Tuning of β-glucosidase and α-galactosidase inhibition by generation and in situ screening of a library of pyrrolidine-triazole hybrid molecules

Article abstract of DOI:10.1016/j.ejmech.2017.06.055

The preliminary screening of two libraries of epimeric (pyrrolidin-2-yl)triazoles (14a-s and 22a-s), generated via click chemistry, allowed the rapid identification of four α-galactosidase (coffee beans) inhibitors (22b,k,p,r) and two β-glucosidase (almond) inhibitors (14b,f) in the low μM range. The additional biological analysis of 14b,f towards β-glucocerebrosidase (human lysosomal β-glucosidase), as target enzyme for Gaucher disease, showed a good correlation with the inhibition results obtained for the plant (almond) enzyme. Surprisingly, although these compounds showed inhibition towards β-glucocerebrosidase as acid hydrolase, they did not inhibit bovine liver β-glucosidase as neutral hydrolase. In contrast to what was observed for β-glucosidase inhibition, the coffee bean α-galactosidase inhibitors of the epimeric library (22b,k,p,r) only showed weak inhibition towards human lysosomal α-galactosidase.

Full text of DOI:10.1016/j.ejmech.2017.06.055

Accepted Manuscript
Tuning of β-glucosidase and α-galactosidase inhibition by generation and in situ
screening of a library of pyrrolidine-triazole hybrid molecules
Macarena Martínez-Bailén, Ana T. Carmona, Elena Moreno-Clavijo, Inmaculada
Robina, Daisuke Ide, Atsushi Kato, Antonio J. Moreno-Vargas
PII:
S0223-5234(17)30509-3
10.1016/j.ejmech.2017.06.055
EJMECH 9550
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 April 2017
Revised Date: 17 May 2017
Accepted Date: 26 June 2017
Please cite this article as: M. Martínez-Bailén, A.T. Carmona, E. Moreno-Clavijo, I. Robina, D. Ide, A.
Kato, A.J. Moreno-Vargas, Tuning of β-glucosidase and α-galactosidase inhibition by generation and
in situ screening of a library of pyrrolidine-triazole hybrid molecules, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.055.
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ACCEPTED MANUSCRIPT
Tuning of β-glucosidase and α-galactosidase inhibition by generation and
in situ screening of a library of pyrrolidine-triazole hybrid molecules
Macarena Martínez-Bailén,a Ana T. Carmona,a Elena Moreno-Clavijo,a Inmaculada Robina,a,*
Daisuke Ide,b Atsushi Katob and Antonio J. Moreno-Vargasa,*
aDepartment of Organic Chemistry, Faculty of Chemistry, University of Seville, C/Prof. García González, 1,
b
41012-Seville, Spain. Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194,
Japan. e-Mail: robina@us.es, ajmoreno@us.es
Abstract
The preliminary screening of two libraries of epimeric (pyrrolidin-2-yl)triazoles (14a-s and 22a-
s), generated via click chemistry, allowed the rapid identification of four α-galactosidase
(coffee beans) inhibitors (22b,k,p,r) and two β-glucosidase (almond) inhibitors (14b,f) in the
low μM range. The additional biological analysis of 14b,f towards β-glucocerebrosidase
(human lysosomal β-glucosidase), as target enzyme for Gaucher disease, showed a good
correlation with the inhibition results obtained for the plant (almond) enzyme. Surprisingly,
although these compounds showed inhibition towards β-glucocerebrosidase as acid hydrolase,
they did not inhibit bovine liver β-glucosidase as neutral hydrolase. In contrast to what was
observed for β-glucosidase inhibition, the coffee beans α-galactosidase inhibitors of the
epimeric library (22b,k,p,r) only showed weak inhibition towards human lysosomal α-
galactosidase.
Keywords: iminosugars; glycosidase inhibitors; pyrrolidines; triazole; click reaction.
Introduction
Iminosugars, carbohydrate mimics containing a nitrogen atom instead of an oxygen in the ring
system, constitute the most important class of glycosidase inhibitors.1 Among them, the search
for β-glucosidase and α-galactosidase inhibitors is attracting the attention of many researchers
of the area in the last fifteen years due to the potential use of these compounds in the therapy
of two lysosomal storage diseases (LSDs), Gaucher and Fabry disease.2 In the case of Gaucher
disease, surprisingly, some competitive lysosomal β-glucosidase (β-glucocerebrosidase)
inhibitors at sub-inhibitory concentrations can act as pharmacological chaperones enhancing
the activity of deficient β-glucocerebrosidase in cultured fibroblasts from patients with this
disease, thus hydrolyzing the excess of β-glucosides stored in the lysosome.3 A similar effect
was observed for some competitive lysosomal α-galactosidase (α-galactosidase A) inhibitors in
the Fabry disease.4
Unfortunately, after the enormous effort made in the last fifteen years in the search for
pharmacological chaperones in LSDs, only 1-deoxygalactonojirimycin (DGJ, Galafold®) has
reached all the clinical trials and has recently been approved by the European Commission as a
drug for Fabry disease.5 The most common problems found in the search for drug candidates
for these diseases are related with the cell and ER permeability,2 and with the lack of selectivity
that could result in considerable side-effects in the clinic.6 Thus, the search of low-molecular-

