding analysis using QCM instrumentation. Frequency responses of:
a) injections with Con A in the presence of methyl α-D-mannopyranoside 5; b) injections with
methyl α-D-mannopyranoside in the absence of Con A (higher ligand concentrations lead to
higher frequency shifts); c) corrected response curves (ligand frequency effect subtracted
from original frequency shifts; higher ligand concentrations lead to lower lectin binding and
lower frequency shift).
The results from the binding study are presented in Figure 3 and Table 1. The stable surfaces
allowed for reproducible binding over time, thus generating inhibitory response curves which
fitted well to the binding data. Furthermore, the EC50-values obtained for methyl α-D-
mannopyranoside 5 were used as reference points, calibrating the surfaces between batches.
Compound 5 was also used as reference in the affinity estimations, using a reported Kd of 122
µM [60]. As can be seen, the α1-6-O-linked dimannoside 1 and the α1-2-O-linked
dimannoside 3 correlated well with previously reported Kd-values, allowing for simplified
comparison between the inhibitors [60]. Contrary to previously published studies, however,
the α1-6-O-linked dimannoside 1 showed almost twice as high inhibition as the
monosaccharide in the present case. Comparison of the α1-6-linked dimannosides 1 and 2
indicated slightly weaker binding of the S-linked dimannoside; results that are in correlation
with previous studies of S-linked disaccharides [10,13]. Furthermore, the results showed a
steeper inhibitory response curve for compound 2, with a corresponding Hill coefficient of
1.8, indicating apparent positive cooperativity. More in-depth analysis is required to elucidate
the reason behind this effect. The α1-2-O-linked dimannoside 3 and the α1-2-S-linked analog
4 both displayed Kd-values of around 3.0 µM, making them the strongest inhibitors in the
study, with 23 times higher affinities than compound 1. No significant increase of the Hill
coefficient was however observed for the α1-2-S-linked dimannoside.
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Figure 3. Inhibitory dose-response curves for interactions of dimannosides 1-4 with
Concanavalin A. Data normalized with respect to frequency response, and estimated EC50-
values of the reference ligand (EC50,ref) used to correlate experiments based on different
surfaces for visualization purposes (cf. Supporting Information). Man = D-mannose; Gal = D-
galactose.
Table 1. Binding data; ITC data taken from [42] and [60]; H = Hill coefficient; RP = relative
potency; Man = D-mannose
Compound
K
d,QCM (µM)
69.3
95% CI (µM) RPQCM HQCM Kd,ITC (µM) RPITC
(58.5-80.4)
(133-182)
(2.4-3.55)
(2.54-3.57)
(493-1280)
1
0.45
23
1
1.8
1
123
-
1
-
1
2
155
3.05
7.09
-
17
-
3
3.02
23
1.2
1
4
Man
795
0.087
452
0.1
3. CONCLUSIONS
A series of O- and S-linked carbohydrates have been designed and synthesized for the
evaluation of S-glycosides in lectin binding. Glycosyl acceptors possessing a single
hydroxyl/sulfhydryl group were synthesized in few steps in generally high yields. The O-
linked disaccharides were obtained with thioethyl-linked glycosyl donors under NIS/TfOH
conditions. The same methodology however failed for the S-linked disaccharides, likely
because of low selectivity between the sulfur entities. The synthesis of the α1-6-S-linked
dimannoside was instead completed through glycosylation with a 1-O-acetyl-glycosyl donor
under Lewis acid catalysis, and the α1-2-S-linked structure was formed from the reaction
between the unprotected 1-thiomannose donor and a triflate acceptor. The O-linked
disaccharides were deprotected using classical catalytic hydrogenation whereas the S-linked
disaccharide was deprotected using Birch reduction.
A competition binding assay was performed using a QCM flow-through instrumentation with
prefabricated mannose-functionalized surfaces and the model lectin Con A. The methodology
proved valid and the acquired Kd-values correlated well with reported affinity values for the
tested O-linked mannosides using isothermal titration calorimetry. The relationship between
12

ACCEPTED MANUSCRIPT
the studied mannosides followed the expected trends, and the α1-6-S-linked dimannoside
displayed similar quantitative inhibition as the α1-6-O-linked dimannoside, however with a
slightly higher Kd-value and a clear apparent positive cooperativity. The highest affinities of
the series were obtained for the α1-2-linked dimannosides, both of which demonstrated Kd-
values in the lower micromolar range. The binding effects were in this case very similar
between the O- and the S-linked structures. These data demonstrates the potential for the S-
linked carbohydrate structures to serve as substitutes for their O-linked counterparts in
biomedical applications.
4. EXPERIMENTAL SECTION
General Methods: All commercially available starting materials were of reagent grade and
1
13
used as received. H and C Nuclear Magnetic Resonance (NMR) data were recorded on a
Bruker Avance 400 instrument at 400 MHz (1H) or a Bruker DMX 500 instrument at 500
MHz (1H) or 125 MHz (13C). Chemical shifts are reported as δ values (ppm) with either
13
1
CHCl3/CDCl3 (1H: δ = 7.26, C = 77.16) or HDO (1H: δ = 4.79) as internal standards. H
1
peak assignments were made by first order analysis of the spectra supported by standard H-
1H correlation spectroscopy (COSY). Thin layer chromatography (TLC) was performed on
precoated Cromatofolios AL Silica gel 60 F254 plates (Merck). Flash column
chromatography was performed on silica gel 60, 0.040-0.063 mm (SDS). Spincoating was
performed using a Cookson Electronics Specialty Coating Systems Spincoater model
P6708D. Photoreactions were performed at 254 nm using a photochemical reactor from
Rayonet Srinivasan - Griffin. Details of all synthetic protocols are provided in the Supporting
information, including a list of abbreviations of reagents and solvents.
4.1. General surface functionalization
QCM analyses were performed using flow-through Attana A200 or A100 C-Fast QCM
systems. Gold-plated and polystyrene-coated [38], 10 MHz QCM crystals were obtained from
Attana AB. The polystyrene surfaces were functionalized with α-D-mannopyranoside
monosaccharides by Photo-Click functionalization methodologies previously developed in
our
laboratory
[39–41].
A
continuous
flow
of
running
buffer
tris(hydroxymethyl)aminomethane (Tris) 10 mM or PBS 10 mM, pH 7.4, 25 µL/min) was
used throughout the experiments, and samples of Con A were prepared in the same buffer.
The
crystals
were
washed/equilibrated
with
buffer
solution
prior
to
manipulations/measurements.
4.2. QCM analysis
Concanavalin A (Con A) and bovine serum albumin (BSA) were purchased from Sigma-
Aldrich. After equilibration of the crystals in the flow-through system, they were subjected to
repeated injections of BSA, followed by injections of low pH buffer (pH 1.5) and finally
additional injections of BSA to fully block any non-functionalized surfaces. Binding to the
surfaces was monitored by frequency logging with Attester 1.1 (Attana), and
adsorption/desorption to the surface recorded as the resulting frequency shifts. Solutions of
the lectins were then injected on the system, where the resulting shifts in frequency
correspond to binding to the surfaces. Bound lectins were released from the surfaces between
measurements by two successive injections of low pH buffer (pH 1.5). The procedure was
then repeated at least two times for each carbohydrate inhibitor concentration to give an
average value and determine the surface stability over time.
ACKNOWLEDGMENTS
13

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This study was supported in part by the Swedish Research Council, the National Institutes of
Health (R01GM080295 and 2R15GM066279, to MY), the National Nature Science
Foundation of China (Nos. 21272083), the Royal Institute of Technology and Attana AB. NT
thanks the Wenner-Gren Foundation for a postdoctoral fellowship grant.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/
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Highlight:
1. Synthetic routes towards α1-2- and α1-6- dimannosides with S- or O-glycosidic bonds.
2. Their recognition properties assessed in competition binding assays with Con A.
3. The role of sulfur in glycosidic bonds has been evaluated using QCM methodology.
4. The binding effects were very similar between the O- and the S-linked structures.

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Supporting Information
Synthesis and Binding Affinity Analysis of α1-2- and α1-6-O/S-linked Dimannosides for the
Elucidation of Sulfur in Glycosidic Bonds using Quartz Crystal Microbalance Sensors
Oscar Norberg,a Bin Wu,b Niranjan Thota,a Jiantao Ge,b Germain Fauquet,a Ann-Kathrin Saur,a
Teodor Aastrup,c Hai Dong,b* Mingdi Yan,a,d* and Olof Ramström a,d*
a Department of Chemistry, KTH - Royal Institute of Technology, Teknikringen 30, S-10044, Stockholm, Sweden.
b School of Chemistry & Chemical Engineering, Huazhong University of Science & Technology, Luoyu Road 1037,
Wuhan, 430074, P. R. China.
c Attana AB, Björnnäsvägen 21, S-11419, Stockholm, Sweden.
d Department of Chemistry, University of Massachusetts, Lowell, 1 University Ave., Lowell, MA 01854, USA.
