α-d-Mannose derivatives as models designed for selective inhibition of Golgi α-mannosidase II

Article abstract of DOI:10.1016/j.ejmech.2011.01.012

Human Golgi α-mannosidase II (hGM) is a pharmaceutical target for the design of inhibitors with anti-tumor activity. Nanomolar inhibitors of hGM exhibit unwanted co-inhibition of the human lysosomal α-mannosidase (hLM). Hence, improving specificity of the

Full text of DOI:10.1016/j.ejmech.2011.01.012

European Journal of Medicinal Chemistry 46 (2011) 944e952
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
a- -Mannose derivatives as models designed for selective inhibition
D
of Golgi
a
-mannosidase II
,
a
b
a
ꢀa
Monika Poláková a , Sergej Sesták , Erika Lattová , Ladislav Petrus , Ján Mucha ,
ꢀ
*
a
a,
**
ꢀ
ꢀ
Igor Tvaroska , Juraj Kóna
a Institute of Chemistry, Center for Glycomics, GLYCOMED, Slovak Academy of Sciences, Dúbravska cesta 9, 845 38 Bratislava, Slovakia
b University of Manitoba, Department of Chemistry, 144 Dysart Road, Winnipeg, Manitoba R3T 2N2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Human Golgi
tumor activity. Nanomolar inhibitors of hGM exhibit unwanted co-inhibition of the human lysosomal
-mannosidase (hLM). Hence, improving specificity of the inhibitors directed toward hGM is desired in
order to use them in cancer chemotherapy. We report on the rapid synthesis of -mannose derivatives
having one of the RS-, R(SO)- or R(SO2)- groups at the -anomeric position. Inhibitory properties of
thirteen synthesized -mannopyranosides were tested against the recombinant enzyme Drosophila
a-mannosidase II (hGM) is a pharmaceutical target for the design of inhibitors with anti-
Received 24 November 2010
Received in revised form
21 December 2010
Accepted 8 January 2011
Available online 15 January 2011
a
D
a
a-D
melanogaster homolog of hGM (dGMIIb) and hLM (dLM408). Derivatives with the sulfonyl [R(SO2)-]
group exhibited inhibitory activities at the mM level toward both dGMIIb (IC50 ¼ 1.5e2.5 mM) and
dLM408 (IC50 ¼ 1.0e2.0 mM). Among synthesized, only the benzylsulfonyl derivative showed selectivity
toward dGMIIb. Its inhibitory activity was explained based on structural analysis of the built 3-D
complexes of the enzyme with the docked compounds.
Keywords:
Synthetic
Oxidation
Mannopyranosyl sulfones
Mannopyranosyl sulfoxides
a-D-mannopyranosides
a
-Mannosidase inhibitors
Ó 2011 Elsevier Masson SAS. All rights reserved.
Molecular modeling
1. Introduction
phenocopy of the lysosomal storage disease called a-mannosidosis.
This phenomena is caused by the additional targeting of LM by
swainsonine, and limits its use in the clinical cancer chemotherapy
[3]. Therefore the finding of a selective GM inhibitor with mini-
mized unwanted co-inhibition of LM has become challenge for
a number of researchers [4e19].
The natural indolizidine swainsonine and some related pyrro-
lidines (Scheme 1) are nanomolar inhibitors of retaining glycoside
hydrolases belonging to the family 38, Golgi
a-mannosidase II (GM)
(E.C.3.2.1.114) and lysosomal -mannosidase (LM) (E.C.3.2.1.24) [1].
a
It was previously shown that treatment of the animals with swai-
nsonine induced a strong immunostimulatory activity that resulted
in an anti-cancer effect [2] and significantly blocked growing tumor
in the lung, liver, and spleen. It was demonstrated that its activity
was associated with the modification of N-glycan biosynthesis in
which GM is reversibly blocked. However, swainsonine causes a
Up to date, dozens of novel swainsonine analogs were proposed
and tested [1]. However, most potent inhibitors of GM exhibit
negligible or no selectivity. To design an effective selective inhibitor
of GM is a major problem, mainly because of the structural simi-
larities of the active sites in GM and LM. The both enzymes are from
ꢁ
the same family with the almost identical structures in radius 10 A
around Zn2þ ion co-factor, which resides at the bottom of the active
site and is essential for catalytic activity of the enzymes as well as
for strong binding of the inhibitors [20]. This is the main reason
why binding affinities of small organic inhibitors such as swain-
sonine are similar for both enzymes with no selectivity observed.
Rose and co-workers [21,22] proposed the design for a selective
inhibitor which mimics a binding position of non-reducing-
terminal N-acetylglucosamine (GlcNAc) residue of the GM subs-
trate. Such type of inhibitor would be capable to attach to GM at its
binding subsite which is missing in LM. Moreover, this residue is
essential for substrate specificity since GM cleaves substrate
without terminal GlcNAc 80-fold more slowly. On the other side,
Abbreviations: GM, Golgi
II; dGM, Drosophila melanogaster
Drosophila melanogaster Golgi -mannosidase II, recombinant enzyme homolog of
human Golgi -mannosidase II; LM, lysosomal -mannosidase; hLM, human lyso-
somal -mannosidase; bLM, Bos taurus lysosomal -mannosidase; dLM408, cata-
lytic domain of Drosophila melanogaster lysosomal -mannosidase, recombinant
enzyme homolog of human lysosomal -mannosidase.
a-mannosidase II; hGM, human Golgi
a-mannosidase
a
-mannosidase II; dGMIIb, catalytic domain of
a
a
a
a
a
a
a
* Corresponding author. Tel.: þ421 2 59410272; fax: þ421 2 59410222.
** Corresponding author. Tel.: þ421 2 59410203; fax: þ421 2 59410222;
E-mail addresses: monika.polakova@savba.sk (M. Poláková), chemkona@savba.
