CuAAC click chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol provides access to triazole-linked piperidine and azepane pseudo-disaccharide iminosugars displaying glycosidase inhibitory properties

Article abstract of DOI:10.1016/j.carres.2016.04.020

Protecting group-free synthesis of 1,2:5,6-di-anhydro-D-mannitol, followed by ring opening with propargylamine and subsequent ring closure produced a separable mix of piperidine N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and azepane N-propargyl 1,6-dideo

Full text of DOI:10.1016/j.carres.2016.04.020

Accepted Manuscript
Title: CuAAC click chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-
gulitol and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol provides access to
triazole-linked piperidine and azepane pseudo-disaccharide iminosugars
displaying glycosidase inhibitory properties
Author: Luís O.B. Zamoner, Valquíria Aragão-Leoneti, Susimaire P. Mantoani,
Michael D. Rugen, Sergey A. Nepogodiev, Robert A. Field, Ivone Carvalho
PII:
S0008-6215(16)30116-1
DOI:
Reference:
http://dx.doi.org/doi: 10.1016/j.carres.2016.04.020
CAR 7183
To appear in:
Carbohydrate Research
Received date: 7-2-2016
Revised date: 16-4-2016
Accepted date: 19-4-2016
Please cite this article as: Luís O.B. Zamoner, Valquíria Aragão-Leoneti, Susimaire P. Mantoani,
Michael D. Rugen, Sergey A. Nepogodiev, Robert A. Field, Ivone Carvalho, CuAAC click
chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-propargyl 1,6-dideoxy-1,6-
imino-D-mannitol provides access to triazole-linked piperidine and azepane pseudo-disaccharide
iminosugars displaying glycosidase inhibitory properties, Carbohydrate Research (2016),
http://dx.doi.org/doi: 10.1016/j.carres.2016.04.020.
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CuAAC click chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol
and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol provides access to
triazole-linked piperidine and azepane pseudo-disaccharide iminosugars
displaying glycosidase inhibitory properties
Luís O. B. Zamoner,a Valquíria Aragão-Leoneti,a Susimaire P. Mantoani,a Michael D.
Rugen,b Sergey A. Nepogodiev,b Robert A. Fieldb and Ivone Carvalhoa*
a
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n,
Monte Alegre, 14040-930, Ribeirão Preto, Brazil.
b
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4
7UH, UK
Corresponding author e-mail: carronal@usp.br
Keywords: Pseudo-disaccharide iminosugars, CuAAC reaction, Click chemistry, glycosidase
inhibition
Highlights



Eight triazole-linked piperidine and azepane pseudo-disaccharide iminosugars were
successful synthesised from a non-protecting 1,2:5,6-di-anhydro-D-mannitol scaffold, and
using Click Chemistry approach;
Interesting α-glucosidase inhibitory activity (53 μM) was achieved for one of the pseudo-
disaccharide iminosugar, containing piperidine core with inverted configuration at C-2 and C-
5 with respect to DNJ (180 μM);
Molecular modeling studies showed binding interactions of (15) and (17) with both
glucosidase catalytic residues (Glu771 and Asp568) in comparison with the less
potent (16) which interacted only with Glu771.
Graphical abstract
O
N
HO
OH
RO
RO
OR
O
N3
OR
O
OMe
AcO
OMe
OAc
Click chemistry
AcO
OR
OR
O
RO
N
N
RO
N
OR
N
RO
OR
IC50= 53 M against yeast -glucosidase
3 fold lower than that of DNJ (IC50 180 M).
Page 1 of 21

Abstract
Protecting group-free synthesis of 1,2:5,6-di-anhydro-D-mannitol, followed by ring opening with
propargylamine and subsequent ring closure produced a separable mix of piperidine N-propargyl 1,5-
dideoxy-1,5-imino-D-gulitol and azepane N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol. In O-
acetylated form, these two building blocks were subjected to CuAAC click chemistry with a panel of
three differently azide-substituted glucose building blocks, producing iminosugar pseudo-
disaccharides in good yield. The overall panel of eight compounds, plus 1-deoxynojirimycin (DNJ) as
a benchmark, were evaluated as prospective inhibitors of almond -glucosidase, yeast -glucosidase
and barley -amylase. The iminosugar pseudo-disaccharides showed no inhibitory activity against
almond -glucosidase; while the parent N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-
propargyl 1,6-dideoxy-1,6-imino-D-mannitol likewise proved to be inactive against yeast α-
glucosidase. Inhibitory activity could be reinstated in the former series by appropriate substitution on
nitrogen. The greater activity of the piperidine could be rationalised based on docking studies.
Further, potent inhibition of -amylase was observed with compounds from both the piperidine and
azepane series.
Introduction
Polyhydroxylated piperidines, well known potent competitive inhibitors of α-
glucosidases, preserve the overall configuration of D-glucose and are characterized by
bioisosteric replacement of the heterocyclic oxygen of a pyranose by nitrogen. Their ability to
be protonated on nitrogen renders these compounds potential mimics of the oxacarbenium
ion-like transition state employed by glucosidases, resulting in potent inhibition.1-4 Arguably
the most studied α-glucosidase inhibitor is 1-deoxynoirimycin (DNJ; 1,5-dideoxy-1,5-imino-
D-glucitol), which was first isolated from Morus alba (mulberry)5 and Streptomyces strains.6
Modified by N-alkylation of the endocyclic nitrogen provides access to more hydrophobic
derivatives with improved potency and selectivity.1-4
The efficient and stereo-controlled synthesis of iminosugars can be achieved by
several methods using carbohydrate as starting materials (reviewed in references 7-9), such as
intramolecular C–N bond formation involving reductive amination,10,11 ring-closing
metathesis followed by oxidation,12,13 intramolecular nucleophilic displacement of alkyl
tosylates/mesylates or bromides,14 in some cases used in combination with Mitsunobu
reaction,15 or even from non-sugar precursor, illustrated by asymmetric conversion of
functionalized pyridine into DNJ,16 among others. The C5-N and C1-N bonds of substituted
piperidines, along with the selective generation of a stereogenic center, are readily
synthesised by a classical double reductive amination of dicarbonyl carbohydrate-based
starting materials, as chiral precursors that contain the same hydroxyl group configuration as
DNJ.17-19 Alternatively, aminocyclization of protected bis-epoxides accessed from cheap and
readily available D-mannitol can be applied to DNJ analogue synthesis; these reactions are
Page 2 of 21

accompanied by concomitant formation of azepane derivatives that are easily separated from
their DNJ counterparts.20,21
Substitution of iminosugars on nitrogen can have a profound impact on their
potency;1-4 this effect has been exploited in the generation of anti-diabetic drug miglitol (N-
hydroxyethyl DNJ),22 for instance. Data for libraries of N-substituted iminosugars reinforce
the notion that inhibitor potency and selectivity against glucosylceramide synthase can be
manipulated by N-substitution18 while more broadly we have shown that the substitution of
sugars with the aid of CuAAC click chemistry23 can be used to enhance inhibition of
Trypanosoma cruzi trans-sialidase,24 yeast -glucosidase and almond -glucosidase.25 In
addition, N-propargylated DNJ and an isomeric L-ido-azepane have been deployed in
CuAAC to generate triazole-linked pseudo-disaccharide iminosugars.21
In order to assess the potential for enhancing the glycosidase inhibitory potency
and/or selectivity of the piperidine and azepane cores, we set out to synthesise iminosugars
based on either the L-gulo-piperidine (1,5-dideoxy-1,5-imino-D-gulitol) or the D-manno-
azepane (1,6-dideoxy-1,6-imino-D-mannitol) scaffold. We then explored CuAAC click
chemistry in order to append these cores to either the C-1, C-3 or C-6 position of D-glucose
through an N-methylene triazole bridge, generating pseudo-disaccharide iminosugars. Herein
we report chemical syntheses of the prospective inhibitors and associated preliminary
glycosidase inhibition studies, along with rationalization of the data by molecular modelling
studies.
Results and Discussion
Synthesis
The synthesis of a small set of six and seven membered ring-derived iminosugars,
containing DNJ stereoisomers was carried out using a C2-symmetric bis-epoxide opening
strategy, as previously employed by Merrer et al.20 and Wilkinson et al.21 In this approach,
the regiospecific opening of one epoxide group by primary amines produces secondary
amines that result an intramolecular SN2 reaction to give both polyhydroxylated piperidine
and azepane scaffolds by a 6-exo-tet or 7-endo-tet aminocyclization processes, respectively.
Our synthesis initially adopted a conventional protecting group approach: protection of the
3,4-diol of 1,2:5,6-di-O-isopropylidene-D-mannitol with benzyl bromide (80%); hydrolysis
of isopropylidene protecting groups with aqueous acetic acid (65%); and epoxide formation
Page 3 of 21

