Synthesis of modified D-mannose core derivatives and their impact on GH38 α-mannosidases

Article abstract of DOI:10.1016/j.carres.2016.04.004

Nine new compounds having five- and modified six-member carbohydrate core derived from D-lyxose or D-mannose, and non-hydrolysable aglycones (benzylsulfonyl or aryl(alkyl)triazolyl) were synthesised to investigate their ability to inhibit the recombinant

Full text of DOI:10.1016/j.carres.2016.04.004

Carbohydrate Research 428 (2016) 62–71
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of modified D-mannose core derivatives and their impact
on GH38 α-mannosidases
a,
b
a
c
Monika Poláková *, Radim Horák , Sergej Šesták , Ivana Holková
a
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravska cesta 9, SK-845 38 Bratislava, Slovakia
Department of Organic Chemistry, University of Palacky, Tr.17. listopadu 12, CZ-771 46 Olomouc, Czech Republic
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalin cˇ iakova 8, SK-832 32 Bratislava, Slovakia
b
c
A R T I C L E
Article history:
Received 2 December 2015
Received in revised form 4 April 2016
Accepted 6 April 2016
I N F O
A B S T R A C T
Nine new compounds having five- and modified six-member carbohydrate core derived from D-lyxose
or D-mannose, and non-hydrolysable aglycones (benzylsulfonyl or aryl(alkyl)triazolyl) were synthesised
to investigate their ability to inhibit the recombinant Drosophila melanogaster homologs of two human
GH38 family enzymes: Golgi mannosidase II (dGMIIb) and lysosomal mannosidase (dLMII).
Two compounds were weak selective dGMIIb inhibitors showing IC50 at mM level. Moreover, it was
found that another GH38 enzyme, commercial jack bean α-mannosidase, was inhibited by triazole con-
jugates regardless of the carbohydrate core while the corresponding sulfones were inactive.
Available online 11 April 2016
Keywords:
GH38 family
Golgi α-mannosidase
Lysosomal α-mannosidase
Inhibition
©
2016 Elsevier Ltd. All rights reserved.
α-Mannosidase inhibitor
3
4
1
. Introduction
α-glucosidase, as well as glycogen phosphorylase on the glucosyl
triazole anomeric configuration has been proven. On the other hand,
only one of 4 tested glycosidases was affected by anomeric 1,5-
anhydrosugars, which have been shown to be selective inhibitors
It is well known that carbohydrates play an essential role in many
biological events. They are involved in fundamental physiological
processes such as cell–cell communication, immune response, patho-
gen defence, cancerogenesis, cell adhesion and others. Due to the
5
of jack bean α-mannosidase. Recently, endocyclic sulfur or sele-
nium containing carbohydrate mimetics with the positively charged
heteroatom have been reported to act as potent glycosidase
inhibitors, while glycosyl sulfoxides displayed only weak activity
towards glycosidases. Various fuco-configured pyrrolidotriazoles
have been found to exhibit enhanced inhibition of α-fucosidase and
their impact on several α-glucosidases was aglycone dependent.8
Modulation of affinity and selectivity towards various glycosi-
dases by ligand multivalency has been demonstrated for mono-, di-
and tri-valent iminosugars (Fig. 1).
In our ongoing research, synthetic mannose derivatives have been
constructed as models for mycobacterial mannosyltansferase
or eukaryotic α-mannosidases belonging to glycoside
hydrolase families 38 and 47.
inhibit ManT, instead, they served as good acceptor substrates.
They were more potent inhibitors of GH47 than GH38
α-mannosidases.
1
important role of carbohydrates in medicine and biology the de-
velopment and synthesis of novel functional structures with the
desired properties is an expanding area of research that continu-
ously provides a broad scale of new monovalent and multivalent
compounds and glycoconjugates. These carbohydrate derivatives have
been employed in many studies including assays of sugar process-
ing enzymes with the aim to identify potent inhibitors of the
biocatalysts. The enzymes include glycoside hydrolases which are
well known to be affected by numerous natural and synthetic car-
bohydrate mimetics. For example, various glycosyl triazoles have
been used to examine their ability to inhibit commercial glycosi-
dases, such as Escherichia coli β-galactosidase, sweet almond
6
7
9
1
0–12
(ManT)
1
3,14
The model compounds failed to
2
10–12
β-glucosidase and bovine liver β-galactosidase. The dependence of
inhibition of two glucosidases, sweet almond β-glucosidase and yeast
1
3,14
One of the GH38 enzymes, human Golgi α-mannosidase II
hGMII) (EC 3.2.1.114), is a very attractive target. It has been dem-
(
onstrated that known potent hGMII inhibitors induce an
immunostimulatory response that results in an anti-cancer effect.15
However, they also showed an undesired co-inhibition of lyso-
somal α-mannosidase (LM) (EC 3.2.1.24). This limits their use in
*
Corresponding author. Institute of Chemistry, Center for Glycomics, Slovak
Academy of Sciences, Dúbravska cesta 9, SK-845 38 Bratislava, Slovakia. Tel.: +421
59410272; fax: +421 2 59410222.
2
E-mail address: monika.polakova@savba.sk (M. Poláková).
http://dx.doi.org/10.1016/j.carres.2016.04.004
008-6215/© 2016 Elsevier Ltd. All rights reserved.
0

M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
63
Fig. 1. Examples of the compounds tested as inhibitors of various glycosidases.
clinical cancer chemotherapy16 and justifies a continued search for
a selective hGMII inhibitor.
All known inhibitors of hGMII and LM are characterised by cyclic
structures and the most potent of them comprise endocyclic
nitrogen.17–19 On the other hand, to the best of our knowledge the
potency and selectivity of five- and six-member ring compounds
containing endocyclic oxygen, such as derivatives of D-lyxose
and D-mannose, have not been investigated in detail. These

64
M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
carbohydrate cores possess cis-configured OH groups at positions
gave 2 in moderate yield. Subsequent tosyl group reduction with
NaBH provided fully protected benzyl 1-thio-α-D-rhamnoside 3.
Oxidation of rhamnoside 3 thio-functionality with mCPBA gave
sulfone 4. In final step, removal of benzoyl protective groups under
2+
2
and 3, which are necessary for binding to Zn co-factor at the active
4
2
0,21
site of dGMII.
We have previously reported the synthesis of a series of 13 alkyl
and phenylalkyl 1-thio-α-D-mannosides, sulfoxides and sulfones
and the examination of their inhibitory effect towards two
α-mannosidases, Drosophila melanogaster homologs of human Golgi
mannosidase II (dGMIIb) and lysosomal mannosidase (dLMII). Some
derivatives exhibited inhibitory activities at mM level towards both
dGMIIb (IC50 = 1.5–2.5 mM) and dLMII (IC50 = 1.0–2.0 mM). However,
mild conditions (K
2 3
CO , MeOH) provided the desired benzyl α-D-
rhamnosyl sulfone 5.
Other target derivatives, phenyl and benzyltriazolyl rhamnosides
14 and 15, were synthesised by Cu(I) catalysed azide–alkyne cyc-
2
2
loaddition (CuAAC) reaction of azide 10 with the corresponding
alkynes. Synthesis of 10 was accomplished from methyl α-D-
mannopyranoside 6 by the same sequence as reported for 3
(tosylation, benzoylation, reduction), followed by debenzoylation
and acetylation of secondary hydroxyls leading to peracetylated
methyl α-D-rhamnoside 8. Its acetolysis gave donor 9 for glycosylation
13
only benzyl mannopyranosyl sulfone was selective towards dGMIIb.
Subsequently, the phenylalkylsulfonyl group has been replaced
with another non-hydrolysable phenyl- or phenylalkyl-triazolyl
moiety. However, this alteration improved neither potency nor se-
lectivity towards dGMII. In general, these triazole conjugates were
better inhibitors of commercial Canavalia enciformis (jack bean)
α-mannosidase (JBMan) (EC 3.2.1.24, GH38 family member) than
2
3
of TMSN
3 4
catalysed by SnCl , yielding 10. The coupling of azide 10
with alkynes 11a and 11b carried out in DMF/H
2
O (3:1) solvent
mixture using copper(II) sulfate and sodium ascorbate1
1,14,24
af-
1
4
dGMIIb and dLMII.
forded 12 and 13 in moderate yields. Triazole conjugates 14 and 15
were obtained after saponification of acetyl protective groups.
The second group consisted of compounds having D-mannose
unit elongated at C-6 (Scheme 2). Mannoside 6 was transformed
over 5 steps to known intermediate 16 which was identified by NMR
With the aim to improve the selectivity towards dGMIIb, further
modifications of the carbohydrate core of potential inhibitors of this
enzyme were carried out, while keeping the same non-hydrolysable
aglycones (benzylsulfonyl, phenyl- and benzyl-triazolyl). These modi-
fications included elongation or deoxygenation at C-6 atom of
D-mannose unit and alteration of D-mannose with D-lyxose. This
paper deals with the synthesis of nine new derivatives and exam-
ination of their ability to inhibit the GH38 family enzymes: dGMIIb,
dLMII and JBMan.
25
spectroscopy as the S-isomer at C-6. Compound 16 was then con-
2
6
4
verted to peracetylated donor 19 in high overall yield. SnCl -
catalysed glycosylation of benzylmercaptan with 19 afforded benzyl
thioglycoside 20, which was by the same reaction sequence as de-
scribed for 3 converted to the required (6S) benzyl 6-C-methyl-α-
D-mannopyranosyl sulfone 22. The same approach reported for
conjugates 14 and 15 was used also in the synthesis of triazoles 26
and 27. Their synthesis commenced from azide 23 prepared from
2. Results and discussion
2.1. Synthesis
3
glycosyl donor 19 and TMSN .