ꢀ
sk (J. Kóna).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.012

M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
945
Scheme 1. Nanomolar inhibitors of
a-mannosidases belonging to the family 38.
LM is not able to cleave the GM substrate with terminal GlcNAc
residue at the same concentration as GM does [21]. Recently, the
GlcNAc binding subsite at the active site of Drosophila melanogaster
GM (dGM) was explored by crystallographic approach [21,22]. The
regions of the active sites, at which selective inhibitors as well as
moiety of the substrate responsible for the substrate specificity,
were characterized based on crystal structures of dGM in combi-
nation with native substrate [21,22,29], swainsonine [20] and its
5-substituted derivatives [4].
For purpose of finding a selective inhibitor of GM and better
understanding the mechanism of action, some swainsonine deri-
vates were synthesized and their biological activities were evalu-
ated [1,4,13e15,18,19,23]. It was observed that 3- and 5-substituted
swainsonine derivatives exhibited a weak selectivity for GM
(IC50 > 3e20-fold) at the nanomolar and micromolar levels, while
6- and 7-substituted derivatives exhibited even poorer activities
(300e9000 less) at the micromolar and milimolar levels [14,15,18].
However, using of swainsonine and its derivatives as models of GM
selective inhibitors or model compounds for the research of their
action modes is complicated mainly because of the demands on
their syntheses [18,19,24].
Fig. 1. The first set of the evaluated glycosides.
mixture of sulfoxides in moderate yield. Benzyl sulfoxide 6 was
purified solely by a column chromatography while additional recr-
ystallization was required to obtain pure major sulfur epimer of
2-phenylethyl sulfoxide 7. Since under conditions used both sulfox-
ides were obtained as the mixtures of (SS) and (RS) epimers, it was
necessary to distinguish their configuration which was established
from 1H NMR spectra. In accordance with previous results [28], the
diastereomeric ratio RS/SS was found to be 10:1. In case of the sulf-
oxide 7, the structure of major (RS) epimer was also confirmed by
X-ray crystallography (unpublished data). Taking into account simi-
larity in the synthesis and chemical shifts in the 1H NMR spectra, the
same configuration is proposed for the sulfoxide 6.
Following our procedure described recently [26], oxidation of
the thioglycosides carried out at room temperature with an excess
of mCPBA smoothly provided sulfones 10 and 11. The deacetylation
under mild conditions using K2CO3 in MeOH with subsequent pu-
rification by column chromatography, afforded the target compo-
unds 4 [25], 5 [25], 8, 9, 12 and 13.
Transformation of the thioglycosides to the sulfoxides and the
sulfoneswasclearlyevident fromtheir1Hand 13C NMR spectra. In1H
NMR spectra signals for H-1 of major acetylated sulfoxide and
sulfone were upfield shifted in comparison with that of the starting
thioglycoside; and the signal of the sulfoxide appeared at the lowest
chemical shift from these sulfur-containing compounds. In 13C NMR
spectra C-1 signal of the acetylated S-glycoside was the most upfield
shifted, while C-1 signals of the oxidized compounds were observed
at markedly higher values. The same trend in the chemical shifts for
H-1 and C-1 was observed for series of deprotected compounds. All
prepared derivatives discussed herewere characterized by NMR and
HRMS. Data are presented in the Experimental section.
In this paper we describe the rapid synthesis of
D-mannose
derivatives substituted at the -anomeric position as starting
a
models for selective inhibitors of GM. Although it is known that six-
member ring structures are not potent inhibitors of GM [1], the
main goal of our study was to modify the aglycone unit of the
mannosides to improve their selectivity directed toward GM. For
their preparation we used the structural motif of an optimized
synthesis described in our previous paper [25]. Moreover,
a-D-
mannose moiety is essential part of the native substrate of GM and
its binding with the Zn2þ ion co-factor at the active site of dGM has
been previously characterized by crystallographic measurements
[21,22]. A reference structure used for our inhibition assays, a-D-
mannose, does not bind at the active site of the mannosidases (no
inhibition activity up to 5 mM), thus an observed activity of the
compounds can be easily assigned to a functional group attached at
the a-anomeric position of the mannose unit.
2. Chemistry
The novel compounds of interest 8, 9, 12 and 13 were subjected
to MS/MS analysis to verify their compositions. In tandem mass
spectra all examined derivatives produced fragment ions corre-
sponding to a number of cross-ring cleavages and total loss of
monosaccharide (Y, Z - type ion cleavages) or phenylalkyl residue
(B, C - type ion cleavages). However, their ratio significantly
differed mainly in dependence on sulfur type linked on the agly-
cone of mannose. Generally, very similar patterns were observed in
spectra recorded from sulfones 12 and 13 and differed from those
obtained for sulfoxides 8 and 9, suggesting different fragmentation
pathways between mannose sulfoxides and sulfones with phe-
nylalkyl function at anomeric position. Fig. 2 shows the represen-
tative tandem mass spectra acquired from 8 and 12. The most
abundant ions in spectra of deprotected sulfoxides were observed
at m/z 234.02 (C1) and corresponded to the loss of phenylalkyl
residue from sulfur [Fig. 2, a)]. On the other side, the most
A glycosylation of the corresponding alcohols and thioalcohols
with mannose pentaacetate under a promotion of Lewis acid
(BF3$OEt2) was used in this study for a coupling of the donor 1 with
benzyl and 2-phenylethyl thioalcohols, yielding thioglycosides 2
and 3 [25]. The synthesis of other compounds (14e20) depicted in
Fig. 1 with similar reaction conditions (BF3$OEt2 or SnCl4 as Lewis
acid) has been reported recently [26].
Thioglycosides are useful intermediates due to the presence of
sulfur atom which can be oxidized to a various extent, in depen-
dence of reaction conditions used. Both thioglycosides 2 and 3 were
subjected to an oxidation which resulted in their conversion to
sulfoxides 6 and 7, or sulfones 10 [27] and 11 (Scheme 2).