by Mitsunobu reaction through treatment with DIAD and Ph3P (70%). Subsequent
aminocyclization with propargylamine and CuAAC coupling reactions with azido-D-glucose
derivatives then gave adducts that required two deprotection steps for cleavage of benzyl
(iminosugar) and acetyl (glucose) groups. This resulted in a rather cumbersome process
overall, which prompted us to consider a shorter route in which a unprotected bis-epoxide (1)
was obtained in two steps by direct tosylation of both primary alcohols of unprotected D-
mannitol (71%), followed by base-promoted cyclization to give bis-epoxide 1 (28%) (Scheme
1).
The nucleophilic ring opening of homochiral C2-symmetric, unprotected bis-epoxide
(1) by propargylamine and further aminocyclization proceeded smoothly under microwave
irradiation to produce six (2) and seven-membered ring (3) iminosugars bearing the required
alkyne functional group for further transformation. The separation and purification of
isomeric derivatives (2) and (3) was easily accomplished by per-O-acetylation of the crude
mixture (Scheme 3). Thus, per-O-acetylated iminosugars (4) and C2-symmetrical per-O-
acetyl D-manno-configured azepane (5) containing the terminal alkyne function, were
isolated in similar yields, 32 and 35%, respectively, with no apparent influence of the free
3,4-diol in bis-epoxide (1) over the regioselectivity of nitrogen heterocycle formation (viz. 6
vs 7 membered ring).20
Coupling of N-propargylated iminosugars (4) and (5) with sugar derivatives bearing,
an azide groups at C-1 (6), C-3 (7)26 or C-6 (8)27, respectively, was achieved using CuAAC
catalyzed by the in situ generation of Cu(I).28,29 This gave six triazole-linked pseudo-
disaccharide iminosugars, in which the L-gulo-piperidine series (9)-(11) was isolated in 50%
to quantitative yield and the D-manno-azepanes (12)-(14) in 73-79% yield. This shortened
synthetic strategy proved to be advantageous, since the final deprotection step was achieved
by a single step de-O-acetylation reaction in high yield, giving pseudo-disaccharide
iminosugars (15)-(20) (Table 1).
Glycosidase inhibitory activity
In order to assess the impact of iminosugar stereochemistry, ring size and N-
substitution on enzyme inhibition, preliminary assessment was made of compounds (2), (3),
(15)-(20) and DNJ against yeast -glucosidase, almond -glucosidase, barley -amylase
Page 4 of 21

using standard methods (p.nitrophenol release from the corresponding glucosides for the
glucosidases, reducing sugar release from soluble starch for amylase).25,30,31 Prospective
inhibitors were initially assessed at 1.0 mM and 0.1 mM concentrations.
Assays with almond -glucosidase showed no notable inhibition by compounds (2)-
(3) or (15)-(20) at up to 1.0 mM concentration under conditions where DNJ proved to be
inhibitory (75 % inhibition at 1.0 mM; 22 % at 0.1 mM). In contrast, a number of the triazole-
linked pseudodisaccharide iminosugars proved to be inhibitors of yeast -glucosidase.
Inhibitor dilution series were assessed to generate IC50 data, which are reported in Table 2.
The parent N-propargylated piperidine and azepane compounds (2) and (3),
respectively, proved to be inactive against the yeast enzyme, even at 1.0mM concentration. In
the piperidine series, inhibitory activity was achieved by CuAAC derivatization of (4), with
N,6-linked compound (17) (IC50 53 μM), the most active compound in the current study,
displaying an IC50 ~3 fold lower than that of DNJ (IC50 180 μM). This was followed by N,1-
linked compound (15) (IC50 175 μM), which displayed similar activity to DNJ and then N,3-
linked(16) (IC50 940 μM), which displayed much weaker activity. We can therefore conclude
that in this series of compounds, the glucose derivatives clicked to the iminosugar are making
additional interactions with the active site of the -glucosidase and that the nature and site of
substitution of this glucose unit is important. Regarding the azepane series, only N,1-linked
compound (18), (IC50 340 μM) showed moderate activity towards -glucosidase, with N,3-
and N,6-linked azepanes (19) and (20) showing only poorly activity (IC50s of 2400 and 1020
μM, respectively).
In order to assess the pseudo-disaccharide nature of the compounds synthesised, they
were also assessed as potential inhibitors of barley -amylase. Physiologically this enzyme
removes maltose disaccharide from the non-reducing termini of amyloses (see reference 25).
Seven of the nine compounds outlined in Table 2 showed less than 50% inhibition at 1mM
concentration. However, pseudo-disaccharide iminosugars (16) and (19) both displayed
complete inhibition at 1.0 mM, with the latter azepane proving more potent that the former
piperidine at 0.1 mM (90% vs 70% inhibition). Further analyses of these inhibitors are
ongoing and will be reported in due course.
Molecular modeling studies
Page 5 of 21

In order to better understand the -glycosidase inhibition noted above, docking
studies were performed with the more potent pseudo-disaccharide -glucosidase inhibitors
(15) and (17), as well as (16) for comparison.32 These were restricted to -glucosidase, since
sugar binding to -amylase is complicated by its’ multiple carbohydrate binding sub-sites
(two upstream of the catalytic residues; at least 4 downstream); studies with -amylase will
be pursued separately by X-ray crystallography.25
The recently described X-ray crystal structure of a soluble Saccharomyces cerevisiae
α-glucosidase Cwht1p (PDB code 4J5T) was employed; this protein has high identity to
human α-glucosidase (34 to 59% in proposed catalytic domain).33 Based on structural
similarity, catalytic residues in Cwht1p were proposed to be a glutamate (Glu771) and an
aspartate (Asp568).32 Docking of pseudo-iminosugars (15)-(17) was performed at Cwht1p
active site, in parallel to MDL 73945, a known potent glucosidase inhibitor (IC50 0.2, 1.0,
5.0 and 8.0 µM against rat sucrase, maltase, glucoamylase and isomaltase, respectively),
which contains a 1-deoxynojirimycin unit linked to the N-glycosyl core through the C-6
position of 6-deoxyglucoside (Figure 1).34
In general, pseudo-disaccharide iminosugars (15)-(17) preserved some typical DNJ
hydrogen bonds of MDL 73945 at the glucosidase active site and additional binding is
achieved due to the positioning of the glucose core by the triazole ring (Table 3).
It was observed that interaction of MDL 73945 with catalytic Glu771 occurs through
the DNJ unit, while iminosugars (15)-(17) interact mainly through the glucose unit (Figure
2). Moreover, the presence of triazole ring promotes a binding mode for pseudo-disaccharide
iminosugars (15)-(17) which is distinct to that of the reference inhibitor, and also allowed
several hydrophobic interactions with Phe444. Despite the similar orientation of N,1-linked
compound (15) and N,3-linked compound (16) in active site of Cwht1p, the N,1-linked
substitution enabled 15 to make an additional interaction with catalytic Asp568 through the
glucose group (6-OH). On other hand, N,6-linked compound (17) showed an orientation
distinct than the other compounds, but it also interaction with catalytic Asp568 through
iminosugar unit (4-OH). In this way, molecular docking calculation showed that (16)
interacts with just one catalytic residue, Glu771, and derivatives (15) (Glc and iminosugar
Page 6 of 21