The last group included derivatives comprising a five member
D-lyxose ring (Scheme 3) possessing cis-OH group arrangement at
positions 2 and 3. Sulfone 30 and triazoles 35 and 36 were ob-
tained from the glycosyl acceptors (TMSN and BnSH) and 1–acylated
3
donors 28 or 31, respectively, using analogous reaction sequences
The first series of prepared compounds (Scheme 1) included
D-rhamnosides, i.e. those having D-mannose unit deoxygenated at
C-6 atom. Synthesis of benzyl α-D-rhamnosyl sulfone 5 com-
menced from benzyl 1-thio-α-D-mannopyranoside 1.13 One pot
tosylation of primary hydroxyl group of 1 followed by benzoylation
as depicted in Schemes 1 and 2.
Scheme 1. Reagents and conditions: (a) 1. TsCl, py/CH2Cl2, rt, 16 h; 2. BzCl, rt, 16h; 2 (50%), 7 (30%); (b) NaBH4, DMF, 65 °C, 6 h; 34%; (c) mCPBA, CH2Cl2, rt, 3 h; 95%; (d)
K2CO3, MeOH, rt; 5 (69%), 14 (83%), 15 (60%); (e) 1. NaBH4, DMF, 65 °C, 4 h; 2. 0.02M MeONa, MeOH, 20 h, rt; 3. Ac2O, py, 16 h, rt; 50% over 3 steps; (f) Ac2O, AcOH, H2SO4(cat.),
1
h, 0 °C; 93%; (g) TMSN3, SnCl4, CH2Cl2, rt, 2 h; 85%; (h) 11a or 11b, CuSO4, Na ascorbate, DMF/H2O, 16 h, rt; 12 (90%), 13 (91%).

M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
65
Scheme 2. Reagents and conditions: (a) H2, 10% Pd/C, MeOH, rt, 4 h; 98%; (b) Ac2O, py, 16 h, rt; 98%; (c) Ac2O, AcOH, H2SO4(cat.), 1 h, 0 °C; 99%; (d) BnSH, BF3·OEt2, CH2Cl2,
rt, 16 h; 70%; (e) mCPBA, CH2Cl2, rt, 3 h; 85%; (f) K2CO3, MeOH, rt; 22 (85%), 26 (80%), 27 (82%); (g) TMSN3, SnCl4, CH2Cl2, rt, 2 h; 88%; (h) 11a or 11b, CuSO4, Na ascorbate,
DMF/H2O, 16 h, rt; 24 (91%), 25 (76%).
1
The structures of the targeted compounds and the intermedi-
ates were determined by NMR spectroscopy. The first series of
compounds (structures 3–6, 8–15), featuring deoxygenation at C-6,
were confirmed based on their characteristic shifts in the C NMR
spectra. For example, the C-6 signal of rhamnoside 3 appeared at
δ = 17.0. The C-6 atom of mannoside 2 showed a significantly higher
chemical shift (δ = 68.6). Moreover, the C-6 signals in all rhamnosides
were easily identified in the 1H NMR spectra (doublet at 1.2–
tified in the H NMR spectra by the presence of a singlet originating
from hydrogen of triazolyl ring (NC=CH). This singlet appeared at
7.93–7.96 and 7.34–7.38 ppm for protected phenyl and benzyl
triazoles, respectively. After deprotection of the saccharide core, the
singlet resonance was shifted to higher values by about 0.5 ppm (8.50
and 7.85 ppm).
In summary, 9 new synthetic conjugates 5, 14, 15, 22, 26, 27, 30,
35 and 36 were prepared by multistep synthesis in moderate to high
yields.
1
3
3
1.4 ppm, J 6.2–6.5 Hz). Similarly, the doublet of C–7 (CH function)
of mannosides 16–27 (elongated at C-6) was observed in the same
region and had a similar coupling constant. Oxidation of
thioglycosides to the corresponding sulfones resulted in a lower
chemical shift of the H-1 signal of the sulfones compared to the shifts
of the thioglycosides. On the contrary, the C-1 signal of the sul-
fones had a significantly (by about 5–6 ppm) higher chemical shift
than the same carbon of the thioglycosides. The opposite shift of
anomeric signals was observed when glycosyl azides were coupled
with alkynes providing glycosyl triazoles. The anomeric proton of
the azide had a slightly lower chemical shift than that of the triazole
derived thereof. Formation of glycosyl triazoles can also be iden-
2.2. Biological assay with the GH38 enzymes13,14
Compounds 5, 14, 15, 22, 26, 27, 30, 35 and 36 were assayed
towards dGMIIb, dLMII and JBMan (Table 1). None of the mannoside
analogs served as a substrate of these enzymes or an inhibitor of
dLMII. In contrast, the activity of dGMIIb, the most important
enzyme, was negatively affected by benzyl α-D-rhamnosyl sulfone
5 (IC50 = 2.0 mM) and benzyl triazole 27 (IC50 = 3.0 mM) elongated
at C-6 of the D-mannose unit, suggesting that these conjugates may
work as selective dGMIIb inhibitors. Deoxygenation at C-6 of
Scheme 3. Reagents and conditions: (a) 1. BnSH, BF3·OEt2, CH2Cl2, rt, 16 h, 2. mCPBA, CH2Cl2, rt, 3 h; 66%; (b) K2CO3, MeOH, rt; 30 (80%), 35 (88%), 36 (89%); (c) TMSN3, SnCl4,
CH2Cl2, rt, 2 h; 78%; (d) 11a or 11b, CuSO4, Na ascorbate, DMF/H2O, 16 h, rt; 33 (91%), 34 (72%).

66
M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
Table 1
Measured IC50 values for dGMIIb, dLMII and JBMan (in mM)
3. Conclusion
Compound
dGMIIb
dLManII
JBMan
All phenyl- and benzyl-triazolyl conjugates act as selective JBMan
inhibitors. Compounds with five- and modified six-member car-
bohydrate cores having endocyclic oxygen and either benzylsulfonyl
or phenyl- and benzyl-triazolyl non-hydrolysable aglycones are in-
active or poor inhibitors of dGMIIb. Alterations of D-mannose core,
carried out with the intention to search for more efficient dGMIIb
inhibitors, did not result in improved features of the tested com-
pounds with respect to selectivity and potency towards this enzyme.
Therefore, our current effort is focused on molecular modelling-
assisted design of new carbohydrate core(s) of dGMIIb inhibitors
that would better meet the requirements.
5
2.0
n.i.
n.i.
n.i.
n.i.
3.0
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
2.3
2.5
n.i.
1.2
1.2
n.i.
2.7
2.8
1
4
1
5
2
2
2
6
2
7
30
35
36
a
a
b
3
3
7
2.0
n.i.
2.0
8b
5.0
0.5
1.1
n.i. - no inhibition or inhibiton < 10% at 5 mM.
a
13
IC50 values taken from Poláková et al.
b
IC50 values taken from Poláková et al.14
4. Experimental
4.1. General methods
TLC was performed on aluminium sheets precoated with silica
gel 60 F254 (Merck). Flash column chromatography was carried out
on silica gel 60 (0.040–0.060 mm, Merck) with distilled solvents
(hexanes, ethylacetate, acetonitrile, methanol). Dichloromethane was
3
D-mannose led to a replacement of hydroxymethyl with a CH -
functionality in rhamnosyl sulfone 5; however, both 5 and
mannopyranosyl sulfone 3713 (IC50 = 2.0 mM, Fig. 2) exhibited the
dried (CaH
2
) and distilled before use. All reactions containing sen-
same selectivity and inhibitory activity towards dGMIIb. On the other
hand, elongation at C-6 of D-mannose in sulfone 22 resulted in its
inactivity towards dGMIIb. These observations indicate that the
potency of benzyl sulfones is influenced by the nature and size of
the C-6 substituent of the mannose core.
1
sitive reagents were carried out under an argon atmosphere. H NMR
13
and C NMR spectra were recorded at 25 °C with a VNMRS 400 MHz
Varian spectrometer, chemical shifts are referenced to either TMS
1
1
(δ 0.00, CDCl
3
for H) or HOD (δ 4.87, CD
3
OD) for H, and to inter-
13
nal CDCl
3
3
(δ 77.23) or CD OD (δ 49.15) for C. Optical rotations were
In general, the triazoles were less potent inhibitors of dGMIIb
1
4
measured on a Jasco P2000 polarimeter at 20 °C. High resolution
mass determination was performed by ESI–MS on a Thermo Sci-
entific Orbitrap Exactive instrument operating in positive mode.
The conjugates 5, 14, 15, 22, 26, 27, 30, 35 and 36 used in bio-
logical tests were lyophilised before the use. p-Nitrophenyl α-D-
mannopyranoside (pNP-Manp), jack bean α-mannosidase and
alkynes 11a and 11b were purchased from Sigma; and swainsonine
was purchased from Calbiochem. Inhibition assays for GH family
than sulfones with the exception of C-6-chain elongated com-
pounds. In this case, benzyl triazole 27 was better than the
corresponding sulfone 22 and it was also a slightly better inhibi-
1
4
tor of dGMIIb (IC50 = 3.0 mM) than benzyl mannosyl triazole 38
IC50 = 5.0 mM).
A more specific influence of the tested compounds on JBMan was
(
observed. While all synthesised derivatives having a benzylsulfonyl
aglycone were inactive, all glycosyl triazoles acted as JBMan inhibi-
tors (IC50 = 1.2–2.8 mM). It is noteworthy that compounds 26 and
3
8 enzymes were performed using a microplate reader (Epoch,
5
TM
BioTek and Gen
data collection and analysis software).
27 elongated at C-6 atom of D-mannose were approximately two
times more efficient than triazoles 14, 15 and 35, 36. These data are
in line with our previous observation that triazoles with a non-
modified mannose moiety are stronger JBMan inhibitors than the
corresponding phenylalkyl mannosyl sulfones that also affect
4
.2. Chemistry
.2.1. Synthesis
4
JBMan.14 All the modification of D-mannose core performed in this
4.2.1.1. General procedure for oxidation of thioglycoside (Method A). To
a stirred and at 0 °C cooled solution of corresponding thioglycoside
study thus resulted in the inactivity of the benzylsulfonyl deriva-
tives towards JBMan while inhibitory activity of the triazoles was
not affected significantly.