The treatmentof thioglycosideswithequimolaramountof mCPBA
at low temperature (ꢀ78 ꢁC to ꢀ55 ꢁC) afforded diastereomeric

946
M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
Scheme 2. Reagents and conditions. a) BF3$OEt2, alcohol, CH2Cl2, 24 h, rt; b) mCPBA, CH2Cl2, 45 min, ꢀ78 ꢁC to ꢀ55 ꢁC; c) mCPBA, CH2Cl2, 60 min, rt; d) K2CO3, MeOH, 10e30 min, rt.
Fig. 2. MALDI-MS/MS spectra of novel mannose derivatives recorded for MNaþ precursor ions at m/z: a) 325.07 (compound 8); and b) 341.07 (compound 12).

M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
947
Table 1
structure not to be hydrolyzed by the enzyme. We suppose that the
inactive glycosides and thioglycosides either weakly bind into the
active site or after binding they can be immediately hydrolyzed by
the enzyme. When the sulfanyl group was oxidized to a sulfoxide
(compound 9) or sulfone ones (compounds 19 and 20), the inhi-
bition of the enzyme became observable at the milimolar level.
Another essential condition for proper binding of a structure into
the active site of the enzyme is stabilization of a reactive confor-
mation of the mannose ring upon binding. It was demonstrated by
means of crystallographic measurements [21,22] that a conforma-
tional interconversion of the manose ring from the stable chair 4C1
to the reactive skew boat B2,5 conformation occurs upon binding
substrate (or a mannoside inhibitor) to the active site of the
enzyme. This reactive conformation of the mannosidase substrate
[21,22] and substrate-enzyme covalent intermediate [29] are
essential for the catalysis and a strong interaction with the Zn2þ ion
co-factor at the active site. Consequently, we can suppose that the
oxidized forms of the thioglycosidic bond, mainly the sulfone one,
in our active compounds prevent enzymatic hydrolysis; moreover
it can preserve the mannose ring in a required reactive conforma-
tion upon binding to the active site.
Measured IC50 values for both Golgi (dGMIIb) and lysosomal (dLM408)
a-mannosidases.
Compound
GM (IC50/mM)
LM (IC50/mM)
Swainsonine
Mannostatin A
5.0 ꢂ 10ꢀ6
11.0 ꢂ 10ꢀ6
0.15 ꢂ 10ꢀ3
n.i
3.0 ꢂ 10ꢀ3
n.i
a
4
5
8
9
-D
-mannosea
n.i.
n.i.
n.i.
2.5
2.0
1.5
n.i.
n.i.
n.i.
n.i.
n.i.
2.5
n.i.
n.i.
n.i.
1.5
n.i.
1.0
n.i.
n.i.
n.i.
n.i.
n.i.
2.0
12
13
14
15
16
17
18
19
20
2.5
2.0
n.i. e No inhibition or inhibition <10% at 5 mM.
a
A mixture of a/b z 95:5.
prominent peaks in derivatives bearing sulfone group were
consistent with a cleavage of the bond between a carbon of man-
nose and sulfur and these fragment ions appeared in 12 at m/z
179.02 (Y1) and m/z 185.05 (B1) [Fig. 2, b)].
To confirm this hypothesis we docked the sulfonyl derivatives as
well as a nanomolar pyrrolidine inhibitor [(2R,3S,4S,5S)-3,4-dihy-
droxy-5-methylpyrrolidinium-2-sulfonate; Scheme 1] to the active
site of dGM. As can be seen from Fig. 3, sulfonyl groups in both
docked structures interact by hydrogen bonding with catalytic acid
Asp341 and by dipoleeion interactions with Arg228 [d(SO2-
3. Biological evaluation and docking
ꢁ
ꢁ
ꢁ
Asp341) ¼ 1.80 A and 2.02 A; and d(SO2-Arg228) ¼ 3.22 A and
The IC50 values (concentrations of inhibitor at which 50% of
activity remains) were evaluated for two sets of the mannopyr-
anosides (Table 1). The first set (Fig. 1) included 3 types of
compounds (glycosides, thioglycosides and sulfones derived
ꢁ
2.67 A for the pyrrolidine derivative and the mannoside, respec-
tively]. It should be noted that Asp341 plays a role of the acid
catalyst donating a proton onto the glycosidic oxygen of the sub-
strate facilitating its hydrolysis. Thus, the sulfone group at the
thereof) selected from recently prepared a-D-mannopyranosides:
a
-anomeric position can block its catalytic function and stabilize
linear alkyl glycosides (compounds 14 and 15), cyclohexylalkyl
glycosides (compounds 16 and 17), and linear alkyl thioglycosides
and glycosyl alkyl sulfones (the compounds 18, 19 and 20) [26]. For
the sulfones 19 and 20 inhibition activities toward both dGMIIb and
dLM408 were found at the mM level (IC50 ¼ 2.0e2.5 mM, Table 1)
with no selectivity observed. The glycosides and thioglycosides did
not show any activity in concentrations of the measurements
(1e5 mM).
the mannose ring of the inhibitor in the reactive conformation
necessary for an effective binding with the Zn2þ ion co-factor.
Indeed, after the post-docking minimization of the sulfonyl deriv-
ative in the active site, its mannose ring maintained the reactive
skew boat conformation [Fig. 3, b)].