units) and (17) (iminosugar core) interact with the two catalytic residues (i.e. Asp568 and
Glu771), while MDL 73945 has a different binding at glucosidase active site than the
tested compounds (15)-(17). Thereby, the diminosugar core of (17) is more strongly linked to
catalytic site of α-glucosidase.
Conclusions
In conclusion, we have demonstrated the synthesis of a series of novel pseudo-
disaccharide iminosugars (15)-(20), based on the isomeric 1,5-dideoxy-1,5-imino-D-gulitol
(piperidine) and 1,6-dideoxy-1,6-imino-D-mannitol (azepane) cores. These compounds, were
prepared from a readily accessible D-mannitol-derived bis-epoxide (1) in a concise manner.
Opening the epoxides with propargylamine and per-O-acetylation gave straightforward
access to the N-propargylated versions of these cores, (4) and (5), which were linked by
CuAAC click chemistry to azido-substituted D-glucose scaffolds (6)-(8) via different
positions (C-1, C-3 or C-6) through triazole bridges. The resulting pseudo-disaccharide
iminosugars showed no inhibitory activity against almond -glucosidase, and the parent N-
propargyl 1,5-dideoxy-1,5-imino-D-gulitol (4) and N-propargyl 1,6-dideoxy-1,6-imino-D-
mannitol (5) likewise proved to be inactive against yeast α-glucosidase.
Despite the loss of inhibitory activity toward yeast α-glucosidase described for 1,5-
dideoxy-1,5-imino-D-iditol (with inverted configuration at C-5 with respect to DNJ),35 for
1,5-dideoxy-1,5-iminoxylitol (lacking the C5 hydoxymethyl group of DNJ)36 and for 1,5-
dideoxy-1,5-imino-D-gulitol (with inverted configuration at C-2 and C-5 with respect to
DNJ) (Table 2), we found that inhibitory activity could be reinstated in the latter series by
appropriate substitution on nitrogen. That is, the pseudo-disaccharide iminosugar (17) has
marked -glucosidase inhibitory activity. However, the reinstatement of activity by N-
Page 7 of 21

substitution was generally more pronounced in the piperidine series (15)-(17) than in the
azepane series (18)-(20). The greater activity of the piperidine could be rationalised based on
docking studies, which showed binding interactions of (15) and (17) with both glucosidase
catalytic residues (Glu771 and Asp568) in comparison with the less potent (16) which
interacted only with Glu771.
In summary, this study illustrates the impact of iminosugar ring size, stereochemistry
and N-substitution on glycosidase inhibition. More detailed analyses, including structural
studies, will be required to define the relative contributions of these features.
Experimental
All chemicals were purchased as reagent grade and used without further purification.
Solvents were dried by standard methods. Reactions were monitored by thin layer
chromatography (TLC) on 0.25 nm pre-coated silica gel plates (Whatman, AL SIL G/UV,
aluminum backing) with the indicated eluents. Compounds were visualized under UV light
(254 nm) and/or by dipping into ethanol/sulfuric acid (95:5, v/v) or orcinol (2%) in aqueous
sulfuric acid (10%), followed by heating the plate for a few minutes. Column
chromatography was performed on silica gel 60 (Fluorochem, 35–70 mesh) or on a Biotage
Horizon TM FLASH Chromatography system with 12 or 25 mm flash cartridges and the
indicated eluents. The microwave assisted reactions were carried out in a CEM® Discover
System, using sealed tubes. NMR spectra were recorded with Bruker Advance DPX 300,
DRX 400 or DRX 500 spectrometers. Chemical shifts () are given in parts per million
downfield from tetramethylsilane. Assignments were made with the aid of HMQC and COSY
experiments. Accurate mass electrospray ionization mass spectra [HRMS (ESI)] were
obtained in positive or negative ionization modes with a Bruker Daltonics MicroOTOF II
ESI-TOF mass spectrometer. Specific optical rotations were determined in Jasco P-2000
Polarimeter, using sodium lamp and wavelength 589 nm at 25 oC. Absorbance readings were
performed using a FLUOstar Omega (BMG labtech) plate reader in absorbance mode at
wavelength 405 nm (glucosidase assays) or 540 nm (amylase assay), using Nunclon Delta 96
well plates (Thermo).
-D-Glucopyranosyl azide (6) was purchased from Aldrich Chemical Co. Ltd.; 3-
azido-3-deoxy-D-glucopyranose (7)26 and methyl 6-azido-6-deoxy--D-glucopyranoside
(8)27 were prepared according to the literature.
Page 8 of 21

1,2:5,6-Di-anhydro-D-mannitol (1):20 D-Mannitol (5.05 g, 27.4 mmol) was solubilised in
pyridine (25,0 mL) and heated to 120o C for 15 minutes. After that, the solution was
refrigerated to 0oC and treated dropwise with tosyl chloride solution (13.1 g, 69 mmol/ 10 mL
pyridine) over 1 hour and stirred at 0oC for 3 hours, then at r.t. for 1 hour. The mixture was
co-evaporated with toluene and the solid was diluted in dichloromethane and washed with
dilute HCl and saturated aqueous NaHCO3 solution The organic layer was dried over MgSO4
and concentrated. The crude mixture of 1,6-di-O-tosyl-D-mannitol was solubilised in a
mixture of acetonitrile and water (38.0 mL, 2:1, v/v), and a small portion of phenolphthalein
o
was added to it. The mixture was stirred at 35-40 C and titrated with NaOH 5 mol.L-1, until
the solution remained pink. After that, a mixture of Na2CO3 (87.4 g) in ethyl acetate (324
mL) was added and stirred vigorously. The solid was filtrated and washed with ethyl acetate.
The organic solution was dried over MgSO4, filtered and concentrated. The resulting mixture
was purified by flash chromatography (hexane and ethyl acetate, 7:3, v/v) to yield 1 (1.12 g,
28 %). 1H NMR (400 MHz, CD3OD): δ 2.77 (2H, m, H-1a, H-6a), 2.82 (2H, m, H-1b, H-6b),
13
3.11-3.17 (2H, m, H-2, H-5), 3.46-3.52 (2H, m, H-3, H-4). C NMR (100 MHz, CDCl3): δ
46.1 (C-1, C-6), 52.8 (C-2, C-5), 73.3 (C-3, C-4).
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (4) and N-propynyl-
2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol
(5):
1,2:5,6-dianhydro-D-
mannitol (0.46 g, 3.1 mmol) was solubilized in methanol (11.4 mL) and to the solution was
o
added propargylamine (0.86 g, 15.6 mmol). The mixture was heated at 90 C under
microwave irradiation by 5 minutes (150 W) in a sealed vessel. After that, the solvents were
concentrated under reduced pressure and the solid was treated with pyridine (8.0 mL) and
acetic anhydride (22.8 mL), then the mixture was stirred at r.t. After 3 hours, the mixture was
treated with ice and the product was extracted with ethyl ether, the organic layer was
concentrated, dried with MgSO4 and the solid purified in flash chromatography column
(toluene and ethyl acetate, 3:7, v/v) to yield of (5) (0.99 mmol, 32 %) and of (6) (1.06 mmol,
35 %) derivatives. N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (4):
1H NMR (400 MHz, CDCl3): δ 2.03, 2.09, 2.13, 2.14 (12H, 4s, 4×CH3), 2.33 (1H, t, J 2.3
Hz, ≡CH), 2.80 (1H, dd, J 4.7 Hz, 11.2 Hz, H-1a), 2.97 (1H, dd, J 9.6 Hz, 11.2 Hz, H-1b),
3.28 (1H, ddd, J 3.3 Hz, 5.8 Hz, 10.0 Hz, H-5), 3.44 (1H, dd, J 2.3 Hz, 17.8 Hz, H-7a), 3.67
(1H, dd, J 2.3 Hz, 17.8 Hz, H-7b), 4.20 (1H, dd, J 5.8 Hz, 11.6 Hz, H-6b), 4.24 (1H, dd, J 3.3
Hz, 11.6 Hz, H-6a), 5.20-5.27 (3H, m, H-2, H-3, H-4). 13C NMR (100 MHz, CDCl3): δ 20.8,
Page 9 of 21