(
1 eq) in CH
was added. The reaction mixture was stirred at rt for 3 h, diluted
with CH Cl (20 mL), washed with satd NaHCO (2 × 20 mL) and
water (30 mL). The organic phase was dried (Na SO ), filtered, con-
2 2
Cl (20 mL) mCPBA (2.5 eq, 77% peroxide content)
In summary, the assays towards Drosophila homologs of the mam-
malian GH38 α-mannosidases (dGMIIb and dLMII) revealed that the
synthesised compounds display poor inhibitory activity. Thus, modi-
fied D-mannose units as well as D-lyxose structural moieties are
inappropriate carbohydrate cores for efficient and selective inhibi-
tors of dGMIIb. The capability of all triazole conjugates to inhibit
JBMan suggests that the plant GH38 enzyme is a member of another
subfamily than dGMIIb and dLMII.
2
2
3
2
4
centrated and the crude product was purified by column
chromatography (hexane:EtOAc).
4
.2.1.2. General procedure for azide synthesis (Method B). A solu-
tion of donor (1eq) in CH Cl (10 mL) was cooled at 0 °C and TMSN
followed 1M SnCl in CH Cl were added. After being stirred at rt
for 2 h, the reaction was diluted with DCM (20 mL) and poured into
cold satd. NaHCO (20 mL). After 15 min stirring it was filtered
through Celite, the organic phase was separated, dried (Na SO ), fil-
2
2
3
4
2
2
3
2
4
tered and concentrated. The crude product was purified by column
chromatography (hexane:EtOAc).
4
.2.1.3. General procedure for CuAAC reaction (Method C). To a so-
lution of azide (1 eq) in DMF: H O (1.6 mL, 3:1) alkyne 11a or 11b
1.1 eq) was added followed by sodium ascorbate (0.8 eq) and Cu(II)
sulphate (0.4 eq). The reaction mixture was stirred at rt for 16 h.
The reaction mixture was poured into satd. NH Cl (20 mL) and ex-
tracted with EtOAc (3 × 20 mL). The organic extracts were combined,
2
(
4
Fig. 2. Compounds previously tested as inhibitors of GH38 mannosidases.13,14

M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
67
washed with water, dried and concentrated. The crude product was
purified by column chromatography (hexane:EtOAc).
1H, H-5), 4.58 (d, 1H, J 14.4 Hz, SCH
SCH Ph), 1.41 (d, 3H, J5,CH3 6.2 Hz, CH
δ 165.8, 165.1, 165.0 (3 × PhCO), 133.8–127.1 (Ar), 87.1 (C-1), 72.2
2
Ph), 4.37 (d, 1H, J 14.4 Hz,
); C NMR (100 Mz, CDCl ):
3 3
1
3
2
4
.2.1.4. General procedure for deprotection (Method D). Protected com-
pound (1eq) was dissolved in dry methanol (3–5 mL) and K CO
0.5 eq) was added thereto. The reaction mixture was stirred at rt
until disappearance of starting material, then filtered and concen-
trated. Purification by column chromatography (CH CN:MeOH or
(C-5), 70.7 (C-4), 69.9 (C-3), 60.5 (SO
2
CH
2
Ph), 18.3 (CH
3
). HRMS
+
2
3
(MALDI): m/z calcd for [C34
H
40
O
9
S]Na : 637.1508. Found: 637.1515.
(
4.2.1.8. Benzyl 6-deoxy-α-
D
-mannopyranosyl sulfone (5).
3
Deprotection of compound 4 (0.065 g, 0.11 mmol) according to
EtOAc:MeOH), evaporation and lyophilisation gave the target com-
pound for biological evaluation.
general procedure (Method D) and purification by column chro-
matography (CH
foam, [α]
3
CN:MeOH 1:0→15:1) gave 5 (0.022 g, 69%). White
1
D
+ 85 (c 0.6, MeOH); H NMR (400 MHz, CDCl
3
): δ 7.50–
4
.2.1.5. Benzyl 2,3,4-tri-O-benzoyl-6-O-tosyl-1-thio-α-
mannopyranoside (2).
To a stirred solution containing 113 (0.56 g, 1.95 mmol) in CH
pyridine (1:1, 15 mL) cooled down on an ice bath tosyl chloride
0.48 g, 2.53 mmol) was added. The resulting mixture was brought
to rt and the stirring was continued for 16 h. The reaction mixture
was diluted with CH Cl (25 mL), washed with 1M HCl (5 × 10 mL),
satd NaHCO (3 × 10 mL), water (20 mL), dried with anhydrous
Na SO , filtered and concentrated. The crude product was dis-
D
-
7.40 (m, 5H, Ar), 4.75 (d, 1H, J1,2 1.2 Hz, H-1), 4.62 (d, 1H, J 14.1 Hz,
SO
SO
2
CH
CH
2
Ph), 4.44 (dd, 1H, J2,3 3.6 Hz, H-2), 4.38 (d, 1H, J 14.1 Hz,
2
Ph), 4.25 (m, 1H, H-5), 3.93 (dd, 1H, J3,4 9.4 Hz, H-3), 3.49 (t,
2 2
Cl :
2
13
1H, J4,5 9.4 Hz H-4), 1.36 (d, 3H, J5,CH3 6.2 Hz, CH
3
); C NMR (100 Mz,
(
CDCl ): δ 137.1–131.4 (Ar), 89.9 (C-1), 73.7 (C-5), 71.4 (2×) (C-3, C-4),
3
65.5 (C-2), 56.4 (SCH
2
Ph), 17.2 (CH
3
). HRMS (MALDI): m/z calcd for
+
2
2
[C13H
18
O
6
S]Na : 325.0722. Found: 325.0745.
3
2
4
4.2.1.9. Methyl 2,3,4-tri-O-benzoyl-6-O-tosyl-α-D-mannopyranoside
(7).
solved in pyridine (7 mL) and cooled down on an ice bath. Benzoyl
chloride (0.64 g, 0.53 mL, 4.54 mmol) was added dropwise. The re-
sulting mixture was brought to rt and the stirring was continued
for 16 h. The reaction mixture was diluted with water (10 mL) and
CH
HCl (3 × 15 mL), satd NaHCO
Na SO , filtered and concentrated. The crude product was purified
To a cooled (0 °C) solution of methyl-α-D-mannopyranoside 6
(1.18 g, 6.1 mmol) in dry pyridine (7 mL), TsCl (1.28 g, 6.7 mmol) in
dry pyridine (5 mL) was added over 15 minutes. The reaction mixture
was then stirred overnight at rt, the solvent was evaporated and
the residue was purified by column chromatography (hexane:EtOAc
1:1→EtOAc) to afford methyl-6-O-tosyl-α-D-mannopyranoside
(0.90 g, 2.6 mmol), which was dissolved in dry pyridine (12 mL). BzCl
(1.5 mL, 12.9 mmol) was added dropwise over 20 minutes at 0 °C
to precooled solution. After stirring at rt overnight, the reaction
mixture was poured on ice cold water (100 mL) and extracted with
DCM (2 × 20 mL). The combined organic layers were washed
with aqueous 1M HCl (40 mL), water (40 mL), brine (40 mL), dried
Cl
2 2
(30 mL). The organic phase was separated, washed with 1M
3
(3 × 10 mL), dried with anhydrous
2
4
by column chromatography (hexanes:EtOAc 10:1→3:1) to give 2
1
(
(
0.73 g, 50% over 2 steps) as an oil. [α]
400 MHz, CDCl
D
+16 (c 0.5, CHCl
3
); H NMR
3
): δ 8.03–7.17 (m, 24H, Ar), 5.80–5.75 (m, 2H, H-3,
H-4), 5.69 (dd, 1H, J2,3 3.1 Hz, H-2), 5.28 (d, 1H, J 1,2 1.4 Hz, H-1), 4.69
m, 1H, H-5), 4.24 (dd, 1H, J 5,6b 5.8 Hz, H-6b), 4.17 (dd, 1H, J 5,6a 2.5 Hz,
6a,6b 12.1 Hz, H-6a), 3.88 (d, 1H, J 13.5 Hz, SCH Ph), 3.80 (d, 1H, J
3.5 Hz, SCH Ph), 2.33 (s, 3H, CH ):
(Ts)); 13C NMR (100 Mz, CDCl
(
J
1
2
2
3
3
2 4
with Na SO , filtered and concentrated. The residue was washed with
δ 165.8, 165.4, 165.3 (3 × PhCO), 137.1–128.0 (Ar), 81.8 (C-1), 71.8
ethanol and the solvent was evaporated. Ethanol was added again
and crystals were filtered, washed with small amount of cold EtOH
(
2
7
C-2), 70.2 (C-3), 68.9 (C-5), 68.6 (C-6), 66.7 (C-4), 34.9 (SCH
2
Ph),
+
0.9 (CH
3
(Ts)). HRMS (MALDI): m/z calcd for [C41
H
36
O
10
S
2
]Na :
and dried under reduced pressure to obtain 7 (1.2 g, 30% over 2 steps)
1
75.1648. Found: 775.1661.
as white crystals. [α]
D
—108 (c 0.5, CHCl
3
). H NMR (400 MHz, CDCl
3
):
δ 8.09–7.19 (m, 19H, Ar), 5.83 (dd, 1H, J3,4 9.8 Hz, H-3), 5.76 (t, 1H,
4,5 9.6 Hz, H-4), 5.62 (dd, 1H, J2,3 3.1 Hz, H-2), 4.91 (d, 1H, J1,2 1.8 Hz,
H-1), 4.28–4.24 (m, 3H, H-5, H-6a, H-6b), 3.49 (s, 3H, OCH ), 2.35
): δ 165.4(2×), 165.3
(3 × PhCO), 144.8–128.0 (Ar), 98.5 (C-1), 70.3 (C-2), 69.7 (C-3), 68.7
4
.2.1.6. Benzyl 2,3,4-tri-O-benzoyl-6-deoxy-1-thio-α-
D
-
J
mannopyranoside (3).