Based on the above-mentioned results obtained from the evalu-
ation of the alkyl sulfones 19 and 20 and crystallographic measure-
ments of complexes of dGM with 5-substituted aryl derivatives of
swainsonine selective inhibitors [4], the second set of the evaluated
compounds (Scheme 2, Table 1) consisted of mannosides (thio-
glycosides 4 and 5, sulfoxides 8 and 9, sulfones 12 and 13) having
aromatic aglycone connected to the sulfur atom through a short alkyl
In general,
-mannosidase substrate and does not exhibit a significant inhib-
itory activity against the -mannosidases. As the first step in their
conversion to inhibitors of the retaining -mannosidases, the gly-
cosidic bond of a potential inhibitor should be converted to the
a-D-mannopyranoside is a structural moiety of the
a
a
a
Fig. 3. a) A nanomolar inhibitor [(2R,3S,4S,5S)-3,4-dihydroxy-5-methylpyrrolidinium-2-sulfonate)] docked at the active site of dGM. The sulfonate group interacts with the catalytic
acid (Asp341) and Arg228. b) Compound 12 docked at dGM. The same type of the interactions between the sulfonyl group and Asp341 and Arg228 is predicted by the modeling.

948
M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
linker (one or two CH2 groups). The main goal was to modify the
aglycone unit of the glycosides to improve their selectivity toward
GM. Therefore, we focused our attention on both groups of deriva-
tives accessible from thioglycosides by oxidation because of inhibi-
tory activity of sulfones found in the first set of the compounds.
The structures 9, 12 and 13 exhibited inhibitory activities at the
milimolar level (IC50 ¼ 1.0e2.5 mM). The benzylsulfonyl derivative
(12) selectively inhibited dGMIIb (IC50 ¼ 2 mM), while no inhibition
to dLM408 was observed in the measured range of 1e5 mM. The
elongation of the aglycone linker by one CH2 group from the ben-
zylsulfone to the 2-phenylethylsulfone group (compound 13)
resulted in the loss of the selectivity. This derivative exhibited
similar inhibiting properties toward both enzymes [IC50(dGMIIb) ¼
1.5 mM and IC50(dLM408) ¼ 1.0 mM]. We docked 12 and 13 to the
active sites of dGM and Bos taurus LM (bLM) to spot differences in
binding posed and energies (Fig. 4 and Table S1 of Supplementary
data). The derivative 12 prefers binding poses at which the agly-
cone chain interacts with a backbone and side chains of conserved
Arg876 and Tyr267 in dGM [Fig. 4, a)] The identical binding pose of
the aromatic chain was found for the docked 5-benzyl swainsonine
at dGM (unpublished results). A similar binding position of an
aromatic chain of nanomolar inhibitors close to Arg876 was rec-
ently described in the crystallographic measurements of 5-subs-
tituted aryl swainsonines with the dGM [4]. The interaction of 12
with tyrosine is missing in bLM because it is not conserved in
known sequences of the lysosomal a-mannosidases [30]. Therefore,
12 prefers at bLM a pose in which the aglycone chain does not
interact with the side chain of Arg823 (analogous to Arg876 in
Fig. 4. Structures 12 and 13 docked into the active site of dGM and bLM (10 poses with the best docking Glide SP score, see also Table S1 in Supplementary data). a) 12 prefers at
dGM specific positions at which a phenyl group interacts with the backbone and side chains of Arg876 and with the side chain of Tyr267. b) At hLM 12 prefers less ordered binding
because of the missing Tyr residue and a different 3-D structure of the active site of bLM compared with dGM. c) and d) The elongation of an alkyl linker allowed a higher flexibility
of the aglycone in 13 and caused less ordered binding at both dGM and bLM.

M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
949
dGM), and various poses are predicted by the program (Fig. 4b). The
5.1.2. General procedure for glycosylation
binding energy for 12 is about 1.56 kcal molꢀ1 (Glide SP docking
score, Table S1, see Supplementary data) and 1.26 kcal molꢀ1 (Glide
XP docking score, Table S2, see Supplementary data) higher (more
negative) in dGM vs bLM. This could explain the observed selec-
tivity in the inhibition measurements for 12. The elongation of an
alkyl linker in 13 allowed a higher flexibility of the aglycone chain
and its less ordered binding at both dGM and bLM [Figs. 4, c) and
d)]. The binding energy for 13 is about 1.67 kcal molꢀ1 (Glide SP
docking score) and 1.12 kcal molꢀ1 (Glide XP docking score) higher
(more negative) in dGM than for bLM. The docking calculations
predict the stronger binding of 12 and 13 toward dGM. The theo-
retical results predict both derivatives as selective inhibitors of GM,
however according the experimental assay only 12 exhibited the
selective binding against dGMIIb.
To a stirred solution containing 1,2,3,4,6-penta-O-acetyl-
D
-man-
nopyranose (1) (2.5 g, 6.40 mmol, 1 eq) in CH2Cl2 (30 mL) the corre-
sponding alcohol (1.25 eq) was added. The reaction mixture was stirred
for 15 min, cooled down on an ice bath and BF3$OEt2 (3.18 g, 2.85 mL,
22.41 mmol, 3.5 eq) was added dropwise. The resulting mixture was
stirred for 15 min, brought to rt and stirring was continued for 24 h. The
reaction mixture was diluted with CH2Cl2 (150 mL) and poured into ice
cold water (250 mL) under stirring. The organic phase was separated,
washed with satd NaHCO3 (3 ꢂ 100 mL), water (100 mL), dried with
anhydrous Na2SO4, filtered and concentrated. The crude product was
purified by column chromatography (hexane:EtOAc, 5:1 /3:1).
5.1.2.1. Benzyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyranoside (2).
Yield 2.51 g; 86%; white crystals; mp 139e140 ꢁC; lit [25] mp
27
137e138.5 ꢁC; [
a
]D ¼ þ158 (c ¼ 0.5, CHCl3); lit [25] [
a
]
¼ þ154 ꢃ 0.9
4. Conclusion
(c ¼ 1.0, CHCl3); HRMS: m/z: calcd for C21H26O9S [M þDNa]þ: 477.1195;
found: 477.1201.