20.9 (CH3), 43.9 (C-1), 49.6 (C-7), 55.5 (C-5), 61.4 (C-6), 66.5 (C-3, C-4), 68.7 (C-2), 77.2
(≡CH), 169.3, 169.7, 170.5 (C=O). ESI HRMS: [M+H]+ calcd for C17H24NO8 370.1496;
found 370.1496. N-Propynyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol
1
(5): H NMR (400 MHz, CDCl3): δ 2.05, 2.12 (12H, 2s, 4×CH3), 2.25 (t, J 2.3 Hz, ≡CH),
2.91 (2H, dd, J 5.6 Hz, 13.8 Hz, H-1a, H-6a), 2.99 (2H, dd, J 4.4 Hz, 13.8 Hz, H-1b, H-6b),
3.39 (1H, dd, J 2.3 Hz, 17.3 Hz, H-7a), 3.45 (1H, dd, J 2.3 Hz, 17.3 Hz, H-7b), 5.40 (2H, dt,
J 1.2 Hz, 4.6 Hz, H-2, H-5), 5.48 (2H, t, J 1.2 Hz, H-3, H-4). 13C NMR (100 MHz, CDCl3): δ
20.8, 21.0 (CH3), 47.9 (C-7), 54.1 (C-1, C-6), 69.9 (C-2, C-5), 70.9 (C-3, C-4), 77.2 (≡CH),
169.9, 170.1 (C=O). ESI HRMS: [M+H]+ calcd for C17H24NO8 370.1496; found 370.1535.
General procedure for removal of acetyl groups
Compounds 4-5 and 7-12 were dissolved in methanol (1.0 mL) and NaOMe (1 mM in
methanol) was added dropwise to achieve a pH of 9.0. After half hour, when TLC showed
total consumption of starting material, the mixture was neutralized with ion exchange resin
DOWEX® 50WX4-50, filtered through a Celite® pad and concentrated under reduced
pressure.
N-Propynyl-1,5-dideoxy-1,5-imino-L-gulitol (2)
1
80% from 4 (0.0082 g, 0.041 mmol): H NMR (500 MHz, D2O): δ 2.71-2.77 (2H, m, H-1a,
≡CH), 2.85 (1H, t, J 10.9 Hz, H-1b), 2.88-2.93 (1H, m, H-5), 3.46 (1H, d, J 17.6 Hz, H-7a),
3.68 (1H, d, J 17.6 Hz, H-7b), 3.83 (1H, dd, J 5.4 Hz, 11.7 Hz, H-6a), 3.89 (1H, dd, J 3.9 Hz,
13
11.7 Hz, H-6b), 3.91-3.94 (1H, m, H-3), 4.05-4.14 (2H, m, H-2, H-4). C NMR (125 MHz,
D2O): δ 42.5 (C-7), 51.5 (C-1), 57.2 (C-5), 60.1 (C-6), 65.3 (C-2), 69.9 (C-3), 70.8 (C-4), 77.2
+
(≡CH). ESI HRMS: [M+H] calcd for C9H15NO4 202.1074; found 202.1076. = 41.1 (c
0.29, H2O).
N-Propynyl-1,6-dideoxy-1,6-imino-D-mannitol (3)
1
86% from 5 (0.0088 g, 0.044 mmol): H NMR (500 MHz, D2O): δ 2.66 (1H, t, J 2.2 Hz,
≡CH), 2.85 (4H, m, H-1a, H-1b, H-6a, H-6b), 3.37 (1H, d, J 17.0 Hz, H-7a), 3.41 (1H, d, J
17.0 Hz, H-7b), 3.89-3.91 (2H, m, H-3, H-4), 4.12 (2H, t, J 4.8 Hz, H-2, H-5). 13C NMR (100
MHz, D2O): δ 47.3 (C-7), 55.6 (C-1, C-6), 69.0 (C-2, C-5), 72.3 (C-3, C-4), 77.4 (≡CH). ESI
+
HRMS: [M+H] calcd for C9H15NO4 202.1074; found 202.1073. = -11.7 (c 0.67, H2O).
Page 10 of 21

CuAAC – General procedure
To a microwave flask equipped with stirring bar, containing alkyne 4 or 5 (1 eq.) and azide 6,
7 or 8 (1.1 eq.) in DMF, was added CuSO4 (0.03 eq) and sodium ascorbate (0.1 eq). The flask
was sealed and allowed to warm at 70oC for 10 minutes under microwave irradiation (150
W). After that, the product was extracted with ethyl acetate e washed with sat. NaHCO3,
dried with MgSO4, concentrated and purified by flash chromatography.
[1-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl]-N-(2,3,4,6-tetra-
O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol)-methane (9)
Compound 6 (0.03 g, 0.075 mmol) was reacted as described in general procedure. The
product 9 was obtained as a colorless amorphous solid (0.075 mmol) in quantitative yield
1
after silica-gel chromatography column. H NMR (300 MHz, CDCl3): δ 1.87, 2.03, 2.05,
2.07, 2.09, 2.10 (24H, 6s, 8×CH3), 2.75 (1H, dd, J 6.1 Hz, 13.1 Hz, H-1a), 2.96 (1H, dd, J
2.9 Hz, 13.1 Hz, H-1b), 3.38 (1H, dd, J 4.8 Hz, 11.2 Hz, H-5), 3.96-4.07 (3H, m, H-5’, H-7a,
H-7b), 4.14 (1H, dd, J 2.0 Hz, 12.6 Hz, H-6’a), 4.26 (1H, dd, J 4.8 Hz, 11.8 Hz, H-6a), 4.33
(1H, dd, J 5.0 Hz, 12.6 Hz, H-6’b), 4.46 (1H, dd, J 6.9 Hz, 11.8 Hz, H-6b), 5.17 (1H, dd, J
3.3 Hz, 8.2 Hz, H-3), 5.21-5.27 (2H, m, H-2, H-4’), 5.32 (1H, dd, J 4.4 Hz, 8.2 Hz, H-4),
13
5.33-5.47 (2H, m, H-2’, H-3’), 5.86 (1H, d, J 9.0 Hz, H-1’), 7.77 (1H, s, triazole). C NMR
(75 MHz, CDCl3): δ 20.0, 20.5, 20.6, 20.7, 20.8, 20.9, 20.9 (CH3), 48.3 (C-1), 49.4 (C-7),
57.4 (C-5), 60.0, 61.5 (C-6 or C-6’), 67.6, 67.9, 68.0, 68.1 (C-2, C-3, C-4, C-4’), 70.4 (C-3’),
72.4 (C-2’), 75.2 (C-5’), 85.9 (C-1’), 120.9 (=CH triazole), 145.6 (Cquat triazole), 168.8,
169.3, 169.7, 169.8, 170.0, 170.4, 170.5 (C=O). ESI HRMS: [M+H]+ calcd for C31H42N4O17
743.2623; found 743.2617.
[1-(1,2,4,6-Tetra-O-acetyl-3-deoxy-D-glucopyranosid-3-yl)-1H-1,2,3-triazol-4-yl]-N-
(2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol)-methane (10)
Compound 7 (0.032 g, 0.083 mmol) was reacted as described in general procedure. The
product 10 was obtained as a colorless amorphous solid in (0.042 mmol) 50% yield after
1
silica-gel chromatography column as α/β mixture (1.0:0.5). H NMR (500 MHz, CDCl3): δ
major isomer β: 1.86, 1.89, 2.03, 2.06, 2.08, 2.10, 2.11, 2.24 (24H, 8s, 8×CH3), 2.72 (1H, dd,
J 6.6 Hz, 12.8 Hz, H-1a), 2.93 (1H, dd, J 3.2 Hz, 12.8 Hz, H-1b), 3.28-3.30 (1H, m, H-5),
3.99 (1H, d, J 14.8 Hz, H-7a), 4.00-4.03 (1H, m, H-5’), 4.02 (1H, d, J 14.8 Hz, H-7b), 4.18-
4.45 (3H, m, H-4’, H-6’a, H-6’b), 4.46 (2H, m, H-6a,H-6b), 4.92 (0.5H, t, J 10.7 Hz, H-3’),
5.12-5.53 (6H, m, H-2, H-3, H-4, H-4’), 5.54 (1H, dd. J 3.6, 11.6 Hz, H-2’α), 5.85 (0.5H, d, J
Page 11 of 21