3
1
3
Compound 2 (0.65 g, 0.86 mmol) was dissolved in DMF (8 mL)
and sodium borohydride (0.16 g, 4.23 mmol) was added slowly. The
resulting mixture was stirred at 65 °C for 6 h. The reaction mixture
was cooled to rt, neutralised with glacial AcOH and diluted with
3 3
(s, 3H, CH (Ts)); C NMR (100 MHz, CDCl
(C-5), 68.4 (C-6), 66.8 (C-4), 55.6 (OMe), 21.6 (CH
3
(Ts)). HRMS
+
32
(MALDI): m/z calcd for [C35H O11S]Na : 683.1563. Found: 683.1553.
CH
NaHCO
2
Cl
2
(30 mL). The organic phase was separated, washed with satd
(3 × 20 mL), water (2 × 20 mL), dried with anhydrous Na SO
3
2
4
,
4.2.1.10. Methyl 2,3,4-tri-O-acetyl-α-D-rhamnopyranoside (8).
filtered and concentrated. The crude product was purified by column
To a solution of 7 (0.87 g, 1.32 mmol) in DMF (15 mL) NaBH
4
chromatography (hexanes:EtOAc 1:0 →12:1) to give 3 (0.17 g, 34%)
(0.25 g, 6.60 mmol) was added and the suspension was stirred at
65 °C under inert atmosphere for 4 h. Then the reaction mixture was
cooled to 0 °C, diluted with DCM (50 mL) and glacial acetic acid
(1.8 mL) was added. It was stirred until evolution of hydrogen ceased.
The solution was shaken with water (50 mL) and aqueous layer ex-
tracted with DCM (50 mL). The organic layers were combined,
1
D 3 3
as an oil. [α] —36.7 (c 0.3, CHCl ); H NMR (400 MHz, CDCl ): δ 8.07–
7
J
(
.22 (m, 20H, Ar), 5.77–5.74 (m, 2H, H-2, H-3), 5.68 (t, 1H, J3,4 6.4 Hz,
4,5 6.4 Hz, H-4), 5.35 (d, 1H, J1,2 1.5 Hz, H-1), 4.51 (dq, 1H, H-5), 3.92
d, 1H, J 9.0 Hz, SCH Ph), 3.85 (d, 1H, J 9.0 Hz, SCH Ph), 1.32 (d, 3H,
): δ 165.8, 165.4, 165.3
3 × PhCO), 137.8–127.4 (Ar), 81.8 (C-1), 72.2 (2×) (C-2, C-4), 70.7
2
3
2
1
J
3 3
5,CH3 6.2 Hz, CH ); C NMR (100 Mz, CDCl
(
(
3
washed with water (3 × 50 mL), satd. aqueous NaHCO (50 mL), brine
C-3), 67.6 (C-5), 35.4 (SCH
2
Ph), 17.0 (CH
S]Na : 605.1610. Found: 605.1607.
3
). HRMS (MALDI): m/z calcd
(1 × 50 mL), dried with Na SO and concentrated. The residue was
2
4
+
for [C34
H
30
O
7
purified by column chromatography (hexane:EtOAc 8:1→5:1) to
afford methyl 2,3,4-tri-O-benzoyl-α-D-rhamnopyranoside (0.40 g,
62%).
4
.2.1.7. Benzyl 2,3,4-tri-O-benzoyl-6-deoxy-α-
D
-mannopyranosyl
sulfone (4).
Methyl 2,3,4-tri-O-benzoyl-α-D-rhamnopyranoside (0.40 g,
0.816 mmol) was dissolved in 0.02 M methanolic MeONa
(10 mL) and stirred for 20 h at rt. The suspension was neutralised
with AcOH, concentrated and the residue was purified by column
chromatography (EtOAc:MeOH 10:1→5:1) yielding methyl α-D-
Oxidation of compound 3 (0.09 g, 0.15 mmol) according to general
procedure (Method A) and purification by column chromatogra-
phy (hexanes:EtOAc 15:1→5:1) gave 4 (0.09 g, 95%) as an oil.
1
D
[α] —50.0 (c 0.2, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 8.07–7.22 (m,
2
5
0H, Ar), 6.33 (dd, 1H, J2,3 3.8 Hz, H-2), 6.09 (dd, 1H, J3,4 10.0 Hz, H-3),
.72 (t, 1H, J4,5 9.9 Hz, H-4), 4.97 (d, 1H, J1,2 1.5 Hz, H-1), 4.88 (dq,
rhamnopyranoside (0.13 g, 96%). Ac
was added dropwise to the cooled (0 °C) solution of methyl
2
O (0.31 mL, 3.29 mmol)

68
M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
α-D-rhamnopyranoside (0.13 g, 0.73 mmol) in pyridine (2 mL). The
solution was stirred at rt overnight and then poured on ice (25 mL),
extracted with DCM (2×), the organic layers were combined and
(t, 1H, J4,5 8.9 Hz, H-4), 4.12–4.08 (m, 2H, CH
2
Ph), 3.75 (dq, 1H, J5,CH3
CO), 1.23 (d,
): δ 169.9, 169.7, 169.3
CH), 138.4, 128.7, 126.6 (Ar), 121.5 (NC CH),
83.5 (C-1), 70.9 (C-4), 69.9 (C-5), 68.9 (C-3), 68.6 (C-2), 32.1 (CH Ph),
). HRMS (MALDI): m/z calcd for
6.3 Hz, H-5), 2.17, 2.06, 2.04 (each s, each 3H, 3× CH
3
1
3
3H, CH
3 3
); C NMR (100 MHz, CDCl
washed with 1 M HCl (2×), dried with Na
2
SO
D
4
and concentrated to
+ 49.7 (c 1.1, CHCl );
): δ 5.29 (dd,
(3 × CH CO), 148.3 (NC
3
=
=
2
afford 8 (0.20 g, 90%) as a colourless oil. [α]
3
2
7
1
lit [α]
D
+ 53.7 (c 1.3, CHCl
3
). H NMR (400 MHz, CDCl
3
20.7(2×), 20.6 (3 × CH
3
CO), 17.2 (CH
3
+
1
9
H, J3,4 10.2 Hz, H-3), 5.24 (dd, 1H, J2,3 3.5 Hz, H-2), 5.07 (t, 1H, J4,5
.8 Hz, H-4), 4.63 (d, 1H, J1,2 1.6 Hz, H-1), 3.86 (dq, 1H, J5,CH3 6.3 Hz,
[C21H
25
N
3
O
7
]Na : 454.1590. Found: 454.1580.
H-5), 3.39 (s, 1H, OCH
3
), 2.15, 2.05, 1.99 (each s, each 3H, 3× CH
3
CO),
): δ 170.1, 170.0(2×)
CO), 98.5 (C-1), 71.1, 69.8, 69.1, 66.2 (C-2, C-3, C-4, C-5),
5.1(OMe), 20.9, 20.8, 20.7 (3 × CH CO), 17.4 (CH ). HRMS (MALDI):
]Na : 327.1056. Found: 327.1059.
4.2.1.15. 1-(α-D-Rhamnopyranosyl)-4-phenyl-1,2,3-triazole (14).
Deprotection of 12 (59 mg, 0.141 mmol) according to general pro-
cedure (Method D) and purification by column chromatography
1
3
1.24 (d, 3H, CH
3
); C NMR (100 MHz, CDCl
3
(
3 × CH
3
5
3
3
(EtOAc) gave 14 as a colourless oil (34 mg, 83%). [α]
D
+ 49.4 (c 0.2,
+
1
m/z calcd for [C13
H
20
O
8
MeOH). H NMR (400 MHz, CD
3
OD): δ 8.47 (s, 1H, CHN), 7.86–7.83
(
m, 2H, Ar), 7.46–7.42 (m, 2H, Ar), 7.38–7.33 (m, 1H, Ar), 6.01 (d,
28
4
.2.1.11. 1,2,3,4-Tetra-O-acetyl-α-D-rhamnopyranoside (9) .
To the solution of 8 (0.20 g, 0.65 mmol) in AcOH (3.5 mL) and
O (1 mL) cooled to 0 °C was added conc. H SO (0.15 mL). After
O (80 mg) was added and mixture
1H, J1,2 2.5 Hz, H-1), 4.72 (dd, 1H, J2,3 3.4 Hz, H-2), 4.13 (dd, 1H, J3,4
8.7 Hz, H-3), 3.59 (t, 1H, J4,5 8.7 Hz, H-4), 3.42 (dq, 1H, J5,CH3 6.3 Hz,
H-5), 1.33 (d, 3H, CH OD): δ 149.2
(NC CH), 131.6, 130.1, 129.7, 126.9 (Ar), 122.2 (NC=CH), 88.7 (C-
Ac
1
2
2
4
3
); 13C NMR (100 MHz, CD
3
h of stirring at 0 °C, NaOAcꢀ3H
2
=
was concentrated. The residue was purified by column chromatog-
1), 73.8(2×) (C-4, C-5), 72.6 (C-3), 69.3 (C-2), 18.1 (CH
3
). HRMS
+
raphy (hexane:EtOAc 4:1→2:1) providing 9 (0.20 g, 93%) as a
17 3 4
(MALDI): m/z calcd for [C14H N O ]Na : 314.1117. Found: 314.1119.
1
colourless oil. H NMR (400 MHz, CDCl
3
): δ 6.02 (d, 1H, J1,2 2.0 Hz,
H-1), 5.31 (dd, 1H, J3,4 10.2 Hz, H-3), 5.25 (dd, 1H, J2,3 3.5 Hz, H-2),
4.2.1.16. 1-(α-D-Rhamnopyranosyl)-4-benzyl-1,2,3-triazole (15).