Table of Contents
Abbreviations of reagents and solvents .............................................................................................S1
Synthesis of 6-hydroxyl and 6-sulfhydryl glycosyl acceptors...........................................................S1
Synthesis of 2-hydroxyl and 2-sulfhydryl glycosyl acceptors...........................................................S3
Synthesis of glycosyl donors..............................................................................................................S5
Glycosylation reactions......................................................................................................................S6
Binding analysis.................................................................................................................................S9
References........................................................................................................................................S10
NMR spectra ....................................................................................................................................S11
Abbreviations of reagents and solvents
AcOH: acetic acid; Ac2O: acetic anhydride; BnBr: benzyl bromide; DCM: dichloromethane;
DIBAL-H: diisobutylaluminum hydride; DIPEA: diisopropylethylamine; DMAP: 4-
dimethylaminopyridine; DMF: dimethylformamide; Hex: hexanes; KSAc: potassium thioacetate;
MeCN: acetonitrile; MS: molecular sieves; NaOMe: sodium methoxide; NIS: N-iodosuccinimide;
OTf: triflate; rt: room temperature; TBANO2: tetrabutylammonium nitrite; THF: tetrahydrofuran;
TMSCl: trimethylsilyl chloride; TMSOTf: trimethylsilyl triflate; Tf2O: triflic anhydride; TsCl: tosyl
chloride; TsOH: tosylic acid
Synthesis of 6-hydroxyl and 6-sulfhydryl glycosyl acceptors
Methyl 2,3:4,6-di-O-benzylidene-α-D-mannopyranoside (6) (Ennis et al., 2002). Commercially
available methyl α-D-mannopyranoside (5, 1.95 g, 10.0 mmol) was dissolved in dry acetonitrile (20
mL) and stirred at rt. Benzaldehyde dimethyl acetal (3.22 mL, 22.1 mmol) and I2 (765 mg, 3.01
mmol) was added and the mixture was stirred at rt for 5.5 h. The mixture was then diluted with
DCM and washed with water containing Na2S2O5 (950 mg, 5.00 mmol). The organic phase was
dried over MgSO4 and evaporated under reduced pressure giving 3.9 g of crude product. The crude
product was separated by flash column chromatography with solvent system DCM:MeOH 12:1
1
giving pure compound 6 (3.43 g, 92%). TLC (Hex/EtOAc 3:1): Rf 0.51; H-NMR (500 MHz,
CDCl3): 7.55-7.55 (m, 2 H, ArH), 7.48-7.50 (m, 2 H, ArH), 7.37-7.43 (m, 6 H, ArH), 6.32 (s, 1 H,
PhCH), 5.67 (s, 1 H, PhCH), 5.05 (as, 1 H, H-1), 4.66 (dd, J = 5.6 Hz and 8.0 Hz, 1 H, H-3), 4.36-
4.42 (m, 1 H, H-6), 4.17 (d, J = 5.4 Hz, 1 H, H-2), 3.93 (at, J = 8.7 Hz, 1 H, H-4), 3.83-3.90 (m, 2 H,
H-5, H-6’) 3.44 (s, 1 H, OMe).
Methyl 2/3-O-benzyl-(R)-4,6-O-benzylidene-α-D-mannopyranoside (7a,b) (Tanaka et al., 2008).
Compound 6 (985 mg, 2.66 mmol) was dissolved in dry DCM (40 mL) and stirred at 0 °C. Then
AlCl3 (18 mg, 0.133 mmol) was added and mixture was stirred under nitrogen while DIBAL-H (5.9
1

ACCEPTED MANUSCRIPT
mL, 1M in hexane) was added drop wise. Mixture was then stirred at 0 °C for 2 h before quenching
by addition of MeOH. The reaction mixture was then diluted with 1M HCl (aq) and extracted with
DCM (2 × 50 mL). The combined organic phases were washed with brine, dried over MgSO4 and
evaporated under reduced pressure giving 1.03 g of crude product. The crude product was separated
by flash column chromatography with solvent system Hex:EtOAc 5:1 giving pure compounds 7a
and 7b, which could both be used in the next step (921 mg, 93%). 7a: TLC (Hex/EtOAc 5:1): Rf
0.24; 1H-NMR (500 MHz, CDCl3): 7.48-7.51 (m, 2 H, ArH), 7.28-7.41 (m, 8 H, ArH), 5.62 (s, 1 H,
PhCH), 4.85 (d, J = 11.9 Hz, 1 H, PhCH2), 4.77 (d, J = 1.1 Hz, 1 H, H-1), 4.72 (d, J = 11.9 Hz, 1 H,
PhCH2), 4.28 (dd, J = 4.4 Hz and 9.7 Hz, 1 H, H-6), 4.10 (at, J = 9.3 Hz, 1 H, H-4), 4.05-4.06 (m, 1
H, H-2), 3.91 (dd, J = 3.5 Hz and 9.6 Hz, 1 H, H-3), 3.87 (at, J = 10.0 Hz, 1 H, H-6), 3.79-3.84 (m,
1 H, H-5), 3.38 (s, 3 H, OMe), 2.64 (d, J = 1.2 Hz, 1 H, OH). 13C-NMR (125 MHz, CDCl3): 138.2,
137.7, 129.1, 128.6, 128.4, 128.1, 128.0, 126.2, 101.8, 101.2, 79.0, 75.7, 73.2, 70.1, 69.1, 63.3, 55.1.
1
7b: TLC (Hex/EtOAc 5:1): Rf 0.13; H-NMR (500 MHz, CDCl3): 7.47-7.50 (m, 2 H, ArH), 7.31-
7.39 (m, 8 H, ArH), 5.58 (s, 1 H, PhCH), 4.74-4.77 (m, 2 H, H-1, PhCH2), 4.69 (d, J = 11.6 Hz, 1 H,
PhCH2), 4.26 (dd, J = 4.4 Hz and 9.8 Hz, 1 H, H-6), 4.06-4.10 (m, 1 H, H-3), 3.91 (t, J = 9.6 Hz, 1
H, H-4), 3.75-3.85 (m, 3 H, H-2, H-5, H-6’), 3.37 (s, 3 H, OMe), 2.34 (d, J = 8.0 Hz, 1 H, OH). 13C-
NMR (125 MHz, CDCl3): 137.8, 137.5, 129.3, 128.8, 128.4, 128.3, 128.1, 126.4, 102.3, 99.6, 79.7,
78.6, 73.9, 69.0, 68.9, 63.5, 55.1.
Methyl 2,3-di-O-benzyl-(R)-4,6-O-benzylidene-α-D-mannopyranoside (8) (Crich et al., 2004).
Compound 7a and 7b (865 mg, 2.32 mmol) was dissolved in dry DMF and stirred at 0 °C. Then
NaH (370 mg, 9.29 mmol) was added and mixture was stirred under nitrogen for 1 h before drop
wise addition of benzyl bromide (300 µL, 2.56 mmol). Mixture was stirred for 2.5 h after which it
was poured on ice water and extracted with diethyl ether twice. The combined organic phases were
dried over MgSO4 and evaporated under reduced pressure giving 1.06 g of pure product 8 (99%).
1
TLC (Hex/EtOAc 3:1): Rf 0.47; H-NMR (500 MHz, CDCl3): 7.50-7.51 (m, 2 H, ArH), 7.27-7.39
(m, 8 H, ArH), 5.65 (s, 1 H, PhCH), 4.80-4.84 (m, 2 H, PhCH2), 4.74 (d, J = 12.4 Hz, 1 H, PhCH2),
4.69 (d, J = 1.2 Hz, 1 H, H-1), 4.65 (d, J = 12.3 Hz, 1 H, PhCH2), 4.22-4.27 (m, 2 H, H-4, H-6),
3.94 (dd, J = 3.3 Hz and 10.1 Hz, 1 H, H-3), 3.89 (at, J = 10.3 Hz, 1 H, H-6’), 3.83-3.84 (m, 1 H, H-
13
2), 3.77 (dt, J = 4.6 Hz and 9.8 Hz, 1 H, H-5), 3.32 (s, 3 H, OMe). C-NMR (125 MHz, CDCl3):
138.9, 138.3, 137.9, 129.0, 128.5, 128.4, 128.3, 128.2, 127.9, 127.6, 127.6, 126.2, 101.6, 100.7,
79.3, 76.5, 76.4, 73.8, 73.2, 69.0, 64.2, 55.0.