A rapid synthesis of a series of sulfur-containing
a-D-man-
nopyranosides having the phenylalkyl functional group with one
or two methylene group(s) long linker between the sulfur atom
and the aromatic ring as the aglycone is described herein. Among
the synthesized compounds, 6, 7, 8, 9, 11, 12 and 13 are novel
derivatives, first time described in this study. Five compounds
showed the weak inhibitory activity directed toward Golgi and
5.1.2.2. 2-Phenylethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyrano-
side (3). Yield 2.25 g; 75%; yellowish solid; mp 82e83 ꢁC; lit [25] mp
27
85.5e86.8 ꢁC; [
a
]D ¼ þ86 (c ¼ 0.5, CHCl3); lit [25] [
a
]
¼ þ96 ꢃ 0.5
D
(c ¼ 0.9, CHCl3); HRMS: m/z: calcd for C22H28O9S [M þ Na]þ:
491.1352; found: 491.1365.
lysosomal
tion showed the selectivity against the former one. Because the
reference -mannose does not exhibit any binding affinity in the
range of the measurement, the inhibition observed by the sulfonyl
derivatives can mainly be assigned to the sulfonyl group attached
a
-mannosidases and benzylsulfonyl derivative in addi-
5.1.3. General procedure for oxidation of phenylalkyl 2,3,4,6-tetra-
O-acetyl-1-thio-a-D-mannopyranosides to sulfoxides
a-
D
A solution of compound (2) (0.2 g, 0.44 mmol, 1 eq) or (3) (0.4 g,
0.85 mmol,1 eq) in CH2Cl2 (10 mL) was cooled at ꢀ78 ꢁC and treated
with mCPBA (1 eq based on 77% peroxide content). The reaction
mixture was stirred and allowed gradually to warm to ꢀ55 ꢁC over
45 min period, then poured into ice cold satd. NaHCO3 (20 mL),
diluted with CH2Cl2 (20 mL). The organic phase was separated,
washed with satd NaHCO3 (2 ꢂ 15 mL) and water (20 mL), dried with
anhydrous Na2SO4, filtered and concentrated. The crude product was
purified by column chromatography (hexane:EtOAc, 3:1 /1:1.5).
at the
data obtained from the theoretical modeling, the sulfonyl group at
the -anomeric position can interact with the catalytic acid of GM
a-anomeric position at the D-mannose moiety. Based on the
a
and block its catalytic function. It can stabilize the mannose ring in
the reactive skew boat conformation which is necessary for an
effective binding with the Zn2þ ion co-factor. However, for better
understanding the mechanism of selective binding of the benzyl-
sulfonyl chain as aglycone in the mannoside, more precise calcu-
lations and crystallographic measurements are planned to be done.
5.1.3.1. (RS)-benzyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl sulf-
oxide (6). Yield 0.14 g, 66%; white solid; mp 162e65 ꢁC; [
a
]
¼ þ34
D
(c ¼ 0.5, CHCl3); HRMS: m/z: calcd for C21H26O10S [M þ Na]þ:
5. Experimental
493.1144; found: 493.1191.
5.1. Chemistry
5.1.3.2. (RS)-2-phenylethyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl
sulfoxide (7). The title product (7) was obtained by crystalisation
(EtOH) of the S-epimeric mixture of the sulfoxides obtained by
column chromatography. Yield 0.31 g; 74%; white solid; mp
5.1.1. General methods
Optical rotations were measured on a Perkin Elmer 241 polar-
imeter at 20 ꢁC. Melting points were determined on a Kofler hot
stage and are uncorrected. 1H NMR and 13C NMR spectra were
recorded at 25 ꢁC with VNMRS 400 MHz Varian or INOVA AV
600 MHz spectrometer. Chemical shifts are referenced to either TMS
150e151 ꢁC; [
a
]D ¼ þ56 (c ¼ 0.5, CHCl3); HRMS: m/z: calcd for
C22H28O10S [M þ Na]þ: 507.1301; found: 507.1295.
5.1.4. General procedure for oxidation of phenylalkyl 2,3,4,6-tetra-
O-acetyl-1-thio-a-D-mannopyranosides to sulfones
(
d
0.00, CDCl3 for 1H) or HOD (
d
4.87, CD3OD for 1H), and to CDCl3 (
49.15) for 13C, as internal standards. High reso-
d
77.23) or CD3OD (
d
To a stirred and at 0 ꢁC precooled solution containing compound
(2) (0.2 g, 0.44 mmol, 1 eq) or (3) (0.2 g, 0.43 mmol, 1 eq) in CH2Cl2
(10 mL) mCPBA (2.5 eq based on 77% peroxide content) was added.
The reaction mixture was stirred at rt for 1 h, diluted with CH2Cl2
(20 mL), washed with satd NaHCO3 (2 ꢂ 15 mL) and water (20 mL).
The organic phase was dried with anhydrous Na2SO4, filtered and
concentrated. The crude product was purified by column chroma-
tography (hexane:EtOAc, 3:1 /1.5:1).
lution (HRMS) and tandem mass spectra (MS/MS) were recorded on
the prototype quadrupole/TOF mass spectrometer. The instrument
was calibrated externally using angiotensin I, bombesin, substance P
and ACTH. The samples (w0.5 mL) werespotted ontoa partiallydried
matrix (DHB), predeposited on the surface of a MALDI target. TLC
was performed on aluminium sheets precoated with silica gel 60
F254 (Merck). Flash column chromatography was carried out on silica
gel 60 (0.040e0.060 mm, Merck) with distilled solvents (hexane,
ethylacetate, acetonitrile, methanol). Reaction solvents were dried
and distilled before use. All reactions containing sensitive reagents
were carried out under argon atmosphere. p-Nitrophenyl
mannopyranoside (PNP-Manp) was purchased from Sigma. Swain-
sonine and mannostatin A were purchased from Calbiochem.
5.1.4.1. Benzyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl sulfone
(10). Yield 0.20 g; 96%; white solid; mp 169e172 ꢁC; lit [27] mp
20
a-
D-
174e176 ꢁC; [
a
]D ¼ þ86 (c ¼ 0.5, CHCl3); lit [27] [
a
]
D
¼ þ83.5 ꢃ1
(c ¼ 0.46, CHCl3); HRMS: m/z: calcd for C21H26O11S [M þ Na]þ:
509.1094; found: 509.1105.