Deprotection of 13 (57 mg, 0.132 mmol) according to general pro-
cedure (Method D) and purification by column chromatography
5
2
.13 (t, 1H, J4,5 10.0 Hz, H-4), 3.94 (dq, 1H, J5,CH3 6.2 Hz, H-5), 2.17,
1
3
.16, 2.07, 2.01 (each s, each 3H, 4× CH
3
CO), 1.24 (d, 3H, CH
3
);
C
NMR (100 MHz, CDCl
3
): δ 170.1, 169.8(2×), 168.4 (4 × CH
3
CO), 90.6
(EtOAc:MeOH 10:1→5:1) gave 15 as a colourless oil (24 mg, 60%).
1
(C-1), 70.5 (C-4), 68.8 (C-3), 68.7 (C-5), 68.6 (C-2), 20.9, 20.8, 20.7(2×)
[α]
D
—35.7 (c 0.1, MeOH). H NMR (400 MHz, CD
3
OD): δ 7.84 (s, 1H,
+
(
4 × CH
3
CO), 17.4 (CH
3
). HRMS (MALDI): m/z calcd for [C14
H
20
O
9
]Na :
CHN), 7.31–7.18 (m, 5H, Ar), 5.90 (d, 1H, J1,2 2.5 Hz, H-1), 4.63 (dd,
1H, J2,3 3.5 Hz, H-2), 4.07 (s, 2H, CH Ph), 4.05 (dd, 1H, J3,4 8.8 Hz, H-3),
.54 (t, 1H, J4,5 8.8 Hz, H-4), 3.31 (m, 1H, H-5), 1.27 (d, 3H, J5,6 6.3 Hz,
3
55.1005. Found: 355.1011.
2
3
CH
22
13
4.2.1.12. 2,3,5-Tri-O-acetyl-α-D-rhamnopyranosyl azide (10) .
3
); C NMR (100 MHz, CD
3
OD): δ 148.9 (NC=CH), 140.5, 129.8(2×),
Reaction of 9 (0.2g, 0.60 mmol, 1eq) and TMSN
3
(1 eq) catalysed
127.7 (Ar), 123.7 (NC=CH), 88.5 (C-1), 73.7 (C-5) 73.6 (C-4), 72.6
by 1M SnCl in CH Cl (1 eq) according to general procedure (Method
4
2
2
(C-3), 70.4 (C-2), 32.7 (CH
2
Ph), 18.1 (CH
3
). HRMS (MALDI): m/z calcd
+
B) and purification by column chromatography (hexane:EtOAc
for [C15
H
19 3
N O
4
]Na : 328.1273. Found: 328.1282.
1
4
:1→2:1) gave azide 10 (0.16 g, 85%) as a clear oil. H NMR (400 MHz,
CDCl
5
1
3
): δ 5.32 (d, 1H, J1,2 2.0 Hz, H-1), 5.21 (dd, 1H, J3,4 10.2 Hz, H-3),
.15 (dd, 1H, J2,3 3.5 Hz, H-2), 5.09 (t, 1H, J4,5 9.8 Hz, H-4), 4.04 (dq,
H, J5,CH3 6.2 Hz, H-5), 2.17, 2.06, 1.99 (each s, each 3H, 3 × CH CO),
): δ 169.9, 169.8(2×)
CO), 87.5 (C-1), 70.5 (C-4), 69.5 (C-2), 68.6 (C-5), 68.3 (C-
4.2.1.17. (6S) Methyl 2,3,4-tri-O-benzyl-6-C-methyl-α-D-
mannopyranoside (16).
3
Compound 16 was synthesised by procedure described
1
3
25
1
1.28 (d, 3H, CH
3
); C NMR (100 MHz, CDCl
3
previously. [α]
7.32–7.19 (m, 15H, Ar), 4.90–4.60 (m, 6H, 3× CH
1,2 1.8 Hz, H-1), 4.04–4.00 (m, 2H, H-4, H-6), 3.82 (dd, 1H, J3,4 9.4 Hz,
H-3), 3.71 (dd, 1H, J2,3 3.1 Hz, H-2), 3.32 (dd, 1H, J4,5 9.7 Hz, J5,6 1.8 Hz,
H-5), 3.22 (s, 3H, OCH ), 1.23 (d, 3H, J 6.5 Hz, C-7(CH
)); 13C NMR
(100 Mz, CDCl ): δ 138.5–138.3 (Ar), 128.3–127.5 (Ar), 99.5 (C-1),
80.4 (C-3), 75.4, 75.1, 74.9, 74.4 (C-2, C-4, C-5, CH Ph), 73.0, 72.3
), 20.4 (C-7). HRMS (MALDI): m/z
D
—34.0 (c 0.1, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ
(3 × CH
3
2
Ph), 4.67 (d, 1H,
3), 20.8, 20.7, 20.6 (3 × CH
3
CO), 17.4 (CH
]Na : 338.0964. Found: 338.0956.
3
). HRMS (MALDI): m/z calcd
J
+
for [C12
H
17 3
N O
7
3
3
4
3
.2.1.13. 1-(2,3,4,-Tri-O-acetyl-α-D-rhamnopyranosyl)-4-phenyl-1,2,
-triazole (12).
Reaction of 10 (50 mg, 0.158 mmol) and alkyne 11a according
3
2
(2 × CH
2
Ph), 65.7 (C-6), 54.8 (OCH
3
+
to general procedure (Method C) and purification by column chro-
calcd for [C29
H
34
O
6
]Na : 501.2253. Found: 501.2274.
matography (hexane:EtOAc 6:1) gave 12 as a colourless oil (59 mg,
1
9
1
1
5
1
0%). [α]
D
+ 16 (c 0.25, CHCl
3
). H NMR (400 MHz, CDCl
3
): δ 7.96 (s,
4.2.1.18. (6S) Methyl 6-C-methyl-α-D-mannopyranoside (17).
Compound 16 (2.2 g, 4.60 mmol) was dissolved in MeOH (50 mL)
and 10% Pd/C was added. The solution was stirred under a H at-
2
mosphere for 4 h, the catalyst was then filtered off and washed with
MeOH (30 mL). The solvent was evaporated and purification of the
H, CHN), 7.88–7.86 (m, 2H, Ar), 7.47–7.44 (m, 2H, Ar), 7.40–7.36 (m,
H, Ar), 6.04 (dd, 1H, J2,3 3.7 Hz, H-2), 5.99 (d, 1H, J1,2 2.3 Hz, H-1),
.91 (dd, 1H, J3,4 9.2 Hz, H-3) 5.20 (t, 1H, J4,5 9.0 Hz, H-4), 3.79 (dq,
H, J5,CH3 6.5 Hz, H-5), 2.20, 2.07, 2.06 (each s, each 3H, 3 × CH
3
CO),
): δ 169.9, 169.8, 169.4
CH), 129.9, 128.9, 128.6, 125.9 (Ar), 119.4
CH), 83.8 (C-1), 70.8 (C-4), 70.1 (C-5), 68.9 (C-3), 68.6 (C-2),
0.8(2×), 20.6 (3 × CH CO), 17.3 (CH ). HRMS (MALDI): m/z calcd for
]Na : 440.1434. Found: 440.1426.
1
3
1
(
(
.27 (d, 3H, CH
3 × CH CO), 148.3 (NC
NC
3
); C NMR (100 MHz, CDCl
3
crude product by column chromatography (CHCl
yielded 17 (0.94 g, 98%) as an oil. [α]
(400 MHz, CD
1.9 Hz, J 6.5 Hz, H-6), 3.82–3.79 (m, 2H, H-2, H-4), 3.68 (dd, 1H, J2,3
3.5 Hz, J3,4 9.5 Hz, H-3), 3.38 (s, 3H, OCH ), 3.29 (dd, 1H, J4,5 9.7 Hz,
)); C NMR (100 Mz, CD OD): δ
3
:MeOH 10:1→5:1)
+ 30.0 (c 0.2, MeOH); H NMR
1
3
=
D
=
3 d
OD): δ 4.71 (d, 1H, J1,2 1.8 Hz, H-1), 4.14 (dq, 1H, J
2
3
3
q
+
[C
20
H
23
N
3
O
7
3
1
3
H-5), 1.32 (d, 3H, J 6.6 Hz, C-7(CH
3
3
4
3
.2.1.14. 1-(2,3,4,-Tri-O-acetyl-α-D-rhamnopyranosyl)-4-benzyl-1,2,
-triazole (13).
102.8 (C-1), 75.9 (C-5), 72.9 (C-3), 72.1, 68.3 (C-2, C-4), 65.9 (C-6),
+
3 8 16 6
55.2 (OCH ), 20.2 (C-7). HRMS (MALDI): m/z calcd for [C H O ]Na :
Reaction of 10 (50 mg, 0.158 mmol) and alkyne 11b according
231.0845. Found: 231.0901.
to general procedure (Method C) and purification by column chro-
matography (hexane:EtOAc 5:1→3:1) gave 13 as a colourless oil
4.2.1.19. (6S) Methyl 2,3,4-tri-O-acetyl-6-C-methyl-α-D-
mannopyranoside (18).