Methyl 2,3,4-tri-O-benzyl-α-D-mannopyranoside (9) (Belakhov et al., 2004). Borane (11.2 mL,
1M in THF) was added to compound 8 (1.04 g, 2.25 mmol) under nitrogen. Reaction mixture was
then stirred for 10 min until all s.m. was dissolved. Then Cu(OTf)2 (41 mg, 0.112 mmol) in THF (1
mL) was added drop wise and the reaction mixture was stirred at rt for 2 h. The mixture was then
cooled to 0 °C before slow addition of MeOH (3 mL). The mixture was then evaporated under
reduced pressure giving 1.09 g of crude product which was separated by flash column
chromatography with solvent system Hex:EtOAc 3:1 giving pure compound 9 (904 mg, 87%). TLC
1
(Hex/EtOAc 3:1): Rf 0.09; H-NMR (500 MHz, CDCl3): 7.27-7.37 (m, 15 H, ArH), 4.94 (d, J =
10.9 Hz, 1 H, PhCH2), 4.78 (d, J = 12.4 Hz, 1 H, PhCH2), 4.63-4.71 (m, 5 H, H-1, PhCH2), 3.89 (at,
J = 9.4 Hz, 1 H, H-4), 3.90 (dd, J = 3.1 Hz and 9.5 Hz, 1 H, H-3), 3.83-3.87 (m, 1 H, H-6), 3.75-
3.80 (m, 2 H, H-2, H-6’), 3.62 (m, 1 H, H-5), 3.30 (s, 3 H, OMe). 1.99 (at, J = 6.4 Hz, 1 H, OH).
13C-NMR (125 MHz, CDCl3): 138.6, 138.6, 138.4, 128.6, 128.5, 128.2, 128.0, 127.9, 127.9, 127.7,
99.5, 80.4, 75.3, 75.1, 74.9, 73.1, 72.4, 72.2, 62.6, 54.9.
Methyl 2,3,4-tri-O-benzyl-6-O-tosyl-α-D-mannopyranoside (10) (Kurhade et al., 2011).
Compound 9 (950 mg, 2.05 mmol) was dissolved in pyridine (10 mL) and molecular sieves (4Å)
were added. Mixture was then stirred at 0 °C under nitrogen for 1 h before addition of p-
toluenesulfonyl chloride (1.37 g, 7.16 mmol). The mixture was then stirred for 4 h after which it
was diluted with EtOAc and washed with 1M HCl (aq), sat. NaHCO3 (aq), water and finally dried
over MgSO4 and evaporated under reduced pressure. The crude product was separated by flash
2

ACCEPTED MANUSCRIPT
column chromatography with solvent system Hex:EtOAc 9:1 giving pure compound 10 (1.24 g,
87%). TLC (Hex/EtOAc 3:1): Rf 0.53; 1H-NMR (500 MHz, CDCl3): 7.75-7.77 (m, 2 H, ArH), 7.23-
7.35 (m, 15 H, ArH), 7.16-7.18 (m, 2 H, ArH), 4.87 (d, J = 11.0 Hz, 1 H, PhCH2), 4.69 (d, J = 12.6
Hz, 1 H, PhCH2), 4.63-4.66 (m, 2 H, H-1, PhCH2), 4.54-4.59 (m, 2 H, PhCH2), 4.45 (d, J = 10.9 Hz,
1 H, PhCH2), 4.27 (dd, J = 1.9 Hz and 10.5 Hz, 1 H, H-6), 4.20 (dd, J = 5.5 Hz and 10.4 Hz, 1 H,
H-6’), 3.83 (dd, J = 3.0 Hz and 9.2 Hz, 1 H, H-3), 3.79 (at, J = 9.4 Hz, 1 H, H-4), 3.71-3.74 (m, 2 H,
H-2, H-5), 3.24 (s, 3 H, OMe), 2.39 (s, 3 H, Me). 13C-NMR (125 MHz, CDCl3): 144.7, 138.4, 138.3,
138.2, 133.2, 129.9, 128.5, 128.5, 128.2, 128.1, 127.9, 127.9, 127.8, 127.8, 127.8, 99.0, 80.2, 75.1,
74.5, 74.2, 72.8, 72.2, 70.1, 69.4, 55.0, 21.8.
Methyl 2,3,4-tri-O-benzyl-6-thio-6-S-acetyl-α-D-mannopyranoside (11) (Cumpstey et al., 2010).
Compound 10 (1.27 g, 2.12 mmol) was dissolved in dry DMF. Then KSAc (728 mg, 6.37 mmol)
was added and the solution was stirred at 60°C under nitrogen for 3.5 h. The reaction mixture was
then diluted with water and diethyl ether and the water phase was extracted twice with diethyl ether.
The combined organic phases were washed with water, brine and dried over MgSO4 and evaporated
under reduced pressure giving 1.12 g of crude product. The crude product was separated by flash
column chromatography with solvent system Hex:EtOAc 9:1 giving pure compound 11 (1.02 g,
1
92%). TLC (Hex/EtOAc 2:1): Rf 0.42; H-NMR (500 MHz, CDCl3): 7.27-7.40 (m, 15 H, ArH),
4.94 (d, J = 10.8 Hz, 1 H, PhCH2), 4.75 (d, J = 12.4 Hz, 1 H, PhCH2), 4.71 (d, J = 12.5 Hz, 1 H,
PhCH2), 4.65-4.67 (m, 2 H, H-1, PhCH2), 4.59-4.63 (m, 2 H, PhCH2), 3.86 (dd, J = 3.1 Hz and 9.1
Hz, 1 H, H-3), 3.76-3.79 (m, 2 H, H-2, H-4), 3.69 (dt, J = 2.7 Hz and 8.6 Hz, 1 H, H-5), 3.58 (dd, J
= 2.8 Hz and 13.6 Hz, 1 H, H-6), 3.09 (dd, J = 8.2 Hz and 13.6 Hz, 1 H, H-6’), 3.30 (s, 3 H, OMe),
13
2.35 (s, 3 H, SAc). C-NMR (125 MHz, CDCl3): 195.3, 138.5, 138.4, 138.4, 128.6, 128.5, 128.5,
128.3, 128.0, 127.9, 127.8, 127.8, 127.7, 99.1, 80.2, 77.7, 75.4, 74.8, 72.9, 72.3, 71.0, 54.9, 31.4,
30.7.
Methyl 2,3,4-tri-O-benzyl-6-thio-α-D-mannopyranoside (12). Compound 11 (135 mg, 0.257
mmol) was dissolved in MeOH (15 mL) after which NaOMe (42 mg, 0.772 mmol) was added.
Mixture was then stirred at rt under nitrogen for 5-10 min before evaporation under reduced
pressure. The crude product was separated by flash column chromatography immediately, with
solvent system Hex:EtOAc 9:1 giving pure compound 12 (94 mg, 76%). TLC (Hex/EtOAc 9:1): Rf
1
0.12; H-NMR (500 MHz, CDCl3): 7.27-7.38 (m, 15 H, ArH), 4.96 (d, J = 11.0 Hz, 1 H, PhCH2),
4.77 (d, J = 12.4 Hz, 1 H, PhCH2), 4.70-4.73 (m, 2 H, H-1, PhCH2), 4.59-4.64 (m, 3 H, PhCH2),
3.88 (dd, J = 3.1 Hz and 9.3 Hz, 1 H, H-3), 3.83 (t, J = 9.2 Hz, 1 H, H-4), 3.79-3.80 (m, 1H, H-2),
3.63 (dd, J = 2.3 Hz and 8.7 Hz, 1 H, H-5), 3.35 (s, 3 H, OMe), 2.88-2.93 (m, 1 H, H-6), 2.70-2.76
13
(m, 1 H, H-6’), 1.74 (dd, J = 7.0 Hz and 9.7 Hz, 1 H, SH). C-NMR (125 MHz, CDCl3): 138.5,
138.5, 138.4, 128.6, 128.5, 128.5, 128.1, 128.0, 127.9, 127.8, 127.7, 99.2, 80.5, 77.5, 75.4, 74.8,
+
73.0, 72.8, 72.3, 54.9, 26.8. HRMS (ESI) m/z calcd. for C28H32O5S [M+Na] 503.1868, found
503.1863.