8.1 Hz, H-1’ β-isomer), 6.41 (1H, d, J 3.6 Hz, H-1’α-isomer), 7.68 (0.4H, s, =CH triazole α),
13
7.73 (0.6H, s, =CH triazole β). C NMR (125 MHz, CDCl3): δ 20.0, 20.1, 20.7, 20.8, 20.9,
21.0 (CH3), 48.6, 48.8 (C-1), 49.3, 49.4 (C-7), 56.8 (C-5), 60.3, 61.3 (C-6, C-6’), 60.8 (C-
3’α), 64.3 (C-3’β), 67.0, 67.7, 67.8, 68.2, 70.3, 73.8 (C-2’, C-4’, C-5’, C-2, C-3, C-4), 88.6
(C-1’α), 92.1 (C-1’β), 121.4 (=CH triazole), 144.7 (Cquat triazole), 168.5, 168.6, 169.1, 169.7,
170.0, 170.5, 170.6 (C=O). ESI HRMS: [M+H]+ calcd for C31H42N4O17 743.2623; found
743.2619.
[1-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-N-
(2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol)-methane (11)
Compound 8 (0.012 g, 0.038 mmol) was reacted as described in general procedure. The
product 11 was obtained as a colorless amorphous solid (0.024 mmol) in 62% yield after
1
silica-gel chromatography column. H NMR (400 MHz, CDCl3): δ 2.01, 2.03, 2.05, 2.06,
2.08, 2.09, 2.11 (21H, 7s, 7×CH3), 2.78 (1H, dd, J 6.1 Hz, 12.9 Hz, H-1a), 2.97 (1H, dd, J 2.6
Hz, 12.9 Hz, H-1b), 3.35 (1H, dd, J 5.2 Hz, 10.3 Hz, H-5), 3.43 (3H, s, OCH3), 3.90 (1H,
ddd, J 2.0 Hz, 8.3 Hz, 10.0 Hz, H-5’), 3.97 (1H, d, J 14.8 Hz, H-7a), 4.02 (1H, d, J 14.8 Hz,
H-7b), 4.27 (1H, dd, J 5.0 Hz, 11.7 Hz, H-6a), 4.32-4.41 (2H, m, H-6’a, H-1’), 4.46 (1H, dd,
J 6.4 Hz, 11.7 Hz, H-6b), 4.65 (1H, dd, J 2.0 Hz, 14.4 Hz, H-6’b), 4.88 (1H, t, J 9.7 Hz, H-
4’), 4.97 (1H, dd, J 8.1 Hz, 9.6 Hz, H-2’), 5.15-5.33 (4H, m, H-2, H-3, H-4, H-3’), 7.68 (1H,
s, =CH triazole). 13C NMR (100 MHz, CDCl3): δ 20.5, 20.6, 20.7, 20.8, 20.9 (CH3), 48.5 (C-
1), 49.3 (C-7), 50.9 (C-6’), 57.2, 57.3 (C-5, OCH3), 60.0 (C-6), 67.7, 67.9, 68.3 (C-2, C-3, C-
4, C-3’), 69.8 (C-4’), 71.0 (C-2’), 72.3 (C-5’), 101.6 (C-1’), 124.2 (=CH triazole), 144.8
(Cquat triazole), 169.4, 169.7, 169.9, 170.0, 170.1, 170.5 (C=O). ESI HRMS: [M+Na]+ calcd
for C30H42N4O16 737.2494; found 737.2519.
[1-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl]-N-(2,3,4,6-tetra-
O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol)-methane (12)
Compound 6 (0.041 g, 0.110 mmol) was reacted as described in general procedure. The
product 12 was obtained as a colorless amorphous solid (0.082 mmol) in 73% yield after
1
silica-gel chromatography column. H NMR (300 MHz, CDCl3): δ 1.87, 2.03, 2.05, 2.08,
2.09, 2.10 (24H, 6s, 8×CH3), 2.89 (2H, dd, J 6.0 Hz, 14.1 Hz, H-1a, H-6a), 2.96 (2H, dd, J
4.7 Hz, J 14.1 Hz, H-1b, H-6b), 3.88 (2H, s, H-7a, H-7b), 4.02 (1H, ddd, J 2.0 Hz, J 5.0 Hz, J
10.0 Hz, H-5’), 4.17 (1H, dd, J 2.0 Hz, 12.5 Hz, H-6’a), 4.33 (1H, dd, J 5.1 Hz, 12.6 Hz, H-
6’b), 5.27 (1H, dd. J 9.2 Hz, 10.0 Hz, H-4’), 5.34-5.48 (5H, m, H-2, H-5, H-2’, H-3’), 5.50
Page 12 of 21

(2H, t, J 1.2 Hz, H-3, H-4), 5.86 (1H, d, J 8.9 Hz, H-1’), 7.82 (1H, s, =CH triazole). 13C
NMR (75 MHz, CDCl3): δ 20.0, 20.4, 20.5, 20.6, 20.7, 20.9 (CH3), 53.0 (C-7), 54.2 (C-1, C-
6), 61.5 (C-6’), 67.7 (C-4’), 70.1, 70.5, 72.4 (C-2, C-5, C-2’, C-3’), 71.1 (C-3, C-4), 75.2 (C-
5’), 85.9 (C-1’), 120.9 (=CH triazole), 145.5 (Cquat triazole), 168.8, 169.3, 169.8, 169.9,
170.0, 170.4 (C=O). ESI HRMS: [M+H]+ calcd for C31H42N4O17 743.2623; found 743.2618.
[1-(1,2,4,6-Tetra-O-acetyl-3-deoxy-D-glucopyranosid-3-yl)-1H-1,2,3-triazol-4-yl]-N-
(2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-manitol)-methane (13)
Compound 7 (0.032 g, 0.083 mmol,) was reacted as described in general procedure. The
product 13 was obtained as a colorless amorphous solid (0.066 mmol as α/β mixture
1
(1.0:0.7)) in 79% yield after silica-gel chromatography column. H NMR (400 MHz,
CDCl3): δ 1.88, 1.90, 1.91, 2.05, 2.10, 2.12, 2.25 (24H, 7s, 8×CH3), 2.85-2.90 (4H, m, H-1a,
H-1b, H-6a, H-6b), 3.70-3.90 (2H, m, H-7a, H-7b), 4.01 (0.7H, ddd, J 2.2 Hz, 4.5 Hz, 10.0
Hz, H-5’ β), 4.05-4.47 (4.4H, m, H-5’ α, H-6’a α and β, H-6’b α and β), 4.93 (0.7H, t, J 10.6
Hz, H-3’ β), 5.17 (1H, t, J 10.9 Hz, H-3’ α), 5.30-5.50 (5.7H, m, H-2, H-3, H-4, H-5, H-4’ α
and β), 5.58 (1H, dd, J 3.6, 11.3 Hz, H-2’ α), 5.86 (0.7H, d, J 8.1 Hz, H-1’ β-isomer), 6.43
(1H, d, J 3.6 Hz, H-1’ α-isomer), 7.68 (0.7H, s, =CH triazole α), 7.72 (1H, s, =CH triazole β).
13C NMR (100 MHz, CDCl3): δ 20.1, 20.7, 20.8, 21.0 (CH3), 53.2 (C-7), 54.0 (C-1, C-6),
60.9 (C-3’α), 61.4, 61.5 (C-6’ α and β), 64.4 (C-3’β), 67.1, 67.2 (C-2, C-5), 68.2 (C-2’α),
69.2, 70.3, 70.4 (C-4’ α and β, C-5’α), 71.5 (C-3, C-4), 73.9 (C-5’β), 88.7 (C-1’α), 92.2 (C-
1’β), 121.0, 121.1 (=CH triazole), 145.1 (Cquat triazole), 168.5, 168.6, 169.2, 170.0, 170.1,
170.6, (C=O). ESI HRMS: [M+H]+ calcd for C31H42N4O17 743.2623; found 743.2615.
[1-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-N-
(2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol)-methane (14)
Compound 8 (0.036 g, 0.090 mmol) was reacted as described in general procedure. The
product 14 was obtained as a colorless amorphous solid (0.069 mmol) in 76% yield after
1
silica-gel chromatography column. H NMR (300 MHz, CDCl3): δ 1.99, 2.02, 2.03, 2.06,
2.09 (21H, 5s, 7×CH3), 2.90 (2H, dd, J 5.7 Hz, 13.8 Hz, H-1a, H-6a), 2.98 (2H, dd, J 4.5 Hz,
13.8 Hz, H-1b, H-6b), 3.42 (3H, s, OCH3), 3.82 (1H, d, J 14.4 Hz, H-7a), 3.88 (1H, d, J 14.4
Hz, H-7b), 3.93 (1H, ddd, J 2.3 Hz, 8.6 Hz, 10.0 Hz, H-5’), 4.37 (1H, dd, J 8.6 Hz, 14.5 Hz,
H-6’a), 4.41 (1H, d, J 8.0 Hz, H-1’), 4.65 (1H, dd, J 2.3 Hz, 14.5 Hz, H-6’b), 4.88 (1H, t, J
9.7 Hz, H-4’), 4.97 (1H, dd, J 8.0 Hz, 9.7 Hz, H-2’), 5.23 (1H, t, J 9.7 Hz, H-3’), 5.31 (2H, t,
13
J 5.3 Hz, H-2, H-5), 5.45 (2H, t, J 1.2 Hz, H-3, H-4), 7.59 (1H, s, =CH triazole). C NMR
Page 13 of 21