1
(
62 mg, 91%). [α]
D
+ 48 (c 0.25, CHCl
3
). H NMR (400 MHz, CDCl
3
):
δ 7.35 (s, 1H, CHN), 7.33–7.25 (m, 5H, Ar), 5.93 (dd, 1H, J2,3 3.8 Hz,
H-2), 5.89 (dd, 1H, J3,4 8.8 Hz, H-3), 5.84 (d, 1H, J1,2 2.4 Hz, H-1), 5.15
A solution of compound 17 (0.9 g, 4.32 mmol) in pyridine (15 mL)
was cooled at 0 °C and acetic anhydride (30 mL) was added. The

M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
69
reaction mixture was stirred at rt for 16 h, then diluted with CH
2
Cl
(3 × 30 mL) and 1M HCl
3 × 20 mL). The organic phase was separated, dried with anhy-
SO , filtered and concentrated. The crude product was
2
3, C-4), 66.6 (C-2), 65.9 (C-6), 57.3 (CH
2
Ph), 20.4 (C-7). HRMS (MALDI):
+
(
(
50 mL) and washed with satd. NaHCO
3
m/z calcd for [C14
H
20
O
7
S]Na : 355.0827. Found: 355.0841.
drous Na
2
4
4
.2.1.23. (6S) 2,3,4,6-Tetra-O-acetyl-6-C-methyl-α-D-
mannopyranosyl azide (23).
Reaction of 19 (0.2g, 0.50 mmol, 1eq) and TMSN
with 1M SnCl in CH Cl (0.9 eq) according to general procedure
Method B) and purification by column chromatography
purified by column chromatography (hexanes:EtOAc 10:1→2:1) to
1
give 18 (1.60 g, 98%) as an oil. [α]
400 MHz, CDCl
1.9 Hz, J 6.6 Hz, H-6), 4.78 (d, 1H, J1,2 1.6 Hz, H-1), 3.76 (dd, 1H,
4,5 9.4 Hz, J5,6 2.1 Hz, H-5), 3.40 (s, 3H, OCH ), 2.16, 2.10, 2.01, 1.97
D
+ 45.2 (c 0.4, CHCl
3
); H NMR
3
(2.5eq) catalysed
(
3
): δ 5.31–5.25 (m, 3H, H-2, H-3, H-4), 5.07 (dq, 1H,
4
2
2
J
J
(
(
1
d
q
(
(
[
3
hexane:EtOAc 3:1→1.5:1) provided 23 (0.17 g, 88%) as a clear oil;
13
each s, each 3H, 4 × CH
100 Mz, CDCl ): δ 170.7, 170.2, 170.0, 169.7 (4× CH
), 71.5 (C-5), 69.7, 69.5 (C-2, C-3), 66.5 (C-6), 65.8 (C-4), 55.4 (OCH
1.2, 21.1, 20.8, 20.7 (4 × CH CO), 15.9 (C-7). HRMS (MALDI): m/z calcd
10]Na : 399.1267. Found: 399.1279.
3
CO), 1.35 (d, 3H, J 6.6 Hz, C-7(CH
CO), 99.1 (C-
),
3
)); C NMR
1
α]
D
+ 30.0 (c 0.2, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 5.46 (d, 1H,
3
3
J
1,2 1.8 Hz, H-1), 5.33 (t, 1H, J3,4 10.0 Hz, H-4), 5.23 (dd, 1H, J2,3 3.4 Hz,
H-3), 5.16 (dd, 1H, 1H, H-2), 5.11 (dq, 1H, J 2.1 Hz, J 6.6 Hz, H-6),
.94 (dd, 1H, H-5), 2.18, 2.11, 2.02, 1.98 (each s, each 3H, 4 × CH CO),
): δ 170.5,
CO), 87.9 (C-1), 73.8 (C-5), 69.3 (C-2), 68.7
3
d
q
2
3
3
3
+
for [C16
H
24
O
13
1.36 (d, 3H, J 6.6 Hz, C-7(CH
3
)); C NMR (100 Mz, CDCl
3
1
70.0, 169.9, 169.6 (4× CH
3
4
.2.1.20. (6S) Benzyl 2,3,4-tri-O-acetyl-6-C-methyl-1-thio-α-D-
(C-3), 66.2 (C-6), 65.3 (C-4), 21.1, 21.0, 20.7(2×) (4 × CH
3
CO), 15.9 (C-
+
mannopyranoside (20).
21 9 3
7). HRMS (MALDI): m/z calcd for [C15H O N ]Na : 410.1176. Found:
2
6
To a solution of 19 (prepared in 96% yield from 18 by acetoly-
410.1181.
sis as described for compound 8) (56 mg, 0.14 mmol) in CH
benzyl mercaptan (32.5 μL, 0.28 mmol) and BF OEt
.42 mmol) were added dropwise at 0 °C. After being stirred at rt
for 16 h, the reaction was diluted with DCM (15 mL) and poured
into cold satd. NaHCO (10 mL). The organic phase was separated,
washed with water, dried (Na SO ), filtered and concentrated. The
residue was purified by column chromatography hexane:EtOAc
:1→3:1) to give 20 (45 mg, 70%) as a clear oil. [α] + 132 (c 0.5,
): δ 7.24–7.19 (m, 5H, Ar), 5.32–
.26 (m, 2H, H-2, H-4), 5.18 (dd, 1H, J2,3 3.4 Hz, J3,4 10.0 Hz, H-3), 5.09
d, 1H, J1,2 1.6 Hz, H-1), 5.06 (dq, 1H, J 2.0 Hz, J 6.7 Hz, H-6), 4.19
dd, 1H, J4,5 9.8 Hz, J5,6 2.0 Hz, H-5), 3.68 (d, 1H, J 11.9 Hz, CH Ph),
Ph), 2.06, 2.05, 1.96, 1.89 (each s, each 3H,
2
Cl
2
(3 mL)
3
2
(52.6 μL,
4
.2.1.24. (6S) 1-(2,3,4,6-Tetra-O-acetyl-6-C-methyl-α-D-
mannopyranosyl)-4-phenyl-1,2,3-triazole (24).
0
Reaction of 23 (40 mg, 0.1 mmol) and alkyne 11a according to
3
general procedure (Method C) and purification by column chro-
matography (hexane:EtOAc 4:1→1:1) gave 24 as a colourless oil
2
4
1
(46 mg, 91%). [α]
D
+ 51.6 (c 0.1, CHCl
3
). H NMR (400 MHz, CDCl
3
):
7
D
δ 7.98 (s, 1H, CHN), 7.87–7.83 (m, 2H, Ar), 7.48–7.38 (m, 3H, Ar), 6.13
d, 1H, J1,2 2.4 Hz, H-1), 6.09 (dd, 1H, J2,3 3.8 Hz, H-2), 5.96 (dd, 1H,
3,4 9.3 Hz, H-3), 5.43 (t, 1H, J4,5 9.3 Hz, H-4), 5.11 (dq, 1H, J 2.8 Hz,
1
3 3
CHCl ); H NMR (400 MHz, CDCl
5
(
(
3
(
J
J
d
d
q
6.6 Hz, H-6), 3.64 (dd, 1H, H-5), 2.12, 2.05, 2.04, 2.03 (each s, each
q
13
2
3
H, 4 × CH
CDCl ): δ 170.5, 169.9, 169.7, 169.4 (4 × CH
29.8, 129.1, 128.8, 126.0, 119.7 (Ar, NC CH), 84.2 (C-1), 75.2 (C-
), 69.3 (C-3), 68.4 (C-2), 65.8(2×) (C-4, C-6), 21.0, 20.7, 20.6 (2×)
3 3
CO), 1.22 (d, 3H, J 6.6 Hz, C-7(CH )); C NMR (100 MHz,
.66 (d, 1H, J 11.9 Hz, CH
× CH CO), 1.26 (d, 3H, J 6.6 Hz, C-7(CH
CO), 136.5, 129.1, 128.9, 127.7
Ar), 81.6 (C-1), 72.2 (C-5), 70.9 (C-2), 70.1 (C-3), 66.5 (C-6), 66.0
C-4), 34.6 (CH Ph), 21.2, 21.1, 20.8, 20.7 (4 × CH CO), 16.1 (C-7). HRMS
S]Na : 491.1352. Found: 491.1371.
2
3
3
CO), 148.6 (NC=CH),
13
4
3
3 3
)); C NMR (100 Mz, CDCl ):
1
5
=
δ 170.7, 170.0, 169.9, 169.7 (4× CH
3
(
(
[
4 × CH
C
3
CO), 14.3 (C-7). HRMS (MALDI): m/z: calcd for
(
(
2
3
+
23
H
27
N
3
O
9
]Na : 512.1645. Found: 512.1649.
+
28 9
MALDI): m/z calcd for [C22H O
4
.2.1.25. (6S) 1-(2,3,4,6-Tetra-O-acetyl-6-C-methyl-α-D-
4.2.1.21. (6S) Benzyl 2,3,4,6-tetra-O-acetyl-6-C-methyl-α-D-
mannopyranosyl)-4-benzyl-1,2,3-triazole (25).
mannopyranosyl sulfone (21).
Oxidation of 20 (40 mg, 0.08 mmol) according to general pro-
cedure (Method A) and purification by column chromatography
Reaction of 23 (51 mg, 0.13 mmol) and alkyne 11b according to
general procedure (Method C) and purification by column chro-
matography (hexane:EtOAc 4:1→1:1) gave 25 as a colourless oil
1
(
hexanes:EtOAc 3:1→1.5:1) provided 21 (36 mg, 85%) as a clear oil.