Synthesis of 2-hydroxyl and 2-sulfhydryl glycosyl acceptors
Methyl 2,3,4,6-tetra-O-trimethylsilyl-α-D-glucopyranoside (14) (Wang et al., 2008).
Commercially available 1-O-methyl-α-D-glucopyranoside (13, 1.16 g, 5.98 mmol) and DIPEA (5.2
mL, 30 mmol) was dissolved in dry DCM (30 mL) and stirred at 0 °C under nitrogen. Then, TMSCl
(4.5 mL, 36 mmol) was added dropwise and reaction mixture was stirred for 25 h after which
temperature had reached rt. The mixture was then evaporated after which the crude product shaken
vigorously with hexane (250 mL in portions) and filtered through celite. The combined filtrate was
evaporated under reduced pressure giving pure compound 14 (2.88 g, quant.). TLC (Hex/EtOAc
9:1): Rf 0.53; 1H-NMR (500 MHz, CDCl3): 4.61 (d, J = 3.6 Hz, 1 H, H-1), 3.73-3.77 (m, 2 H, H-3,
H-6), 3.67 (dd, J = 5.4 Hz and 11.3 Hz, 1 H, H-6’), 3.50 (dd, J = 2.0 Hz and 5.4 Hz, 1 H, H-5), 3.47
(dd, J = 3.7 Hz and 9.0 Hz, 1 H, H-2), 3.43 (dd, J = 8.4 Hz and 9.5 Hz, 1 H, H-4), 3.34 (s, 1 H,
3

ACCEPTED MANUSCRIPT
OMe), 0.16 (s, 9 H, Si(CH3)3), 0.16 (s, 9 H, Si(CH3)3), 0.15 (s, 9 H, Si(CH3)3), 0.12 (s, 9 H,
Si(CH3)3). 13C-NMR (125 MHz, CDCl3): 98.8, 74.5, 73.1, 71.4, 71.2, 61.4, 53.7, 0.5, 0.1, -0.3, -1.1.
Methyl 3-O-benzyl-(R)-4,6-O-benzylidene-α-D-glucopyranoside (15) (Wang et al., 2008).
Compound 14 (1.73 g, 3.58 mmol) and PhCHO (380 µL, 3.7 mmol) was dissolved in dry DCM (30
mL) and stirred at -78 °C under nitrogen for 30 min. Then TMSOTf (97 µL, 0.54 mmol) in DCM (1
mL) was added drop wise and the mixture was allowed to stir for 1 h. Then PhCHO (440 µL, 4.30
mmol) and Et3SiH (630 µL, 3.9 mmol) in DCM (2 mL) was added drop wise and the mixture was
stirred for another 3 h. The mixture was then heated to rt, diluted with EtOAc and washed twice
with sat. NaHCO3 (aq), and brine and finally dried over MgSO4 and evaporated under reduced
pressure giving 1.4 g of crude product. The crude product was separated by flash column
chromatography with solvent system Hex:EtOAc 3:1 giving pure compound 15 (1.25 g, 94%). TLC
(Hex/EtOAc 9:1): Rf 0.36; 1H-NMR (500 MHz, CDCl3): 7.45-7.48 (m, 2 H, ArH), 7.23-7.38 (m, 8
H, ArH), 5.55 (s, 1 H, PhCH), 4.75-4.78 (m, 2 H, H-1, PhCH2), 4.27 (dd, J = 4.6 Hz and 10.0 Hz, 1
H, H-4), 3.79-3.84 (m, 2 H, H-3, H-6), 3.69-3.76 (m, 2 H, H-2, H-5), 3.62 (at, J = 9.3 Hz, 1 H, H-
13
6’), 3.43 (s, 3 H, OMe), 2.30 (d, J = 7.4 Hz, OH). C-NMR (125 MHz, CDCl3): 138.6, 137.5,
129.1, 128.5, 128.4, 128.1, 127.8, 126.2, 101.4, 100.0, 82.1, 79.0, 74.9, 72.6, 69.2, 62.7, 55.5.
Methyl 2-O-trifluoromethanesulfonyl-3-O-benzyl-(R)-4,6-O-benzylidene-α-D-glucopyranoside
(16) (Walvoort et al., 2010). Compound 15 (1.30 g, 3.49 mmol) was dissolved in dry DCM (20 mL)
and stirred at -15 °C under nitrogen. Then pyridine (2.2 mL, 28 mmol) was added drop wise
followed by Tf2O (1.2 mL, 7.0 mmol) in DCM (3 mL). The reaction mixture was stirred at -15 °C
for 2.5 h before it was diluted with DCM and poured on ice water. The organic phase was washed
with 1M HCl (aq), sat. NaHCO3 (aq), water and brine, and finally dried over MgSO4 and
evaporated under reduced pressure giving pure product 16 (1.74 g, 99%). TLC (Hex/EtOAc 1:1): Rf
0.86; 1H-NMR (500 MHz, CDCl3): 7.45-7.48 (m, 2 H, ArH), 7.38-7.42 (m, 3 H, ArH), 7.27-7.35 (m,
5 H, ArH), 5.57 (s, 1 H, PhCH), 4.97 (d, J = 3.8 Hz, H-1), 4.86 (d, J = 11.0 Hz, PhCH2), 4.77 (d, J
= 11.0 Hz, PhCH2), 4.74 (dd, J = 3.5 Hz and 9.4 Hz, 1 H, H-2), 4.32 (dd, J = 4.9 Hz and 10.3 Hz, 1
H, H-6), 4.13 (at, J = 9.4 Hz, 1 H, H-3), 3.89 (dt, J = 4.8 Hz and 10.0 Hz, 1 H, H-5), 3.77 (at, J =
13
10.3 Hz, 1 H, H-6’), 3.70 (at, J = 9.5 Hz, 1 H, H-4), 3.48 (s, 3 H, OMe). C-NMR (125 MHz,
CDCl3): 137.5, 137.0, 129.3, 128.5, 128.5, 128.4, 128.1, 126.1, 101.7, 97.8, 83.7, 82.2, 75.4, 75.1,
68.8, 62.4, 55.9.
Methyl 3-O-benzyl-(R)-4,6-O-benzylidene-α-D-mannopyranoside (17) (Ennis et al., 2002).
Compound 16 (496 mg, 0.984 mmol) was dissolved in dry toluene (10 mL). Then TBANO2 (1.42 g,
4.92 mmol) was added and the reaction mixture was refluxed (70 °C) for 21 h. The reaction mixture
was then diluted with DCM, washed twice with water and brine, and dried over MgSO4 and
evaporated under reduced pressure giving 820 mg of crude product. The crude product was
separated by flash column chromatography with solvent system Hex:EtOAc 4:1 giving pure
1
compound 17 (257 mg, 70%). TLC (Hex/EtOAc 4:1): Rf 0.34; H-NMR (500 MHz, CDCl3): 7.49-
7.50 (m, 2 H, ArH), 7.28-7.40 (m, 8 H, ArH), 5.62 (s, 1 H, PhCH), 4.85 (d, J = 11.9 Hz, PhCH2),
4.77 (as, H-1), 4.71 (d, J = 11.9 Hz, PhCH2), 4.28 (dd, J = 4.0 Hz and 9.6 Hz, 1 H, H-6), 4.05-4.13
(m, 2 H, H-2, H-4), 3.91 (dd, J = 3.5 Hz and 9.5 Hz, 1 H, H-3), 3.87 (at, J = 10.1 Hz, 1 H, H-6’),
3.81 (dt, J = 4.4 Hz and 9.5 Hz, 1 H, H-5), 3.38 (s, 3 H, OMe), 2.65 (as, 1 H, OH). 13C-NMR (125
MHz, CDCl3): 138.2, 137.7, 129.1, 128.6, 128.4, 128.1, 128.0, 126.2, 101.8, 101.2, 79.0, 75.7, 73.2,
70.1, 69.1, 63.3, 55.1.