950
M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
Table 2
1H NMR chemical shifts of 2, 3, 6, 7, 10 and 11.
H-60 (J5,6
)
Aglycone (JH,H)
Acetyl groups
0
0
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6
)
H-6 (J6,6
)
2a
3b
6a
5.11 s
5.33e5.26 m
5.33e5.26 m
5.33e5.26 m
4.37 m
(2.2)
3.95 dd
(ꢀ12.1)
4.29 dd
(5.3)
7.32e7.26 m, 5H, Ar,
3.81 d (13.5), 1H, CH2,
3.74 d (13.5), 1H, CH2
7.31e7.19 m, 5H, Ar,
2.95e2.89 m, 3H and
2.86e2.80 m, 1H, CH2CH2
7.37e7.30 m, 5H, Ar,
4.27 d (13.4), 1H, CH2,
4.05 d (13.4), 1H, CH2
7.35e7.24 m, 5H, Ar,
3.19e3.12 m, 4H, CH2CH2
7.43e7.40 m, 5H, Ar,
4.53 d (14.2), 1H, CH2,
4.26 d (14.2), 1H, CH2
7.34e7.22 m, 5H, Ar,
3.46e3.36 m, 2H, CH2,
3.23e3.18 m, 1H, CH2,
3.14e3.09 m, 1H, CH2
2.12, 2.11, 2.04,
1.97 each s
5.27 s
5.34 dd
(3.2)
5.32e5.25 m
5.32e5.25 m
(9.5)
4.35 ddd
(2.2)
4.07 dd
(ꢀ12.2)
4.30 dd
(5.5)
2.17, 2.05 (2x),
1.99 each s
4.54 d
(1.5)
5.83 dd
(3.7)
5.58 dd
(9.8)
5.31 t
(9.6)
4.11 ddd
(2.2)
4.04 dd
(ꢀ12.1)
4.22 dd
(5.6)
2.13, 2.08, 2.03,
2.00 each s
7b
4.67 d
(1.8)
4.68 d
(1.9)
5.86 dd
(3.7)
5.89 dd
(3.7)
5.57 dd
(9.7)
5.59 dd
(9.3)
5.32 t
(9.7)
5.28 t
(9.6)
4.10e4.04 m
4.10e4.04 m
(ꢀ12.4)
4.24 dd
(5.9)
4.28 dd
(5.8)
2.17, 2.05, 2.04,
2.02 each s
2.14, 2.11, 2.07,
1.99 each s
10a
4.72 ddd
(2.3)
4.15 dd
(ꢀ12.5)
11b
4.79 d
(2.2)
5.96 dd
(3.6)
5.59 dd
(9.1)
5.28 t
(9.4)
4.67 ddd
(2.3)
4.15 dd
(ꢀ12.5)
4.26 dd
(6.1)
2.16, 2.07, 2.03,
2.01 each s
a
400 MHz in chloroform-d.
600 MHz in chloroform-d.
b
5.1.4.2. 2-Phenylethyl 2,3,4,6-tetra-O-acetyl-
a
-
D
-mannopyranosyl sulfone
5.1.5.4. (RS)-2-phenylethyl a-D-mannopyranosyl sulfoxide (9). Yield
(11). Yield 0.20 g; 97%; white solid; mp 99e102 ꢁC; [a]D ¼ þ50 (c ¼ 0.5,
CHCl3); HRMS: m/z: calcd for C22H28O11S [M þ Na]þ: 523.1250; found:
523.1254.
80.1 mg; 97%; oil; [
a]
¼ þ110 (c ¼ 0.5, MeOH); HRMS: m/z: calcd
D
for C14H20O6S [M þ Na]þ: 339.0878; found: 339.0910.
1H and 13C NMR data for compounds 2, 3, 6, 7, 10 and 11 are
listed in Tables 2 and 3.
5.1.5.5. Benzyl a-D-mannopyranosyl sulfone (12). Yield 48.3 mg;
87%, yellowish oil; [
a
]D ¼ þ105 (c ¼ 0.5, MeOH); HRMS: m/z: calcd
for C13H18O7S [M þ Na]þ: 341.0671; found: 341.0689.
5.1.5. General procedure for removal of acetyl groups
Acetylated compound was dissolved in MeOH (5 mL) and
K2CO3 (0.5 eq) was added. The reaction mixture was stirred for
at rt (10e30 min), filtered and concentrated. The crude product
was purified by column chromatography (CH3CN:MeOH,
20:1 /9:1).
5.1.5.6. 2-Phenylethyl
a-D-mannopyranosyl sulfone (13). Yield
58.5 mg; 88%; white solid; [
a]
¼ þ86 (c ¼ 0.5, MeOH); HRMS: m/z:
D
calcd for C14H20O7S [M þ Na]þ: 355.0827; found: 355.0854.
1H and 13C NMR data for compounds 4, 5, 8, 9, 12 and 13 are
listed in Tables 4 and 5.
5.1.5.1. Benzyl 1-thio-
a-
D-mannopyranoside (4). Yield 89.8 mg; 95%;
5.2. Biology
pale yellow oil;
[a]
¼ þ344 (c ¼ 0.5, MeOH); lit [25]
D
27
[
a]
¼ þ342 ꢃ1.8 (c ¼ 0.55, MeOH); HRMS: m/z: calcd for
5.2.1. Enzyme preparation
The purification and characterization of recombinant D. mela-
nogasterGolgi(dGMIIb)and lysosomal(dLM408)mannosidases were
D
C13H18O5S [M þ Na]þ: 309.0773; found: 309.0801.