(75 MHz, CDCl3): δ 20.5, 20.7, 20.8, 21.0 (CH3), 50.1 (C-6’), 53.2 (C-7), 54.2 (C-1, C-6),
57.2 (OCH3), 69.9 (C-4’), 70.2 (C-2, C-5), 71.0, 71.1 (C-2’, C-3, C-4), 72.4, 72.5 (C-3’, C-
5’), 101.5 (C-1’), 124.1 (=CH triazole), 144.6 (Cquat triazole), 169.4, 169.7, 169.9, 170.0,
170.1 (C=O). ESI HRMS: [M+H]+ calcd for C30H42N4O16 715.2674; found 715.2681.
[1-(β-D-Glucopyranosyl)-1H-1,2,3-triazol-4-yl]-N-(1,5-dideoxy-1,5-imino-L-gulitol)-
methane (15)
1
90% (0.0065 g, 0.016 mmol): H NMR (400 MHz, D2O): δ 2.46 (1H, t, J 11.0 Hz, H-1a),
2.58-2.64 (1H, m, H-5), 2.70 (1H, dd, J 4.3 Hz, 11.0 Hz, H-1b), 3.56 (1H, t, J 9.3 Hz, H-4’),
3.61-3.75 (3H, m, H-3’, H-3ª, H-6’a), 3.75-3.79 (1H, m, H-5’), 3.82-4.02 (8H, m, H-2, H-4,
H-6a, H-6b, H-2’, H-6’b, H-7a, H-7b), 5.69 (1H, d, J 9.2 Hz, H-1’), 8.18 (1H, s, =CH
13
triazole). C NMR (100 MHz, D2O): δ 47.1 (C-7), 50.7 (C-1), 58.1 (C-5), 59.4 (C-6), 60.4
(C-6’), 68.9 (C-3’), 69.9 (C-5’), 65.6, 70.4, 72.2 (C-2, C-2’, C-4), 75.9, 78.8 (C-3, C-3’), 87.4
(C-1’), 124.7 (=CH triazole), 142.3 (Cquat triazole). ESI HRMS: [M+H]+ calcd for
C15H26N4O9 407.1778; found 707.1777. = 9.8 (c 0.96, H2O).
[1-(3-Deoxy-D-glucopyranosid-3-yl)-1H-1,2,3-triazol-4-yl]-N-(1,5-dideoxy-1,5-imino-L-
gulitol)-methane (16)
79% (0.0134 g, 0.033 mmol, as α/β mixture (1:1): 1H NMR (500 MHz, D2O): δ 2.46 (2H, t,
J 9.6 Hz, H-1a), 2.58-2.66 (2H, m, H-5), 2.70 (2H, d, J 2.7 Hz, H-1b), 3.53-3.59 (1H, m, H-5’
α or β), 3.60-4.05 (21H, m, H-2, H-3, H-4, H-6a, H-6b, H-7a, H-7b, H-2’ β, H-4’ α and β, H-
6’a α and β, H-6’b α and β), 4.13 (1H, dd, J 3.6 Hz, 10.8 Hz, H-2’α), 4.46 (1H, t, J 10.3 Hz,
H-3’β), 4.61 (1H, t, J 10.4 Hz, H-3’α), 4.76 (1H, d, J 7.8 Hz, H-1’ βisomer), 5.27 (1H, d, J
3.6 Hz, H-1’ αisomer), 8.04, 8.05 (2H, 2s, =CH triazole). 13C NMR (100 MHz, D2O): δ 47.1
(C-7), 50.6 (C-1), 56.8 (C-5), 59.3 (C-6), 60.3, 60.5 (C-6’), 65.9 (C-3’α), 68.8 (C-3’β), 65.3,
67.7, 67.9, 69.5, 69.7, 70.4, 71.6, 72.0 (C-2’, C-4’, C-2, C-3, C-4, C-7), 77.0 (C-5’), 91.5 (C-
1’α), 96.3 (C-1’β), 125.9, 126.3 (=CH triazole), 144.7 (Cquat triazole), ESI HRMS: [M+H]+
calcd for C15H26N4O9 407.1778; found 407.1772.
[1-(Methyl 6-deoxy-β-D-glucopyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-N-(1,5-dideoxy-1,5-
imino-L-gulitol)-methane (17)
97% (0.0057 g, 0.013 mmol): 1H NMR (400 MHz, D2O): δ 2.37 (1H, dd, J 10.4 Hz, 11.3 Hz,
H-1a), 2.52-2.58 (1H, m, H-5), 2.63 (1H, dd, J 4.5, 11.3 Hz, H-1b), 3.13-3.20 (2H, m, H-2’,
Page 14 of 21

H-4’), 3.32 (3H, s, OCH3), 3.43 (1H, t, J 9.2 Hz, H-3’), 3.75 (1H, ddd, J 2.6 Hz, 8.2 Hz, 10.5
Hz, H-5’), 3.83-3.99 (7H, m, H-2, H-3, H-4, H-6a, H-6b, H-7a, H-7b), 4.22 (1H, d, J 7.8 Hz,
H-1’), 4.54 (1H, dd, J 8.2 Hz, 14.4 Hz, H-6’a), 4.82 (1H, dd, J 2.6 Hz, 14.4 Hz, H-6’b), 8.07
(1H, s, =CH triazole). 13C NMR (125 MHz, D2O): δ 47.1 (C-7), 50.7 (C-1), 51.0 (C-6’), 57.0
(C-5, OCH3), 58.2 (C-6), 69.8, 70.6, 71.1 (C-2, C-3, C-4), 70.9, 73.3 (C-2’, C-4’), 73.7 (C-
5’), 75.6 (C-3’), 103.1 (C-1’), 126.6 (=CH triazole), 144.8 (Cquat triazole)ESI HRMS: [M+H]+
calcd for C16H28N4O9 421.1934; found 721.1928. = 5.6 (c 0.31, H2O).
[1-(β-D-Glucopyranosyl)-1H-1,2,3-triazol-4-yl]-N-(1,6-dideoxy-1,6-imino-D-mannitol)-
methane (18)
1
95% (0.0126 g, 0.031 mmol): H NMR (400 MHz, D2O): δ 2.68-2.79 (4H, m, H-1a, H-1b,
H-6a, H-6b), 3.40 (1H, t, J 9.2 Hz, H-4’), 3.62-3.74 (3H, m, H-3’, H-5’, H-6’a), 3.77-3.87
(5H, m, H-3, H-4, H-6’b, H-7a, H-7b), 3.93 (1H, t, J 9.2 Hz, H-2’), 4.00 (2H, t, J 4.7 Hz, H-
2, H-5), 5.68 (1H, t, J 9.2 Hz, H-1’), 8.16 (1H, s, =CH triazole). 13C NMR (100 MHz, D2O):
δ 52.3 (C-7), 54.9 (C-1, C-6), 60.4 (C-6’), 68.4 (C-2, C-5), 68.9 (C-4’), 72.2 (C-2’), 72.5 (C-
3, C-4), 75.9 (C-5’), 78.9 (C-3’), 87.4 (C-1’), 125.1 (=CH triazole), 142.5 (Cquat triazole) ESI
+
HRMS: [M+H] calcd for C15H26N4O9 407.1778; found 407.1770. = -7.6 (c 1.83, H2O).
[1-(3-Deoxy-D-glucopyranosid-3-yl)-1H-1,2,3-triazol-4-yl]-N-(1,6-dideoxy-1,6-imino-D-
mannitol)-methane (19)
1
91% (0.0223 g, 0.055 mmol, as α/β mixture (1:1)): H NMR (500 MHz, D2O): δ 2.65-2.80
(8H, m, H-1a, H-1b, H-6a, H-6b), 3.50-4.05 (21H, m, H-2, H-3, H-4, H-5, H-2’ β, H-4’ α and
β, H-5’ α and β, H-6’a α and β, H-6’b α and β, H-7a, H-7b), 4.10 (1H, dd, J 3.3 Hz, 10.3 Hz,
H-2’α), 4.44 (1H, t, J 10.3 Hz, H-3’ β), 4.60 (1H, t, J 10.3 Hz, H-3’α), 4.75 (1H, d, J 7.8 Hz,
13
H-1’β-isomer), 5.25 (1H, d, J 3.3 Hz, H-1’ α-isomer), 8.04, 8.06 (2H, 2s, =CH triazole). C
NMR (100 MHz, D2O): δ 52.3 (C-7), 55.4 (C-1, C-6), 60.3, 60.5 (C-6’ α and β), 65.8 (C-3’α),
68.8 (C-3’β), 69.6 (C-2’α), 67.7, 67.9, 68.9, 71.7, 72.1, 72.4 (C-2, C-3, C-4, C-5, C-4’), 71.5
(C-3, C-4), 77.0 (C-5’), 91.6 (C-1’α), 96.4 (C-1’β), 125.4, 125.7 (=CH triazole), 145.1 (Cquat
triazole),ESI HRMS: [M+H]+ calcd for C15H26N4O9 407.1778; found 407.1772.
[1-(Methyl 6-deoxy-β-D-glucopyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-N-(1,6-dideoxy-1,6-
imino-D-mannitol)-methane (20)
90% (0.0117 g, 0.028 mmol): 1H NMR (400 MHz, D2O): δ 2.73-2.78 (4H, m, H-1a, H-1b, H-
Page 15 of 21