Methyl
2-thio-2-S-acetyl-3-O-benzyl-(R)-4,6-O-benzylidene-α-D-mannopyranoside
(18)
(Cumpstey et al., 2010). Compound 16 (1.12 g, 2.22 mmol) was dissolved in dry DMF (25 mL) and
stirred at rt. Then KSAc (1.27 g, 11.1 mmol) was added and mixture was stirred at at 40 °C under
nitrogen for 16 h. The reaction mixture was then diluted with diethyl ether and water and the water
phase was extracted with diethyl ether twice. The combined organic phases were dried over MgSO4
and evaporated under reduced pressure giving 1.53 g of crude product. The crude product was
separated by flash column chromatography with solvent system Hex:EtOAc 9:1 giving pure
4

ACCEPTED MANUSCRIPT
1
compound 18 (851 mg, 89%). TLC (Hex/EtOAc 6:1): Rf 0.20; H-NMR (500 MHz, CDCl3): 7.49-
7.50 (m, 2 H, ArH), 7.24-7.40 (m, 8 H, ArH), 5.59 (s, 1 H, PhCH), 4.73 (as, H-1), 4.68 (d, J = 12.2
Hz, PhCH2), 4.63 (d, J = 12.2 Hz, PhCH2), 4.38 (ad, J = 5.0 Hz, 1 H, H-2), 4.29 (dd, J = 5.0 Hz and
9.9 Hz, 1 H, H-3), 4.24 (dd, J = 4.2 Hz and 9.9 Hz, 1 H, H-6), 3.85 (dt, J = 4.4 Hz and 9.8 Hz, 1 H,
H-5), 3.79 (at, J = 10.2 Hz, 1 H, H-6’), 3.70 (at, J = 9.6 Hz, 1 H, H-4), 3.36 (s, 3 H, OMe), 2.40 (s,
13
3 H, SAc). C-NMR (125 MHz, CDCl3): 194.3, 138.1, 137.5, 129.1, 128.4, 128.3, 127.8, 127.7,
126.2, 102.6, 101.8, 80.6, 78.2, 72.9, 72.1, 69.0, 63.9, 55.3, 48.2, 30.9, 29.9.
Methyl 2-thio-3-O-benzyl-(R)-4,6-O-benzylidene-α-D-mannopyranoside (19). Compound 18
(105 mg, 0.250 mmol) was dissolved in MeOH (5 mL) after which NaOMe (40 mg, 0.750 mmol)
was added. Mixture was then stirred at rt under nitrogen for 10 min before evaporation under
reduced pressure. The crude product was separated by flash column chromatography immediately,
with solvent system Hex:EtOAc 10:1 giving pure compound 19 (75 mg, 77%). TLC (Hex/EtOAc
1
10:1): Rf 0.19; H-NMR (500 MHz, CDCl3): 7.50-7.53 (m, 2 H, ArH), 7.30-7.42 (m, 8 H, ArH),
5.65 (s, 1 H, PhCH), 4.79-4.81 (m, 2 H, H-1, PhCH2), 4.72 (d, J = 12.3 Hz, PhCH2), 4.21-4.31 (m, 2
H, H-4, H-6), 4.14 (dd, J = 4.7 Hz and 9.6 Hz, 1 H, H-3), 3.86-3.91 (m, 2 H, H-5, H-6’), 3.66 (at, J
= 5.5 Hz, 1 H, H-2), 3.38 (s, 3 H, OMe), 2.01 (d, J = 6.3 Hz, 1 H, SH). HRMS (ESI) m/z calcd. for
C21H24O5S [M+Na] + 411.1242, found 411.1234.
Synthesis of glycosyl donors
Ethyl 1-thio-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (23) (Saksena et al., 2002). α/β-D-
Mannose pentaacetate (22, 786 mg, 2.01 mmol) and ethanethiol (450 µL, 6.0 mmol) was dissolved
in dry DCM (20 mL) and stirred at 0 °C under nitrogen. Then, BF3·Et2O (2.0 mL, 16 mmol) in
DCM (5 mL) was added drop wise. The reaction mixture was then allowed to reach rt and after 24 h,
the reaction was quenched by diluting it with DCM and pouring the mixture on ice water. The
organic phase was washed with sat. NaHCO3 (aq), water and brine, and finally dried over MgSO4
and evaporated under reduced pressure giving 526 mg of crude product. The crude product was
separated by flash column chromatography with solvent system Hex:EtOAc 2:1 giving pure
1
compound 23 (402 mg, 51%). TLC (Hex/EtOAc 1:1): Rf 0.31; H-NMR (500 MHz, CDCl3): 5.34
(dd, J = 1.5 Hz and 3.2 Hz, 1 H, H-2), 5.29-5.33 (m, 2 H, H-1, H-4), 5.27 (dd, J = 3.3 Hz and 9.9
Hz, 1 H, H-3), 4.40 (ddd, J = 2.2 Hz, 5.3 Hz and 9.5 Hz, 1 H, H-5), 4.32 (dd, J = 5.5 Hz and 12.2
Hz, 1 H, H-6), 4.10 (dd, J = 2.3 Hz and 12.3 Hz, 1 H, H-6’), 2.58-2.71 (m, 2 H, SCH2CH3), 2.17 (s,
3 H, OAc), 2.10 (s, 3 H, OAc), 2.05 (s, 3 H, OAc), 1.99 (s, 3 H, OAc), 1.31 (t, J = 7.4 Hz, 3 H,
13
SCH2CH3). C-NMR (125 MHz, CDCl3): 170.8, 170.2, 169.9, 169.9, 82.5, 71.4, 69.7, 69.1, 66.6,
62.6, 25.6, 21.1, 20.9, 20.9, 20.8, 14.9.
Ethyl 1-thio-α-D-mannopyranoside (24) (Saksena et al., 2002). Compound 23 (902 mg, 2.30
mmol) was dissolved in MeOH (20 mL) after which NaOMe (137 mg, 2.53 mmol) was added.
Mixture was then stirred at rt under nitrogen for 3 h after which the solvent was evaporated under
reduced pressure, giving pure product 24 (525 mg, quant.). 1H-NMR (500 MHz, D2O): 5.32 (d, J =
1.1 Hz, 1 H, H-1), 4.04 (dd, J = 1.6 Hz and 3.4 Hz, 1 H, H-2), 4.00 (ddd, J = 2.3 Hz, 6.3 Hz and 9.6
Hz, 1 H, H-5), 3.88 (dd, J = 2.3 Hz and 12.3 Hz, 1 H, H-6), 3.75-3.79 (m, 2 H, H-3, H-6’), 3.66 (t, J
= 9.8 Hz, 1 H, H-4), 2.61-2.73 (m, 2 H, SCH2CH3), 1.27 (t, J = 7.4 Hz, 3 H, SCH2CH3). 13C-NMR
(125 MHz, D2O): 84.5, 72.0, 71.2, 67.4, 73.2, 61.0, 25.5, 14.3.
Ethyl 1-thio-2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (25) (Saksena et al., 2002).
Compound 24 (70 mg, 0.311 mmol) was dissolved in dry DMF (4 mL) and stirred at 0 °C under
nitrogen for 30 min. Then BnBr (160 µL, 1.4 mmol) was added followed by slow addition of NaH
(60% in mineral oil, 124 mg, 3.11 mmol). The reaction mixture was then stirred vigorously for 4 h
before it was diluted with diethyl ether and water and the water phase was extracted with diethyl
ether twice. The combined organic phases were dried over MgSO4 and evaporated under reduced
pressure giving 153 mg of crude product. The crude product was separated by flash column
chromatography with solvent system Hex:EtOAc 7:1 giving pure compound 25 (73 mg, 40%). TLC
5

ACCEPTED MANUSCRIPT
(Hex/EtOAc 3:1): Rf 0.29; 1H-NMR (500 MHz, CDCl3): 7.24-7.39 (m, 18 H, ArH), 7.16-7.18 (m, 2
H, ArH), 5.40 (as, 1 H, H-1), 4.88 (d, J = 10.8 Hz, PhCH2), 4.73 (d, J = 12.5 Hz, PhCH2), 4.65-4.68
(m, 2 H, PhCH2), 4.54-4.60 (m, 2 H, PhCH2), 4.49-4.52 (m, 2 H, PhCH2), 4.13 (dd, J = 4.4 Hz and
10.0 Hz, H-4), 4.03 (t, J = 9.7 Hz, H-3), 3.80-3.85 (m, 3 H, H-2, H-5, H-6), 3.71 (dd, J = 1.2 Hz and
11.0 Hz, H-6’), 2.52-2.67 (m, 2 H, SCH2CH3), 1.24 (t, J = 7.4 Hz, 3 H, SCH2CH3). 13C-NMR (125
MHz, CDCl3): 138.7, 138.5, 138.4, 138.3, 128.5, 128.4, 128.4, 128.1, 128.0, 128.0, 127.9, 127.8,
127.7, 127.6, 82.0, 80.5, 76.5, 75.3, 75.2, 73.5, 72.2, 72.2, 72.1, 69.3, 25.5, 15.1.