(50 mg, 76%). [α] —20.0 (c 0.1, CHCl ). H NMR (400 MHz, CDCl ):
D
3
3
1
[
α]
D
+ 54 (c 0.5, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 7.45–7.40 (m,
δ 7.33–7.22 (m, 6H, CHN, Ar), 6.00 (dd, 1H, J2,3 3.9 Hz, H-2), 5.97 (d,
1H, J1,2 2.2 Hz, H-1), 5.91 (dd, 1H, J3,4 9.3 Hz, H-3), 5.38 (t, 1H, J4,5
9.4 Hz, H-4), 5.05 (dq, 1H, Jd 2.7 Hz, Jq 6.6 Hz, H-6), 4.12 (m, 2H,
2
5
5
4
4
H, Ar), 5.90 (dd, 1H, J2,3 3.7 Hz, H-2), 5.55 (dd, 1H, J3,4 9.8 Hz, H-3),
.36 (t, 1H, J4,5 9.8 Hz, H-4), 5.11 (dq, 1H, J 2.3 Hz, J 6.6 Hz, H-6),
.78 (d, 1H, J1,2 1.7 Hz, H-1), 4.60 (dd, 1H, J4,5 9.9 Hz, J5,6 2.3 Hz, H-5),
.48 (d, 1H, J 14.2 Hz, CH Ph), 4.28 (d, 1H, J 14.2 Hz, CH Ph), 2.12,
.11, 2.03, 1.98 (each s, each 3H, CH CO), 1.44 (d, 3H, J 6.6 Hz,
): δ 170.4, 4 169.6, 169.4, 169.3
CO), 130.9, 129.6, 129.5, 126.7 (Ar), 86.2 (C-1), 76.6 (C-5),
9.3 (C-3), 66.3 (C-6), 64.8(2×) (C-2, C-4), 57.3 (CH Ph), 21.1, 20.8,
CO), 16.2 (C-7). HRMS (MALDI): m/z calcd for
d
q
PhCH ), 3.51 (dd, 1H, H-5), 2.18, 2.08, 2.02, 2.01 (each s, each 3H,
13
2
2
4 × CH CO), 1.13 (d, 3H, J 6.6 Hz, C-7(CH )); C NMR (100 MHz, CDCl ):
3
3
3
2
3
δ 170.4, 170.0, 169.7, 169.4 (4 × CH CO), 148.8 (NC
=CH), 138.6, 129.8,
3
1
3
C-7(CH
3
)); C NMR (100 Mz, CDCl
3
128.9, 128.8, 126.7, 121.8 (Ar, NC=CH), 84.1 (C-1), 74.9 (C-5), 69.3
(4× CH
3
(C-3), 68.5 (C-2), 65.8(2×) (C-4, C-6), 32.2 (CH Ph), 21.1, 20.9, 20.8,
2
6
2
2
20.7 (4 × CH CO), 15.9 (C-7). HRMS (MALDI): m/z: calcd for
3
+
0.7, 20.6 (4 × CH
3
[C24H29N O ]Na : 526.1802. Found: 526.1809.
3
9
+
[
C
22
H
28
O
11S]Na : 523.1250. Found: 523.1257.
4
.2.1.26. (6S) 6-C-Methyl-1-α-D-mannopyranosyl-4-phenyl-1,2,3-
triazole (26).
Deprotection of 24 (45 mg, 0.09 mmol) according to general pro-
cedure (Method D) and purification by column chromatography
4.2.1.22. (6S) Benzyl 6-C-methyl-α-D-mannopyranosyl
sulfone (22).
Deprotection of compound 21 (30 mg, 0.06 mmol) according to
general procedure (Method D) and purification by column chro-
(CH
[α]
3
CN:MeOH 10:1→5:1) gave 26 as white powder (23.6 mg, 80%).
1
matography (CH
foam. [α]
3
CN:MeOH 9:1) afforded 22 (17 mg, 85%) as a white
D
+ 48 (c 0.2, MeOH). H NMR (400 MHz, CD
3
OD): δ 8.49 (s, 1H,
1
D
+ 98 (c 0.4, MeOH); H NMR (400 MHz, CD
3
OD): δ 7.51–
CHN), 7.86–7.84 (m, 2H, Ar), 7.47–7.36 (m, 3H, Ar), 6.13 (d, 1H, J1,2
3.0 Hz, H-1), 4.72 (t, 1H, J2,3 3.3 Hz, H-2), 4.18 (dd, 1H, J3,4 8.0 Hz, H-3),
7.40 (m, 5H, Ar), 4.84 (d, 1H, J1,2 1.6 Hz, H-1), 4.60 (d, 1H, J 14.0 Hz,
CH
2
Ph), 4.41 (dd, 1H, J2,3 3.1 Hz, H-2), 4.36 (d, 1H, J 14.0 Hz, CH
1.8 Hz, J 6.6 Hz, H-6), 4.05–3.96 (m, 3H, H-3, H-4,
H-5), 1.43 (d, 3H, J 6.7 Hz, C-7(CH
32.4, 129.9, 129.8, 126.37 (Ar), 90.6 (C-1), 81.5 (C-5), 73.2, 67.3 (C-
2
Ph),
4.13 (dq, 1H, J
3.14 (dd, 1H, J5,6 2.6 Hz, H-5), 1.22 (d, 3H, J 6.5 Hz, C-7(CH
(100 MHz, CD OD): δ 149.1 (NC CH), 131.4, 130.0, 129.6, 127.8, 121.9
(Ar, NC CH), 88.5 (C-1), 80.4 (C-5), 73.1 (C-3), 70.0 (C-2), 68.7
d q
2.8 Hz, J 6.5 Hz, H-6), 4.01 (t, 1H, J4,5 8.4 Hz, H-4),
13
4
.17 (dq, 1H, J
d
q
3
)); C NMR
13
3
)); C NMR (100 Mz, CD
3
OD): δ
3
=
1
=

70
M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
(
[
C-4), 65.9 (C-6), 20.0 (C-7). HRMS (MALDI): m/z: calcd for
3
(3 × CH CO), 93.0 (C-1), 76.8 (C-4), 75.4 (C-2), 70.7 (C-3), 62.4 (C-
+
C
15
H
19
N
3
O
5
]Na : 344.1222. Found: 344.1230.
5), 20.8, 20.5, 20.4 (3 × CH
3
CO). HRMS (MALDI): m/z: calcd for
+
[
C
11
H
15
N
3
O
7
]Na : 324.0808. Found: 324.0814.
4
.2.1.27. (6S) 4-Benzyl-6-C-methyl-1-α-D-mannopyranosyl-1,2,3-
triazole (27).
Deprotection of 25 (37 mg, 0.073 mmol) according to general pro-
cedure (Method D) and purification by column chromatography
4
3
.2.1.31. 1-(2,3,5-Tri-O-acetyl-α-D-lyxofuranosyl)-4-phenyl-1,2,
-triazole (33).
Reaction of 32 (0.13 g, 0.43 mmol) and alkyne 11a according to
general procedure (Method C) gave 33 after purification by column
(
[
CH
α]
3
CN:MeOH 10:1→5:1) gave 27 as white powder (20 mg, 82%).
1
D
—27.3 (c 0.1, MeOH). H NMR (400 MHz, CD
3
OD): δ 7.81 (s, 1H,
chromatography (hexane:EtOAc 4:1→2.5:1). White powder (0.16 g,
CHN), 7.29–7.18 (m, 5H, Ar), 6.00 (d, 1H, J1,2 2.8 Hz, H-1), 4.66 (t, 1H,
2,3 3.2 Hz, H-2), 4.09–4.05 (m, 4H, H-3, H-6, CH Ph), 3.94 (t,1H, J3,4
.6 Hz, H-4), 2.95 (dd, 1H, J5,6 2.6 Hz, H-5), 1.12 (d, 3H, J 6.5 Hz,
1
9
1
J
1%). [α]
D
+ 25 (c 0.2, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 7.93 (s,
J
8
2
H, CHN), 7.84–7.81 (m, 2H, Ar), 7.47–7.33 (m, 3H, Ar), 6.21 (d, 1H,
1,2 4.6 Hz, H-1), 6.10 (t, 1H, J2,3 4.8 Hz, H-2), 5.87 (t, 1H, J3,4 4.8 Hz,
H-3), 4.83 (dt, 1H, H-4), 4.35 (dd, 1H, J4,5a 4.8 Hz, J5a,5b 11.9 Hz, H-5a),
1
3
C-7(CH
1
3
)); C NMR (100 MHz, CD
3
OD): δ 148.9 (NC
=CH), 140.2,
29.7, 129.6, 127.5, 123.5 (Ar, NC=CH), 88.4 (C-1), 80.1
4
3
(
.29 (dd, 1H, J4,5b 7.1 Hz, H-5b), 2.09, 2.07, 2.04 (each s, each 3H,
(C-5), 73.0 (C-3), 69.9 (C-2), 68.5 (C-4), 65.6 (C-6), 32.5 (PhCH
2
), 19.9
]Na : 358.1379.
13
× CH
3 × CH
=
3
CO); C NMR (100 MHz, CDCl
3
): δ 170.4, 169.3, 169.1
CH), 129.9, 128.8, 128.5, 125.9, 119.2 (Ar,
CH), 89.7 (C-1), 78.4 (C-4), 75.2 (C-2), 71.0 (C-3), 61.9 (C-5),
+
(
C-7). HRMS (MALDI): m/z: calcd for [C16
H
21
N
3
O
5
3
CO), 148.2 (NC=
Found: 358.1387.
NC
2
0.7, 20.4, 20.3 (3 × CH
3
CO). HRMS (ESI-MS): m/z: calcd for
+
[
C
19
H
21
N
3
O
7
]Na : 426.1277. Found: 426.1280.
4
.2.1.28. Benzyl 2,3,5-tri-O-benzoyl-α-D-lyxofuranosyl
sulfone (29).
To a precooled at 0 °C solution of 2829 (0.22 g, 0.44 mmol) in
Cl (5 mL) benzyl mercaptan (68 mg, 64 μL, 0.55 mmol) and
OEt (0.22g, 0.19 mL, 1.53 mmol) were added dropwise. After
being stirred at rt for 16 h, the reaction was diluted with DCM
15 mL) and poured into cold satd. NaHCO (10 mL). The organic
phase was separated, washed with water, dried (Na SO ), filtered
and concentrated. The residue was dissolved in CH Cl (10 mL),
4
3
.2.1.32. 1-(2,3,5-Tri-O-acetyl-α-D-lyxofuranosyl)-4-benzyl-1,2,
-triazole (34).