6a, H-6b), 3.14-3.20 (2H, m, H-2’, H-4’), 3.33 (3H, s, OCH3), 3.43 (1H, t, J 9.2 Hz, H-3’),
3.73 (1H, ddd, J 2.3 Hz, 7.8 Hz, 10.0 Hz, H-5’), 3.80 (2H, s, H-3, H-4), 3.86 (2H, br s,H-7),
4.00 (2H, t, J 4.5 Hz, H-2, H-5), 4.22 (1H, d, J 8.0 Hz, H-1’), 4.55 (1H, dd, J 7.8 Hz, 14.9 Hz,
13
H-6’a), 4.82 (1H, dd, J 2.5 Hz, 14.9 Hz, H6’bb), 7.99 (1H, s, =CH triazole). C NMR (125
MHz, D2O): δ 51.2 (C-6’), 56.2 (C-7), 55.7 (C-1, C-6), 57.3 (OCH3), 69.1 (C-2, C-5), 72.8
(C-3, C-4), 71.2, 73.3 (C-2’, C-4’) 74.0 (C-5’), 72.9 (C-3’), 103.4 (C-1’), 126.6 (=CH
triazole), 142,7 (Cquat triazole). ESI HRMS: [M+H]+ calcd for C16H28N4O9 421.1934; found
421.1931. = -17.5 (c 0.66, H2O).
Enzyme assays
Yeast α-glucosidase, almond -glucosidase and barley β-amylase were assayed
essentially as described previously, except that DMSO and NaN3 were omitted.28,29 In brief,
the activity of sweet almond -glucosidase [37 °C, pH 6.2 in AcONa 20 mM, PIPES 10 mM,
EDTA 0.1 mM], was assessed by monitoring the rate of release of p.nitrophenolate from
p.nitrophenyl -D-glucopyranoside (625 M) over 30 min. Yeast -glucosidase [37 °C, pH
6.8 in AcONa 20 mM, PIPES 10 mM, EDTA 0.1 mM] was assessed by monitoring the rate
of release of p.nitrophenol from p.nitrophenyl -D-glucopyranoside (200 M) over 30 min.
Barley β-amylase (0.5 U ml-1) [37 °C, pH 5.3 in AcONa] was assayed using soluble
starch (from potato, 0.64% w/v) as a substrate. Following a 30 min incubation, the released
reducing ends were quantified by the addition of 100 μL developing buffer [Na carbonate
0.4 M, pH 10.7, CuSO4 2.5 mM, 4,4′-dicarboxy-1,2′-biquinoline 2.5 mM, L-serine 6 mM] to
100 μL assay volume. The mixture was then incubated for 30 min at 80 °C and the
absorbance measured at 540 nm.25,31
Docking Procedures
Ligands were docked in silico into the proposed active site of Cwht1p (PDB code
45T). All docking runs were performed with the GOLD 5.2 software.37,38 For the
calculations, the orientation of highest score (top-ranked) was selected for each of
compounds here investigated. Top-ranked orientations were selected by GOLD via an
empirical energy function (the ChemPLP).39 Based on this function, the software classifies
the orientations of the molecules by a decreasing ordering of affinity (the fitness) with the
binding site of the glucosidase. The simulations were performed inside a sphere of 10 Å
radius in a point centered among Asp568 and Glu771 catalytic residues and Phe385, Phe389,
Page 16 of 21

Asp392, Arg428 and Phe444 residues, with 10 orientations (docking runs) for each molecule.
Acknowledgements
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial
support. Studies at the JIC were supported by the UK BBSRC Institute Strategic Programme
on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes
Foundation.
References
1. Melo, E. B.; Gomes, A. S.; Carvalho, I. Tetrahedron 2006, 62, 10277-10302.
2. Compain, P.; Martin, O. R. Iminosugars: From Synthesis to Therapeutic Applications. Wiley-
VCH: Weinheim, Germany, 2007.
3. Asano, N. Cell. Mol. Life Sci. 2009, 66, 1479-1492.
4. Moorthy, N. S. H. N.; Ramos, M. J.; Fernandes, P. A. Mini-Rev. Med. Chem. 2012, 12, 713-720.
5. Asano, M.; Oseki, K.; Tomioka, E.; Kizu, H. Matsui, K. Carbohydr. Res. 1994, 259, 243-255.
6. Ishida, N.; Kumagai, K.; Niida, T.; Tsuruoka, T.; Yumoto, H. J. Antibiot. 1967, A20, 66-71.
Inouye, S.; Tsuruoka, T.; Ito, T.; Niida, T. Tetrahedron 1968, 23, 2125-2144.
7. Afarinkia, K.; Bahar, A. Tetrahedron: Asymmetry 2005, 16, 1239-1287.
8. Compain, P.; Chagnault, V.; Martin, O. R. Tetrahedron: Asymmetry 2009, 20, 672-711.
9. Zelli, R.; Longevial, J.-F.; Dumy, P.; Marra, A. New J. Chem., 2015, 39, 5050-5074.
10. Baxter, E. W.; Reitz, A. B. J. Org. Chem. 1994, 59, 3175-3185
11. Tamayo, J. A.; Franco, F.; Lo Re, D. Synlett 2010, 9, 1323-1326.
12. Van den Nieuwendijk, A. M. C. H.; Ruben, M.; Engelsma, S. E.; Risseeuw, M. D. P.; Van den
Berg, R. J. B. H. N.; Boot, R. G.; Aerts, J. M.; Brussee, J.; van der Marel, G. A.; Overkleeft, H.
S. Org. Lett. 2010, 12, 3957-3959.
13. Danoun, G.; Ceccon, J.; Greene, A. E.; Poisson J.-F. Eur. J. Org. Chem. 2009, 4221-4224.
14. Xuezheng, S.; Rawle, I. Tetrahedron Lett. 2007, 48, 3115-3118.
15. Zhang, Z-X.; Wu, B.; Wang, B.; Li, T.-H.; Zhang, P.-F.; Guo, L-N.; Wang, W.-J.; Zhao, W.;
Wang P. G. Tetrahedron Lett. 2011, 52, 3802-3804.
16. Comins, D. L.; Fulp, A. B. Tetrahedron Lett. 2001, 42, 6839-6841.
17. Matos, C.; Lopes, R.; Lopes, C. Synthesis 1999, 4, 571–573.
18. Wennekes, T.; Van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland, A.;
Aerts, J. M. F. G.; Overkleeft H. S. J. Org. Chem. 2007, 72, 1088-1097.
19. Csuk, R.; Prell, E.; Korb, C.; Kluge, R.; Strohl. D. Tetrahedron 2010, 66, 467-472.
20. Merrer, Y.; Poitout, L.; Depezay, J.; Dosbaa, I.; Geoffroy, S.; Foglietti, M. Bioorg. Med. Chem.
1997, 5, 519-533.
21. Wilkinson, B. L.; Bornaghi, L. F.; Lopez, M.; Healy, P. C.; Poulsen, S.; Houston, T. A. Aust. J.
Chem. 2010, 63, 821-829.
22. Scott, L. J.; Spencer, C. M. Drugs 2000, 59, 521-549.
Reviewed in: Meldal, M; Tornoe, C. W. Chem. Rev.
, 108, 2952-3015.
2008
23.
Carvalho, I.; Andrade, P.; Campo, V. L.; Guedes, P. M. M.; Sesti-Costa, R.; Silva, J. S.;
Schenkman, S.; Dedola, S.; Hill, L.; Rejzek, M.; Nepogodiev, S. A.; Field, R. A. Bioorg. Med.
24.
Chem.
, 18, 2412-2427. Campo, V. L.; Sesti-Costa, R.; Carneiro, Z. A.; Silva, J. S.;
2010
Schenkman, S.; Carvalho, I. Bioorg. Med. Chem.
, 20, 145-156.
2012
25. Rejzek, M.; Stevenson, C. E.; Southard, A. M.; Stanley, D.; Denyer, K.; Smith, A. M.; Naldrett,
Page 17 of 21