5.1.5.2. 2-Phenylethyl 1-thio-a-D-mannopyranoside (5). Yield 0.12 g;
done with procedures used for relative a-mannosidases [31]. PCR
94%; pale yellow oil;
[
a
]D ¼ þ168 (c ¼ 0.5, MeOH); lit [25]
fragments encoding soluble forms of dGMIIb and dLM408 (i.e., lack-
ing the sequences encoding the putative N-terminal cytoplasmic and
transmembrane domains) were cloned to Pichia pastoris GS115 cells
27
[
a
]
¼ þ198 ꢃ 0.5 (c ¼ 0.9, MeOH); HRMS: m/z: calcd for C14H20O5S
D
[M þ Na]þ: 323.0929; found: 323.0951.
using modified plasmid pICZ
were expressed in a secreted form to condition medium. The super-
natant of condition medium was used as a crude enzyme in -man-
nosidase assay or for further purification of His-tagged enzymes.
aC (Invitrogen). The a-mannosidases
5.1.5.3. (RS)-benzyl a-D-mannopyranosyl sulfoxide (8). Yield 60.1 mg;
94%; oil; [
a
]D ¼ þ61 (c ¼ 0.5, MeOH); HRMS: m/z: calcd for C13H18O6S
a
[M þ Na]þ: 325.0722; found: 325.0740.
Table 3
13C NMR chemical shifts of 2, 3, 6, 7, 10 and 11.
C-1
C-2
C-3
C-4
C-5
C-6
Aglycone
Acetyl groups
2a
81.6
70.8
69.9
66.5
69.2
62.5
137.0, 129.2, 128.9,
127.7 Ar, 34.9 CH2
140.0, 128.8, 128.7,
126.8 Ar, 36.2 CH2, 32.9 CH2
130.6, 129.3, 129.0,
128.7 Ar, 56.2 CH2
138.5, 129.1, 128.8,
127.2 Ar, 52.2 CH2, 28.3 CH2
131.1, 129.6, 129.4,
127.1 Ar, 57.3 CH2
170.8, 170.0 (2x), 169.9 CH3CO,
21.1, 21.0, 20.9, 20.8 CH3CO
170.8, 170.1, 170.0, 169.9 CH3CO,
21.1, 20.9 (2x), 20.8 CH3CO
170.6, 169.8, 169.7, 169.6 CH3CO,
21.0, 20.9, 20.8, 20.7 CH3CO
170.6, 169.8 (2x), 169.6 CH3CO,
21.1, 20.9, 20.8 (2x) CH3CO
170.6, 169.9, 169.4, 169.3 CH3CO,
21.0, 20.9 (2x), 20.7 CH3CO
170.6, 169.8, 169.4 (2x) CH3CO,
20.8 (2x), 20.7, 20.6 CH3CO
3b
82.8
89.9
91.3
86.3
88.2
71.3
67.0
67.2
64.7
64.9
69.6
69.2
69.0
69.1
69.0
66.5
65.9
65.9
65.5
65.6
69.2
74.8
74.7
73.6
73.8
62.6
62.5
62.5
62.8
62.7
6a
7b
10a
11b
137.3, 129.2, 128.6,
127.5 Ar, 52.5 CH2, 28.0 CH2
a
100 MHz in chloroform-d.
150 MHz in chloroform-d.
b

M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
951
Table 4
1H NMR chemical shifts of 4, 5, 8, 9, 12 and 13.
H-60 (J5,6
)
Aglycone (JH,H)
0
0
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6
)
H-6 (J6,6
)
4a
5b
5.08 d (1.0)
3.96e3.74 m
3.96e3.74 m
3.69e3.65 m
3.69e3.65 m
3.96e3.74 m
3.96e3.74 m
7.39e7.23 m, 5H, Ar,
3.96e3.74 m, 2H, CH2
7.29e7.18 m, 5H, Ar,
2.96e2.92 m, 3H and
5.26 d (1.0)
3.93e3.90 m
3.93e3.90 m
3.69e3.65 m
3.69e3.65 m (2.3)
3.85 dd (ꢀ11.9)
3.76 dd (5.9)
2.87e2.84 m, 1H, CH2CH2
7.42e7.38 m, 5H, Ar,
4.45 d (13.6), 1H, CH2,
4.05 d (13.7), 1H, CH2
7.36e7.32 m, 5H, Ar,
3.45e3.41 m, 1H and
3.22e3.10 m, 3H, CH2CH2
7.50e7.38 m, 5H, Ar,
4.65 d (14.0), 1H and
4.40 d (14.0), 1H, CH2
7.35e7.23 m, 5H, Ar,
3.58e3.48 m, 2H and
3.20e3.11 m, 2H, CH2CH2
8b
4.56 d (1.6)
4.74 d (1.3)
4.79 d (1.2)
5.00 d (1.3)
4.33 dd (3.4)
4.43 dd (3.2)
4.42 dd (3.7)
4.56 dd (3.7)
3.86 dd (9.3)
3.87e3.84 m
3.97 dd (9.3)
4.03 dd (9.2)
3.71 t (9.5)
3.72 t (9.5)
3.69 t (9.3)
3.73 t (9.6)
3.60e3.57 m (2.0)
3.45e3.41 m
3.91 dd (ꢀ12.1)
3.87e3.84 m (ꢀ12.2)
3.93 dd (ꢀ12.2)
3.92 dd (ꢀ12.1)
3.68 dd (6.7)
3.65 dd (6.6)
3.72 dd (6.7)
3.71 dd (6.6)
9a
12a
13b
4.26e4.23 m (2.1)
4.23e4.20 m (2.1)
a
400 MHz in chloroform-d.
600 MHz in chloroform-d.
b
5.2.2.
a-mannosidase assay
5.3. Molecular modeling
The supernatants of yeast expressing soluble forms of the
a
-mannosidase were incubated with the substrate PNP-Manp at
The crystal structures of dGM with nanomolar inhibitor
swainsonine (PDB ID: 3BLB) [4] and bLM (PDB ID: 1OD7) [30] were
used as 3-D enzyme models of hGM and hLM for docking of
synthesized mannosides by the GLIDE program [32,33] of the
Schrödinger package [34].