Reaction of 32 (0.13 g, 0.43 mmol) and alkyne 11b according to
CH
BF
2
2
3
2
general procedure (Method C) gave 34 after purification by column
chromatography (hexane:EtOAc 4:1→2.5:1). White powder (0.13 g,
7
(
3
2
4
1
2%). [α]
D
—36 (c 0.25, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 7.34–
2
2
7.22 (m, 6H, CHN, Ar), 6.07 (d, 1H, J1,2 4.5 Hz, H-1), 6.01 (t, 1H, J2,3
cooled at 0 °C and treated with mCPBA (2.5eq) according to general
procedure (Method A). The crude product was purified by column
4
1
4
.7 Hz, H-2), 5.83 (t, 1H, J3,4 4.8 Hz, H-3), 4.76 (dt, 1H, H-4), 4.29 (dd,
H, J4,5a 4.9 Hz, J5a,5b 11.8 Hz, H-5a), 4.24 (dd, 1H, J4,5b 7.2 Hz, H-5b),
chromatography (hexane:EtOAc 6:1→4:1) to afford 29 (0.15 g, 66%
.11 (m, 2H, CH
2
Ph), 2.14, 2.07, 2.06 (each s, each 3H, 3 × CH
C NMR (100 MHz, CDCl ): δ 170.6, 169.5, 169.2 (3 × CH CO), 148.4
NC CH), 138.6, 128.9, 128.8, 126.8, 121.3 (Ar, NC CH), 89.6 (C-
), 78.4 (C-4), 75.3 (C-2), 71.2 (C-3), 62.1 (C-5), 32.3 (CH
0.6, 20.5 (3 × CH CO). HRMS (MALDI): m/z: calcd for [C20
40.1434. Found: 440.1439.
3
CO);
1
over 2 steps, purity > 95%) as a clear oil; [α]
NMR (400 MHz, CDCl
.4 Hz, H-2), 6.15 (dd, 1H, J3,4 5.1 Hz, H-3), 5.14 (m, 1H, H-4), 5.05
d, 1H, J1,2 4.1 Hz, H-1), 4.79 (dd, 1H, J4,5b 6.9 Hz, J5a,5b 11.8 Hz, H-5b),
.62 (dd, 1H, J4,5a 5.1 Hz, H-5a), 4.58 (d, 1H, J 14.0 Hz, CH Ph), 4.30
): δ 166.2, 165.1,
Ph), 90.7 (C-1), 80.2, 72.2,
Ph). HRMS (MALDI): m/z: calcd
D
+ 55 (c 0.3, CHCl
3
); H
1
3
3
3
3
): δ 8.02–7.28 (m, 20H, Ar), 6.40 (dd, 1H, J2,3
(
=
=
5
(
1
2
4
2
Ph), 20.9,
+
3
H
23
N
3
O
7
]Na :
4
2
13
(d, 1H, J 14.0 Hz, CH
2
Ph); C NMR (100 MHz, CDCl
3
1
7
64.6 (3 × PhCO), 133.6–128.4 (PhCO, CH
2
1.1, 60.6, 56.7 (C-2, C-3, C-4, C-5, CH
2
4
.2.1.33. 1-α-D-Lyxofuranosyl-4-phenyl-1,2,3-triazole (35).
Deprotection of 33 (0.12 g, 0.30 mmol) according to general pro-
cedure (Method D) and purification by column chromatography
+
for [C33
H
28
O
9
S]Na : 623.1352. Found: 623.1361.
(
[
CH
α]
3
CN:MeOH 1:0→6.5:1) gave 35. White powder (72 mg, 88%).
4
.2.1.29. Benzyl α-D-lyxofuranosyl sulfone (30).
Deprotection of compound 29 (0.12 g, 0.20 mmol) according to
general procedure (Method D) and purification by column chro-
matography (CH CN:MeOH 10:1→5:1) gave 30 (46 mg, 80%). Yellow
+ 32 (c 0.3, MeOH); 1H NMR (400 MHz, CD
oil, [α] OD): δ 7.76–
.41 (m, 5H, Ar), 4.78 (t, 1H, J3,4 5.1 Hz, H-3), 4.70 (d, 1H, J1,2 5.2 Hz,
H-1), 4.61 (d, 1H, J 12.0 Hz, CH Ph), 4.41 (d, 1H, J 12.0 Hz, CH Ph),
.34–4.32 (m, 1H, H-4), 4.28 (dd, 1H, J2,3 4.8 Hz, H-2), 3.88 (dd, 1H,
1
D
+ 23.3 (c 0.2, MeOH); H NMR (400 MHz, CD
3
OD): δ 8.53 (s,
1
H, CHN), 7.86–7.80 (m, 2H, Ar), 7.45–7.32 (m, 3H, Ar), 6.09 (d, 1H,
1,2 5.8 Hz, H-1), 4.86 (dd, 1H, J2,3 4.4 Hz, H-2), 4.50 (m, 1H, H-4),
4.43 (t, 1H, J3,4 4.1 Hz, H-3), 3.89 (dd, 1H, J4,5a 4.6 Hz, J5a,5b 11.8 Hz,
H-5a), 3.82 (dd, 1H, J4,5b 6.2 Hz, H-5b); C NMR (100 MHz, CD
δ 149.1 (NC CH), 131.5, 130.0, 129.4, 126.8, 121.2 (Ar, NC
4.1 (C-1), 84.3 (C-4), 78.2 (C-2), 72.7 (C-3), 61.7 (C-5).
J
3
D
3
13
3
OD):
CH),
7
=
=
2
2
9
4
+
1
3
15 3 4
HRMS (MALDI): m/z: calcd for [C13H N O ]Na : 300.0960. Found:
3
J
4,5a 4.4 Hz, H-5a), 3.84 (dd, 1H, J4,5b 6.3 Hz, J5a,5b 12.0 Hz, H-5b);
C
00.0969.
NMR (100 MHz, CD
7
m/z: calcd for [C12
3
OD): δ 132.3, 129.8 (CH
2
Ph), 94.3 (C-1), 85.7,
Ph). HRMS (MALDI):
S]Na : 311.0565. Found: 311.0571.
3.1, 72.6 (C-2, C-3, C-4), 61.4 (C-5), 56.8 (CH
2
+
H
16
O
6
4
.2.1.34. 4-Benzyl-(1-α-D-lyxofuranosyl)-1,2,3-triazole (36).
Deprotection of compound 34 (0.10 g, 0.24 mmol) according to
4
.2.1.30. 2,3,5-Tri-O-acetyl-α-D-lyxofuranosyl azide (32).
general procedure (Method D) and purification by column chro-
3
0
Reaction of 31 (0.53g, 1.66 mmol, 1eq) and TMSN
catalysed by 1 M SnCl in CH Cl (1.05eq) according to general pro-
cedure (Method B) and purification by column chromatography
3
(1.3eq)
matography (CH
(62 mg, 89%). [α]
3
CN:MeOH 1:0→6.5:1) gave 36. White powder
1
4
2
2
D
+ 68.6 (c 0.2, MeOH); H NMR (400 MHz, CD
3
OD):
δ 7.90 (s, 1H, CHN), 7.31–7.18 (m, 5H, Ar), 6.00 (d, 1H, J1,2 5.8 Hz, H-1),
4.79 (dd, 1H, J2,3 4.5 Hz, H-2), 4.43 (m, 1H, H-4), 4.38 (t, 1H, J3,4 4.1 Hz,
(hexane:EtOAc 3:1→1.5:1) gave azide 32 (0.39 g, 78%) as a clear oil;
1
[α]
D
+ 82 (c 0.8, CHCl
3
); H NMR (400 MHz, CDCl
3
): δ 5.51 (t, 1H, J3,4
H-3), 4.07 (s, 2H, CH
3.79 (dd, 1H, J4,5b 6.2 Hz, H-5b); C NMR (100 MHz, CD
(NC CH), 140.3, 129.6, 127.5, 122.8 (Ar, NC CH), 94.1 (C-1), 84.2
Ph). HRMS (MALDI):
2
Ph), 3.85 (dd, 1H, J4,5a 4.6 Hz, J5a,5b 11.8 Hz, H-5a),
13
5
.2 Hz, H-3), 5.43 (d, 1H, J1,2 3.0 Hz, H-1), 5.08 (dd, 1H, J2,3 5.0 Hz,
3
OD): δ 148.7
H-2), 4.55 (dt, 1H, H-4), 4.22 (dd, 1H, J4,5a 4.8 Hz, H-5a), 4.17 (dd,
1
3
=
=
H, J4,5b 7.4 Hz, J5a,5b 11.8 Hz, H-5b), 2.05, 2.04, 2.03 (each s, each 3H,
(C-4), 78.1 (C-2), 72.7 (C-3), 61.7 (C-5), 32.6 (CH
2
1
3
+
× CH
3
CO); C NMR (100 MHz, CDCl
3
): δ 170.6, 169.5, 169.3
m/z: calcd for [C14
H
17
N
3
O
4
]Na : 314.1117. Found: 314.1124.

M. Poláková et al. / Carbohydrate Research 428 (2016) 62–71
71
4
.3. Biochemistry
Programme Project No. 2003SP200280203 and by the European
Social Fund (project no. CZ.1.07/2.4.00/31.0130) and IGA
(IGA_PrF_2014026). Dr. Vladimír Puchart is appreciated for helpful
discussion.
4.3.1. Enzyme preparation
The purification and characterisation of recombinant Dro-
sophila melanogaster Golgi (dGMIIb) and lysosomal (dLMII)
3
1
mannosidases was carried out as we described recently.
Supplementary material
4
.3.2. Class II α-mannosidase assay13
The supernatants of yeast expressing soluble forms of the
α-mannosidase were incubated with the substrate pNP-Manp at
7 °C for 2–3 h. The standard assay mixture consisted of 50 mM
1H and 13C NMR spectra of new compounds. Supplementary data
to this article can be found online at doi:10.1016/j.carres.2016.04.004.
3
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Acknowledgment
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This work was supported by the Slovak Research and Develop-
ment Agency (the project APVV-0484-12), the Scientific Grant Agency
of the Ministry of Education of the Slovak Republic and the Slovak
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