M. J.; Lawson, D. M.; Field, R. A. Mol. Biosyst. 2011, 7, 718-730.
Lowary, T. L.; Hindsgaul, O. Carbohydr. Res. , 251, 33-67.
1994
, 102, 169-183.
26.
27.
28.
29.
Baer, H. H.; Hanna, H. R. Carbohydr. Res.
1982
Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
, 67, 3057-3064.
2002
Rostovtsev, V. V.; Green, L. G.; Fokin V. V.; Sharpless K. B. Angew. Chem. Int. Ed.
, 41, 2596-2599.
2002
Dedola, S.; Hughes, D. L.; Nepogodiev, S. A.; Rejzek, M.; Field, R. A. Carbohydr. Res.,
345, 1123-1134.
,
2010
30.
31. Vester-Christensen, M. B.; Abou Hachem, M.; Naested, H.; Svensson, B.; Protein Express.
Purif. 2010, 69, 112-119.
Gomes, A. S.; Silva, C. H. T. P.; Silva, V. B.; Carvalho, I. Curr. Bioact. Compd.
, 5, 99-109.
2009
32.
33.
34.
Barker, M. K.; Rose, D. R. J. Biol. Chem.
, 288, 13563-13574.
2013
Robinson, K. M.; Begovic, M. E.; Rhinehart, B. L.; Heineke, E. W.; Ducep, J.-B.; Kastner, P.
R.; Marshall, F. N.; Danzin, C. Diabetes , 40, 825-830.
1991
Fowler, P. A.; Haines, A. H.; Taylor, R. J. K.; Chrystal E. J. T.; Gravestock M. B. Carbohydr.
35.
Res.
, 246, 377-381.
1993
Bernotas, R. C.; Papandreou, G.; Urbach, J.; Ganem, B. Tetrahedron Lett.
, 31, 3393-3396.
, 267, 727–748.
1990
36.
37.
38.
Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J Mol Biol
1997
Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins.
609-623.
, 52,
2003
Liebeschuetz, J. W.; Cole, J. C.; Korb, O. J. Comput. Aided Mol. Des.
, 26, 737-748.
2012
39.
Figure 1. Structure of MDL 73945, a potent -glucosidase inhibitor.
A
B
Page 18 of 21

C
D
Figure 2. Diagram of MDL 73945 and pseudo-disaccharide iminosugars (15)-(17) docked in
Cwht1p active site. A: MDL 73945 in grey, B: C-1 linked (15) in green, C: C-3 linked -anomer
of (16) in orange, and D: C-6 linked (17) in pink. Interaction colour: in green, hydrogen bond
interactions; in pink, - T-shape interaction; in purple: - interaction.
HO
O
HO
N
N
HO
HO
RO
RO
iii
i, ii
OR
+
RO
OR
OH
OH
OH
OH
O
RO
OR
OR
2: R = H
4: R = Ac
3: R = H
5: R = Ac
iv
iv
v
v
1
Scheme 1. Facile synthesis of N-propargylated six membered ring L-gulo iminosugar (2) and D-
manno-configured azepane (3): i. TsCl, pyr (71%); ii. NaOH, MeCN/H2O (28%); iii. Propargylamine,
MeOH, microwave; iv. Ac2O, pyr (Over two steps 2 -32%, 3 -35%); v. NaOMe, MeOH (quant).
Page 19 of 21

Table 1. Triazole adducts accessed by CuAAC with acetylated L-gulo-DNJ (4) and D-manno-azepane
(5) in conjunction with azidoglucose derivatives (6)-(8)
AcO
AcO
AcO
AcO
N3
AcO
AcO
O
O
O
N3
OAc
AcO
OAc
OMe
OAc
N3
OAc
6
8
7
OR
O
OR
OMe
RO
RO
OR
OR
OR
O
RO
RO
RO
RO
RO
OR
RO
RO
RO
AcO
AcO
O
OR
N
N
N
N
N
OR
N
OR
N
N
N
N
N
N
N
RO
AcO
OR
OR
OR
OAc
10: R= Ac
16: R= H
9: R= Ac
15: R= H
11: R= Ac
17: R= H
4
OR
O
OR
OMe
RO
N
RO
N
OR
OR
OR
O
OR
O
OR
N
RO
RO
RO
RO
RO
RO
AcO
AcO
OR
OR
N
N
N
N
N
N
N
N
N
N
RO
OR
RO
OR
RO
OR
AcO
OAc
12: R= Ac
18: R= H
13: R= Ac
19: R= H
5
14: R= Ac
20: R= H
Table 2. Inhibition (IC50) of yeast -glucosidase by pseudo-disaccharide iminosugars based on N-
propargyl 1,5-dideoxy-1,5-imino-D-gulitol (2) and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol
(3). NI: No inhibition at 1.0 mM concentration. IC50 values are accurate to ca +/- 10 %.
OH
OH
O
OMe
HO
HO
OH
OH
OH
O
O
HO
HO
HO
HO
OH
HO
HO
HO
HO
OH
N
N
N
N
N
N
OH
OH
N
N
N
N
N
N
N
HO
HO
HO
HO
OH
OH
OH
OH
2: NI
HO
HO
HO
16: 940M
15: 175M
17: 53M
NH
OH
OMe
OH
O
HO
N
HO
N
OH
OH
OH
OH
DNJ: 180M
O
O
OH
OH
N
N
N
HO
HO
HO
HO
HO
HO
HO
HO
OH
OH
N
N
N
N
N
N
N
N
HO
OH
19: 2400M
HO
OH
HO
OH
HO
3: NI
OH
18: 340M
20: 1020M
Page 20 of 21

Table 3. Main interactions found for glucose and iminosugar core of pseudo-disaccharide
iminosugars MDL 73945 and (15)-(17) in the docking studies with the proposed active site of
Cwht1p (PDB code 4J5T).
Chemical
group
MDL73945
15
16
17
Glucose
Glu429
Phe444
HB
HB
2-OH
Trp771
Glu771
HB
--
--
Glu429
HB
Arg428
Glu429
Phe444
HB
HB
HB
Glu429
Arg387
HB
HB
3-OH
HB
--
--
4-OH
6-OH
O-ring
1-CH
Trp391
--
HB
--
--
--
HB
--
Glu429
HB
Arg387
HB
--
Glu429
Phe444
HB
HB
Asp568
--
--
--
--
Trp710
--
HB
--
--
--
--
--
--
Hidrofobic
(sigma-π)
Phe444
--
Hidrofobic
(sigma-π)
3-CH
--
--
--
--
Phe444
--
--
Iminosugar
His561
Glu707
HB
HB
2-OH
Arg428
HB
Arg428
Trp391
HB
HB
Glu771
HB
His561
Glu707
HB
HB
Gly566
Trp710
HB
HB
Trp391
Gly566
HB
HB
3-OH
4-OH
Gly566
Asp568
Trp710
HB
HB
HB
--
--
Glu771
HB
Glu771
HB
HB
6-OH
5-CH
--
--
--
--
Asp392
--
HB
--
Asp392
Phe385
Asp392
--
BH
--
Hidrofobic
(sigma-π)
π-π T-
shape
π-π T-
shape
--
--
Phe444
π-π T-shape
Phe444
Phe444
Triazole
Page 21 of 21