37 ꢁC for 2e3 h. The standard assay mixture consisted of 50 mM
sodium acetate buffer pH 5.2 for LM or pH 5.8 for GMIIb, 2 mM
PNP-Manp (from 100 mM stock solution in DMSO), 1e5
(supernatant of the culture medium) and in case of GMIIb final
0.2 mM CoCl2 in the total reaction volume 50 L. A blank sample
contained no enzyme. The samples were prepared in triplicates.
The reactions were terminated by the addition of 0.5 mL of
100 mM Na2CO3 and the absorbances were recorded at 410 nm
(spectrophotometer).
mL enzyme
m
For docking all molecules of water at the active site of dGM
were deleted except one (WAT1820, numbering according to PDB
ID: 3BLB). This water has shown to be conserved in crystal struc-
tures of dGM either with intact substrates and inhibitors
[4,5,21,22]. In all docking calculations the catalytic acid (Asp341 in
dGM and Asp319 in bLM) was modeled in the protonated form in
accordance with its catalytic role as a general acid. The pyrrolidine
derivative from Scheme 1 was docked with the protonated ring
nitrogen and ionized sulfonate group as predicted for pH ¼ 6.5 by
the Epik program [35] of the Schrödinger package. The mannoside
ligands were docked into the active site in the reactive skew boat
B2,5 conformation of the mannose ring. For the docking calcula-
tions default values of parameters were used with selected
constraints that scored only poses at which the interactions
between the Zn2þ ion and 2-OH and 3-OH groups of the manno-
sides were included. The receptor box for the docking conforma-
tional search was centered at the Zn2þ ion co-factor bound at the
5.2.3. Inhibition assays
Inhibition assays of new mannose analogs with LM 408 and
GMIIb were carried out at the conditions outlined above and the IC50
values were determined. Stock concentrations of inhibitors were
made up in DMSO in the concentration of 100 mM and stored at
ꢀ20ꢁC. The inhibitory effect of DMSO was tested for both enzymes.
The 5% DMSO was selected as a maximal final concentration in the
assay. This concentration causes 10% or 15% of inhibition of LM or
GMIIb, eventually. For this reason maximal concentration of the
tested compounds in the reaction was 5 mM. The inhibitory effect of
the compounds was calculated in percentage of inhibition toward
control sample containing the same concentration of DMSO. The
swainsonine and mannostatin A were used as standard mannose
inhibitors.
ꢁ
bottom of the active site with a size of 39 ꢂ 39 ꢂ 39 A using partial
atomic charges for the receptor from the OPLS2005 force field
except for the Zn2þ and side chains of His90, Asp92, Asp204,
Arg228, Tyr269, Asp341 and His471 (analogous residues were
selected for bLM). For these structural fragments the charges were
calculated at the quantum mechanics level with the DFT (Density
Functional Theory) method (M05-2X) [36,37] using hybrid
quantum mechanics/molecular mechanics (QM/MM) model (M05-
2X/LACVP*:OPLS2005) with the Qsite program [38] of the Schrö-
dinger package. The grid maps were created with no Van der Waals
radius and charge scaling for the atoms of the receptor. Flexible
docking in standard (SP) and extra (XP) precision was used. The
potential for nonpolar parts of the ligands was softened by scaling
the Van der Waals radii by a factor of 1.0 for atoms of the ligands
with partial atomic charges less than specified cutoff of 0.15. The
5000 poses were kept per ligand for the initial docking stage with
scoring window of 100 kcal molꢀ1 for keeping initial poses; the
best 400 poses were kept per ligand for energy minimization. The
Table 5
13C NMR chemical shifts of 4, 5, 8, 9, 12 and 13.
C-1
C-2
C-3
C-4
C-5
C-6
Aglycone
4
85.1
73.6/75.2
69.0
73.6
62.9
139.7, 130.4, 129.6,
128.2 Ar, 35.2 CH2
5b
86.6
94.2
95.9
89.7
93.2
73.8/75.1
69.0
67.9
67.8
66.1
67.8
73.3
81.6
81.4
78.5
80.0
62.9
63.2
63.1
61.7
63.2
142.0, 129.7, 129.5, 127.4
Ar, 37.3 CH2, 33.5 CH2
132.1, 130.9, 129.9, 128.6
Ar, 55.5 CH2
140.3, 129.9, 129.8, 127.9
Ar, 52.5 CH2, 29.1 CH2
131.0, 128.4, 128.3, 127.8
Ar, 55.7 CH2
8b
68.2
68.3
65.3
66.9
72.9
9a
72.7
71.3
72.9
12a
13b
139.6, 129.9, 129.7, 127.9
Ar, 52.7 CH2, 28.7 CH2
ꢁ
a
ligand poses with RMS deviations less than 0.5 A and maximum
100 MHz in chloroform-d.
150 MHz in chloroform-d.
b
ꢁ
atomic displacement less than 1.3 A were discarded as duplicates.

952
M. Poláková et al. / European Journal of Medicinal Chemistry 46 (2011) 944e952
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Acknowledgement
This work was supported by the Slovak Research and Develop-
ment Agency (projects APVV-51-046505 and APVV-0117-06),
Scientific Grant Agency of the Ministry of Education of Slovak
Republic and Slovak Academy of Sciences (projects VEGA-2/0199/
09, VEGA-02/0176/09 and VEGA-02/0095/08), by the Research &
Development Operational Programme funded by the ERDF (Center
of Excellence of Methods and Processes of Green Chemistry,
Contract No. 26240120001) and the Slovak State Programme
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Appendix. Supplementary data
1H and 13C NMR spectra of the compounds 2e13. Tables with
docking scores for complexes dGM:12, dGM:13, bLM:12 and
bLM:13. Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.ejmech.2011.01.012.
ꢀ
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