Ethyl 1-thio-2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranoside (27) (Düffels et al., 2000).
Compound 26, synthesized in five steps from D-mannose according to Beignet et al. (Beignet et al.,
2004), (2.00 g, 3.94 mmol) was dissolved in dry MeCN (20 mL). Then molecular sieves (4Å) were
added and the mixture was stirred at rt under nitrogen for 1.5 h. Then, ethanethiol (970 µL, 13
mmol) and HgBr2 (71 mg, 0.20 mmol) in dry MeCN (1 mL) was added to the reaction mixture. The
reaction mixture was then stirred at 60 °C under nitrogen for 24 h after which it was diluted with
DCM and washed with sat. NaHCO3 (aq) and brine, and finally dried over MgSO4 and evaporated
under reduced pressure giving 1.6 g of crude product. The crude product was separated by flash
column chromatography with solvent system Hex:EtOAc 4:1 giving pure compound 27 (1.44 g,
1
68%). TLC (Hex/EtOAc 4:1): Rf 0.28; H-NMR (500 MHz, CDCl3): 7.25-7.36 (m, 13 H, ArH),
7.15-7.17 (m, 2 H, ArH), 5.43 (dd, J = 1.5 Hz and 2.6 Hz, 1 H, H-2), 5.32 (d, J = 1.2 Hz, 1 H, H-1),
4.85 (d, J = 10.8 Hz, PhCH2), 4.67-4.70 (m, 2 H, PhCH2), 4.46-4.53 (m, 3 H, PhCH2), 4.16 (dd, J =
3.4 Hz and 9.1 Hz, H-5), 3.89-3.95 (m, 2 H, H-3, H-4), 3.84 (dd, J = 4.3 Hz and 11.0 Hz, H-6), 3.68
(dd, J = 2.0 Hz and 10.8 Hz, H-6’), 3.69 (dd, J = 1.6 Hz and 10.8 Hz, H-6’), 2.55-2.69 (m, 2 H,
SCH2CH3), 2.16 (s, 3 H, OAc), 1.28 (t, J = 7.4 Hz, 3 H, SCH2CH3). 13C-NMR (125 MHz, CDCl3):
170.6, 138.5, 138.3, 137.9, 128.7, 128.6, 128.5, 128.3, 128.0, 127.9, 127.8, 127.7, 127.1, 82.6, 78.7,
75.3, 74.7, 73.6, 72.0, 72.0, 70.7, 69.0, 25.6, 21.3, 15.0.
1,2-di-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranoside (29) (Mayer and Schmidt, 1999).
Compound 26 (2.30 g, 4.54 mmol) was dissolved in AcOH (35 mL) and water (18 mL) was added.
The reaction mixture was then refluxed (100 °C) for 4 h after which the solvent was evaporated
under reduced pressure giving a yellow syrup. The crude product 28 was then dissolved in pyridine
(20 mL) and Ac2O (4.3 mL, 45 mmol) was added. The reaction mixture was stirred at rt for 2 h after
which the solvent was evaporated under reduced pressure giving 2.4 g of crude product. The crude
product was separated by flash column chromatography with solvent system Hex:EtOAc 5:1 giving
1
pure compound 29 (1.87 g, 77% over two steps). TLC (Hex/EtOAc 3:1): Rf 0.32; H-NMR (500
MHz, CDCl3): 7.26-7.36 (m, 13 H, ArH), 7.16-7.18 (m, 2 H, ArH), 6.12 (d, J = 2.0 Hz, H-1), 5.36
(at, J = 2.4 Hz, H-2), 4.86 (d, J = 10.6 Hz, PhCH2), 4.73 (d, J = 11.1 Hz, PhCH2), 4.68 (d, J = 12.0
Hz, PhCH2), 4.55 (d, J = 11.1 Hz, PhCH2), 4.50-4.52 (m, 2 H, PhCH2), 3.95-4.00 (m, 2 H, H-3, H-
4), 3.80-3.87 (m, 2 H, H-5, H-6), 3.69 (dd, J = 1.6 Hz and 10.8 Hz, H-6’), 2.17 (s, 3 H, OAc), 2.07
(s, 3 H, OAc). 13C-NMR (125 MHz, CDCl3): 170.3, 168.5, 138.3, 137.8, 128.6, 128.5, 128.5, 128.3,
128.1, 128.0, 128.0, 127.9, 127.8, 91.5, 77.8, 75.5, 74.0, 73.9, 73.7, 72.1, 68.6, 67.7, 21.1, 21.1.
Glycosylation reactions
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-
mannopyranoside (30). Compound 9 (311 mg, 0.670 mmol) and compound 27 (467 mg, 0.870
mmol) was dissolved in dry DCM (10 mL). Then molecular sieves (4Å) were added and the
reaction mixture was stirred at -10 °C under nitrogen for 45 min. Then NIS (451 mg, 2.01 mmol)
was added and the reaction vessel was shielded with aluminum foil. Then TfOH (9 µL, 0.1 mmol)
in dry DCM (1 mL) was added drop wise. After 30 min, the reaction mixture was diluted with
DCM and water. Then Na2S2O5 (380 mg, 2.01 mmol) was added and the mixture was shaken until
no red color could be observed. The organic phase was washed twice with sat. NaHCO3 (aq) and
brine, and finally dried over MgSO4 and evaporated under reduced pressure giving 648 mg of crude
product. The crude product was separated by flash column chromatography with solvent system
6

ACCEPTED MANUSCRIPT
1
Hex:EtOAc 3:1 giving pure compound 30 (503 mg, 80%). TLC (Hex/EtOAc 3:1): Rf 0.19; H-
NMR (500 MHz, CDCl3): 7.20-7.38 (m, 28 H, ArH), 7.12-7.14 (m, 2 H, ArH), 5.46-5.47 (m, 1 H,
H-2’), 4.95 (d, J = 1.4 Hz, 1 H, H-1’), 4.91 (d, J = 11.3 Hz, 1 H, PhCH2), 4.85 (d, J = 11.0 Hz, 1 H,
PhCH2), 4.70-4.71 (m, 3 H, H-1, PhCH2), 4.63-4.66 (m, 2 H, PhCH2), 4.56-4.61 (m, 2 H, PhCH2),
4.42-4.49 (m, 4 H, PhCH2), 3.95 (dd, J = 3.3 Hz and 9.4 Hz, 1 H, H-3’), 3.84-3.91 (m, 4 H, H-4’),
3.78-3.80 (m, 2 H, H-2), 3.66-3.72 (m, 3 H), 3.59 (dd, J = 1.4 Hz and 10.7 Hz, 1 H), 3.25 (s, 3 H,
13
OMe), 2.14 (s, 3 H, OAc). C-NMR (125 MHz, CDCl3): 170.5, 138.8, 138.6, 138.6, 138.5, 138.4,
138.0, 128.5, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6, 127.6,
98.9, 98.1, 80.4, 77.7, 75.2, 75.1, 74.8, 74.7, 74.4, 73.5, 72.8, 72.2, 71.5, 71.5, 71.1, 68.9, 68.7, 66.9,
54.8, 21.3. HRMS (ESI) m/z calcd. for C57H62O12 [M+Na] + 961.4139, found 961.4133.
Methyl α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside (1) (Sandström et al., 2004).
Compound 30 (289 mg, 0.308 mmol) was dissolved in MeOH (10 mL). Then NaOMe (9 mg, 0.2
mmol) in MeOH (0.5 mL) was added and the reaction mixture was stirred at rt under nitrogen for
24 h. Then AcOH (70 µL, 1.2 mmol) and Pd (10% on C) (160 mg, 0.15 mmol) was added to and
the reaction mixture was stirred at rt under hydrogen for 72 h. Then, the reaction mixture was
filtered through celite and rinsed with MeOH. The combined filtrate was evaporated under reduced
pressure giving 138 mg of crude product. The crude product was separated by flash column
chromatography with solvent system DCM:MeOH 3:1 giving pure compound 1 (84 mg, 77%). TLC
(DCM/MeOH 3:1): Rf 0.10; 1H-NMR (500 MHz, D2O): 4.90 (d, J = 1.6 Hz, 1 H, H-1’), 4.74 (d, J =
1.6 Hz, 1 H, H-1), 3.96-3.99 (m, 2 H, H-2’), 3.93-3.94 (m, 1 H, H-2), 3.89 (dd, J = 2.0 Hz and 12.0
Hz, 1 H), 3.84 (dd, J = 3.5 Hz and 9.4 Hz, 1 H), 3.68-3.77 (m, 6 H, H-3), 3.65 (at, J = 9.5 Hz, 1 H),
13
3.40 (s, 3 H, OMe). C-NMR (125 MHz, D2O): 101.6, 100.1, 73.3, 71.4, 71.3, 71.2, 70.6, 70.5,
67.3, 67.1, 66.2, 61.5, 55.4.
Methyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-6-thio-α-
D-mannopyranoside (31). Compound 12 (167 mg, 0.348 mmol) and compound 29 (242 mg, 0.452
mmol) was dissolved in dry DCM (10 mL). Then molecular sieves (3Å) were added and the
reaction mixture was stirred at 0 °C under nitrogen for 15 min. Then BF3·Et2O (160 µL, 1.5 mmol)
in DCM (0.5 mL) was added drop wise. The reaction mixture was then stirred for 13 h after which
it was diluted with DCM and poured on ice water. The organic phase was washed with sat.
NaHCO3 (aq) and brine, and finally dried over MgSO4 and evaporated under reduced pressure
giving 385 mg of crude product. The crude product was separated by flash column chromatography
with solvent system Hex:EtOAc 6:1 giving pure compound 31 (1.44 g, 61%). TLC (Hex/EtOAc
1
4:1): Rf 0.39; H-NMR (500 MHz, CDCl3): 7.24-7.38 (m, 28 H, ArH), 7.15-7.16 (m, 2 H, ArH),
5.51-5.52 (m, 1 H, H-2’), 5.41 (as, 1 H, H-1’), 4.92 (d, J = 11.0 Hz, 1 H, PhCH2), 4.84 (d, J = 10.8
Hz, 1 H, PhCH2), 4.71-4.72 (m, 3 H, H-1, PhCH2), 4.64-4.68 (m, 2 H, PhCH2), 4.58-4.60 (m, 3 H,
PhCH2), 4.46-4.51 (m, 2 H, PhCH2), 4.41 (d, J = 12.0 Hz, 1 H, PhCH2), 4.14 (dd, J = 1.9 Hz and
8.2 Hz, 1 H), 3.92-3.99 (m, 2 H, H-3’), 3.85-3.87 (m, 2 H), 3.82 (dd, J = 3.7 Hz and 10.9 Hz, 1 H),
3.73-3.77 (m, 2 H, H-2, H-5), 3.61 (dd, J = 1.2 Hz and 10.7 Hz, 1 H), 3.32 (s, 3 H, OMe), 3.09 (dd,
J = 2.1 Hz and 13.6 Hz, 1 H, H-6a), 2.84 (dd, J = 7.4 Hz and 13.6 Hz, 1 H, H-6b), 2.15 (s, 3 H,
OAc). 13C-NMR (125 MHz, CDCl3): 170.4, 138.6, 138.6, 138.5, 138.4, 138.4, 137.9, 128.5, 128.5,
128.5, 128.4, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 99.0, 83.1, 80.3, 78.8,
77.4, 75.4, 75.2, 74.6, 74.6, 73.5, 72.8, 72.3, 72.1, 72.0, 71.0, 70.5, 68.9, 55.0, 33.0, 21.3. HRMS
(ESI) m/z calcd. for C57H62O11S [M+Na] + 977.3911, found 977.3905.
Methyl
2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-6-thio-α-D-
mannopyranoside (32). Compound 31 (201 mg, 0.211 mmol) was dissolved in dry THF (4 mL)
and added to a prepared solution of NH3 (l) (~10 mL condensed from gas) containing dissolved Li
(100 mg, 15 mmol) at -78 °C under nitrogen. The reaction mixture was stirred for 2 min before
quenching by addition of NH3Cl (until blue color disappeared). The reaction mixture was then
allowed to reach rt and the solvent evaporated at 1 atmosphere. After evaporation, the reaction
mixture was dried under vacuum for 2 h before the addition of pyridine (30 mL) and Ac2O (12 mL)
7

ACCEPTED MANUSCRIPT
giving a milky slurry. The reaction mixture was then stirred at rt under nitrogen for 22 h. After that,
the reaction mixture was diluted with DCM and washed with water, 6 M HCl (aq) and brine. The
organic phase was dried over MgSO4 and evaporated under reduced pressure giving 263 mg of
crude product. The crude product was separated by flash column chromatography with solvent
system Hex:EtOAc 4:1 1:1 giving pure compound 32 (117 mg, 85%). TLC (Hex/EtOAc 1:1): Rf
0.44; 1H-NMR (500 MHz, CDCl3): 5.21-5.36 (m, 7 H, H-1, H-2, H-3, H-4, H-2’, H-3’, H-4’), 4.26
(d, J = 1.6 Hz, 1 H, H-1’), 4.37 (ddd, J = 2.2 Hz, 5.0 Hz and 9.5 Hz, 1 H, H-5’), 4.31 (dd, J = 5.0
Hz and 12.2 Hz, 1 H, H-6a’), 4.09 (dd, J = 2.1 Hz and 12.3 Hz, 1 H, H-6b’), 3.94 (ddd, J = 2.9 Hz,
7.4 Hz and 9.6 Hz, 1 H, H-5), 3.42 (s, 3 H, OMe), 2.85 (dd, J = 2.8 Hz and 14.0 Hz, 1 H, H-6a),
2.72 (dd, J = 7.4 Hz and 14.0 Hz, 1 H, H-6b), 2.17 (s, 3 H, OAc), 2.15 (s, 3 H, OAc), 2.12 (s, 3 H,
13
OAc), 2.05 (s, 6 H, OAc), 1.99 (s, 6 H, OAc). C-NMR (125 MHz, CDCl3): 170.8, 170.3, 170.1,
170.0, 170.0, 169.9, 169.9, 98.6, 82.7, 77.4, 71.0, 69.6, 69.6, 69.5, 69.3, 69.0, 68.9, 66.3, 62.4, 55.6,
32.1, 21.1, 21.0, 20.9, 20.9, 20.8, 20.8. HRMS (ESI) m/z calcd. for C27H38O17S [M+Na] + 689.1722,
found 689.1715.
Methyl α-D-mannopyranosyl-(1→6)-6-thio-α-D-mannopyranoside (2). Compound 32 (218 mg,
0.393 mmol) was dissolved in MeOH (10 mL) and stirred at rt under nitrogen. Then Li (5.5 mg,
0.79 mmol) dissolved in MeOH (1 mL) was added to the reaction mixture and the mixture was
stirred for 2.5 h before evaporation giving the pure product 2 (137 mg, 94%). 1H-NMR (500 MHz,
D2O): 5.34 (d, J = 1.1 Hz, 1 H, H-1’), 4.72 (d, J = 1.4 Hz, 1 H, H-1), 4.06 (dd, J = 1.3 Hz and 3.3
Hz, 1 H, H-2’), 4.00 (ddd, J = 2.1 Hz, 6.0 Hz and 9.9 Hz, 1 H, H-5’), 3.93 (dd, J = 1.6 Hz and 3.3
Hz, 1 H, H-2), 3.88 (dd, J = 2.2 Hz and 12.3 Hz, 1 H, H-4’), 3.70-3.81 (m, 4 H, H-3, H-5, H-3’, H-
6a’), 3.67 (at, J = 9.7 Hz, 1 H, H-6b’), 3.62 (at, J = 9.5 Hz, 1 H), 3.41 (s, 3 H, OMe), 3.17 (dd, J =
2.3 Hz and 14.0 Hz, 1 H, H-6a), 3.80 (dd, J = 8.4 Hz and 14.0 Hz, 1 H, H-6b). 13C-NMR (125 MHz,
D2O): 101.6, 85.1, 73.9, 72.3, 71.7, 71.6, 71.1, 70.5, 70.1, 67.7, 61.5, 55.4, 32.2. HRMS (ESI) m/z
calcd. for C13H24O10S [M+Li] + 379.1245, found 379.1235.
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→2)-3-O-benzyl-(R)-4,6-O-
benzylidene-α-D-mannopyranoside (33). Compound 17 (168 mg, 0.451 mmol) and compound 27
(314 mg, 0.870 mmol) was dissolved in dry DCM (15 mL). Then molecular sieves (4Å) were added
and the reaction mixture was stirred at -10 °C under nitrogen for 20 min. Then NIS (406 mg, 1.80
mmol) was added and the reaction vessel was shielded with aluminum foil. Then TfOH (6 µL, 0.07
mmol) in dry DCM (0.5 mL) was added drop wise. After 30 min, the reaction mixture was diluted
with DCM and water. Then Na2S2O5 (340 mg, 1.80 mmol) was added and the mixture was shaken
until no red color could be observed. The organic phase was washed twice with sat. NaHCO3 (aq)
and brine, and finally dried over MgSO4 and evaporated under reduced pressure giving 405 mg of
crude product. The crude product was separated by flash column chromatography with solvent
system Hex:EtOAc 6:1 giving pure compound 33 (150 mg, 39%). TLC (Hex/EtOAc 6:1): Rf 0.36;
1H-NMR (500 MHz, CDCl3): 7.50-7.52 (m, 2 H, ArH), 7.23-7.40 (m, 21 H, ArH), 7.16-7.18 (m, 2
H, ArH), 5.63 (s, 1 H, PhCH), 5.59 (dd, J = 1.6 Hz and 3.0 Hz, 1 H, H-2’), 5.12 (d, J = 1.3 Hz, 1 H,
H-1’), 4.86 (d, J = 10.7 Hz, 1 H, PhCH2), 4.83 (d, J = 12.3 Hz, 1 H, PhCH2), 4.77 (d, J = 11.0 Hz, 1
H, PhCH2), 4.74 (d, J = 1.5 Hz, 1 H, H-1b), 4.64-4.67 (m, 2 H, PhCH2), 4.56 (d, J = 11.0 Hz, 1 H,
PhCH2), 4.83 (d, J = 12.0 Hz, 1 H, PhCH2), 4.77 (d, J = 11.0 Hz, 1 H, PhCH2), 4.23 (dd, J = 4.6 Hz
and 10.0 Hz, 1 H), 4.01-4.05 (m, 3 H, H-3’, H-2), 3.89-3.94 (m, 2 H), 3.72-3.85 (m, 5 H), 3.23 (s, 3
13
H, OMe), 2.14 (s, 3 H, OAc). C-NMR (125 MHz, CDCl3): 170.2, 138.7, 138.4, 138.3, 138.1,
137.8, 129.0, 128.5, 128.5, 128.3, 128.3, 128.2, 128.0, 127.9, 127.9, 127.8, 127.6, 126.3, 101.7,
100.9, 100.3, 79.1, 78.2, 76.7, 75.7, 75.5, 74.6, 73.6, 73.0, 72.1, 72.0, 69.2, 69.0, 68.6, 63.9, 54.9,
21.3. HRMS (ESI) m/z calcd. for C50H54O12 [M+Na] + 869.3513, found 869.3507.
Methyl α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside (3) (Sandström et al., 2004).
Compound 33 (224 mg, 0.265 mmol) was dissolved in MeOH (15 mL). Then NaOMe (14 mg, 0.27
mmol) in MeOH (0.5 mL) was added and the reaction mixture was stirred at rt under nitrogen for 6
h. Then AcOH (60 µL, 1.1 mmol) and Pd (10% on C) (280 mg, 0.27 mmol) was added to and the
8

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reaction mixture was stirred at rt under hydrogen for 24 h. Then, the reaction mixture was filtered
through celite and rinsed with MeOH. The combined filtrate was evaporated under reduced pressure
giving 178 mg of crude product. The crude product was separated by flash column chromatography
with solvent system DCM:MeOH 3:1 giving pure compound 3 (80 mg, 85%). TLC (DCM/MeOH
3:1): Rf 0.09; 1H-NMR (500 MHz, D2O): 5.01 (d, J = 1.6 Hz, 1 H, H-1’), 4.99 (d, J = 1.5 Hz, 1 H,
H-1), 4.06 (dd, J = 1.7 Hz and 3.2 Hz, 1 H, H-2’), 3.95 (dd, J = 1.7 Hz and 3.3 Hz, 1 H, H-2), 3.82-
13
3.90 (m, 4 H, H-3, H-3’), 3.57-3.78 (m, 6 H), 3.89 (s, 3 H, OMe). C-NMR (125 MHz, D2O):
102.9, 100.0, 79.2, 73.9, 73.2, 70.9, 70.8, 70.6, 67.6, 67.5, 61.8, 61.6, 55.4.
Methyl α-D-mannopyranosyl-(1→2)-2-thio-(R)-4,6-O-benzylidene-α-D-mannopyranoside (34).
1-Thio-α-D-mannopyranose sodium salt, prepared according to Pei et al. (Pei et al., 2006), (180 mg,
1.1 eq.) and compound 21, prepared in two steps from methyl α-D-glucopyranoside 13 according to
Knapp et al. (Knapp et al., 1990), (316 mg, 1.0 eq.) were added to dry DMF (2 mL). The mixture
was stirred at 50 °C under nitrogen atmosphere for 18 hours. After removal of the solvent, the
residue was directly purified by flash chromatography with solvent system CHCl2/MeOH 10:1
giving compound 34 (94 mg, 25%). TLC (DCM/MeOH 5:1): Rf 0.62; The compound decomposed
in solution and was used directly in the next step.
Methyl α-D-mannopyranosyl-(1→2)-2-thio-α-D-mannopyranoside (4). Compound 34 (80 mg)
was dissolved in DCM/MeOH/MeCOOH (1:1:2, 2 mL), and the mixture was stirred at 50 °C under
nitrogen atmosphere overnight. The solvent was removed under reduced pressure and co-
evaporated with toluene. The residue was directly purified by flash chromatography with solvent
1
system CHCl3/MeOH 3:1 giving compound 4 (49 mg, 76%). TLC (DCM/MeOH 3:1): Rf 0.50; H
NMR (600 MHz, D2O) δ = 5.41 (s, 1 H, H-1'), 5.00 (s, 1 H, H-1), 4.19 (dd, J = 9.5, 4.7, 1 H, H-3),
4.14 (dd, J = 3.3, 1.4, 1 H, H-2'), 4.05 – 4.00 (m, 1 H, H-5'), 3.93 (dd, J = 12.3, 2.1, 1 H, H-6a'),
3.89 (dd, J = 12.3, 2.1, 1 H, H-6a), 3.81 – 3.74 (m, 3 H, H-3', H-6b, H-6b'), 3.69 – 3.62 (m, 2 H, H-
5, H-4'), 3.54 (t, J = 9.7, 1 H, H-4), 3.47 (d, J = 4.7, 1 H, H-2), 3.40 (s, 3 H, OMe). 13C NMR (151
MHz, D2O) δ = 102.3, 87.6, 74.2, 73.4, 72.0, 71.5, 70.0, 68.4, 67.8, 61.6, 61.3, 55.4, 52.8. HRMS
(ESI) m/z calcd. for C13H24O10S [M+Na] + 395.0982, found: 395.0959.
Binding analysis
Binding analyses were performed using a QCM flow-through instrumentation (Attana). The
functionalized QCM sensors were initially equilibrated to produce a stable baseline signal, and then
subjected to BSA injections to block nonspecific binding. Competitive binding was subsequently
recorded as shifts in frequency, corresponding to resulting protein surface binding, by injections of
solutions containing Con A (2 µM) and the respective ligands at different concentrations (cf. Figure
2a). Between individual recordings, bound protein was released by successive injections of low pH
buffer (pH 1.5). Frequency effects emanating from the ligand (cf. Figure 2b) were subtracted,
resulting in corrected binding curves (cf. Figure 2c), from which the quantitative binding was
extracted and subjected to nonlinear regression analysis using a dose-response inhibition model
(equation 1).
(1)
Y represents the frequency response (Hz), fmin and fmax are the minimum and maximum frequencies
recorded (Hz), Hill coeff is the Hill coefficient, C is the concentration (M) number, and EC50 is the
half maximal effective concentration (M) number. The Hill coefficient is generally considered a
measure of potential cooperativity effects in proteins composed of several subunits (Gesztelyi et al.,
2012). Kd-values were subsequently estimated using the Cheng-Prusoff relationship (Cheng and
Prusoff, 1973), using the reported value of 122 µM of the reference compound (methyl α-D-
mannopyranoside; Mandal et al., 1994). For visualization purposes, the data from the respective
experiments, including the resulting fitted competition binding curves, were normalized with
9

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respect to frequency response, and the respective estimated EC50-values of the reference ligand
(EC50,ref) were used to correlate experiments based on different surfaces (cf. Figure 3).
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