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weight iminosugars with lipophilic substituents able to cross the cell membranes and
selectively inhibit glycosidases remains as an interesting goal.
The generation and in situ biological screening of a library of glycosidase inhibitors is an
interesting strategy for the rapid discovery of potent inhibitors. The choice of the key reaction
for the generation of the library is crucial for the success of the strategy. Reactions that afford
products in high (or quantitative) yields and do not generate by-products that could interfere
in the further in situ biological analysis, are the most appropriate. In this regard, click reactions
largely fulfill these requirements. This strategy has been previously explored by several authors
using different reactions to generate libraries, such as reactions between an aminomethyl
pyrrolidine and a set of aldehydes or acids leading to imines7 or amides,8 respectively. The
combination of the well-known copper catalyzed azide alkyne cycloaddition (CuAAC), the most
typical click reaction, and the in situ biological screening has been broadly used in the
discovery of a wide range of bioactive molecules.9 However this strategy has been scarcely
explored in the glycosidase inhibition field. As far as we are aware, only two examples have
been reported in the last years. The first one used this methodology for the search of
aminocyclitol derived compounds as β-glucocerebrosidase inhibitors.10 The second one was
reported by our research group for the search of α-
L
-fucosidase inhibitors.11 With this
methodology we have been able to find one of the most potent α-
L
-fucosidase inhibitors
containing a pyrrolidine iminosugar core. The results of this work, and other previous findings
in our research,12 demonstrate that the insertion of aromatic or heteroaromatic moieties into
the pyrrolidine iminosugar core increases the selectivity and potency of the resulting inhibitor
that, simultaneously, becomes more lipophilic. This structural feature is relevant for the
therapeutic potential of pyrrolidine iminosugars. It circumvents the lack of selectivity in
pyrrolidines as glycosidase inhibitors which can be attributed to their high conformational
flexibility.
Based on our successful previous results, and following our interest in the search of new
glycosidase inhibitors, we report herein the extension of this methodology for the rapid
discovery of α-galactosidase and β-glucosidase inhibitors. We have used the CuAAC between
epimeric azidomethyl pyrrolidines and a set of alkynes in combination with the in situ
screening to choose the best triazole-derived aglycone for a five-membered iminosugar
inhibitor. The new compounds are low-molecular-weight inhibitors containing a 3,4-
dihydroxypyrrolidine core. This five-membered iminosugar core was chosen instead the six-
membered one with the aim to preserve the inhibition activity but using a less hydrophilic
iminosugar core that could facilitate the traffic of the inhibitor through the cell membranes.
Additionally, the correlation of the inhibitory activity of the new compounds towards β-
glucosidases and α-galactosidases of different sources (non-mammalian vs human lysosomal)
will be also studied.
Results and discussion
Synthesis and selection of the appropriate pyrrolidine scaffolds.
The first step to successfully achieve our goal was the choice of an adequate pyrrolidine
scaffold for the generation of the library. We planned to use 3,4-dihydroxypyrrolidines carrying
a triazole moiety at C-2 as a model of non-complex small molecules that could act as potent

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and selective inhibitor based on our previous results.11 For this reason, we first synthesized
and explored the biological activity of seven pyrrolidine-triazole hybrids (1-4, ent-1, ent-2, 7) in
order to establish the adequate configuration of the pyrrolidine and the adequate distance
between the pyrrolidine and the triazole moiety that generates the best inhibitors. The effect
on the inhibitory activity provoked by the change of the triazole moiety for an amide or
thiourea group (compounds 5 and 6) was also analyzed.
Figure 1. Pyrrolidine-triazole hybrid molecules and the amide/thiourea analogues synthesized for
biological evaluation and scaffold selection.
For the preparation of pyrrolidine-triazoles 1 and 2, we started from diol 8 that was previously
prepared by Fleet from D
-mannose.13

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Scheme 1. Synthesis of pyrrolidine-triazoles 1 and 2, pyrrolidine-amide 5 and pyrrolidine-thiourea 6.
Protecting group manipulation of N-benzyl pyrrolidine 8 afforded N-Boc pyrrolidine 9 that was
transformed into the aldehyde 10 by oxidative degradation of the diol chain. Reaction of 10
with Bestmann-Ohira reagent afforded alkyne 11 in moderate-to-good yield. Aldehyde 10 was
also transformed into azidomethyl-pyrrolidine 13 through a conventional sequence of
reactions.14 Alkyne 11 reacted with benzyl azide under CuAAC conditions to afford triazole 1
after acidic deprotection. Azide derivative 13 was deprotected to give 14 which was
transformed into triazole 2 by click reaction. Additionally, hydrogenation of azide 13 gave
aminomethyl pyrrolidine 15 that was benzoylated to give 5 in excellent yield after acidic
deprotection. Reaction of 15 with phenyl isothiocyanate followed by deprotection afforded 6
in good yield. Enantiomers ent-1 and ent-2 were prepared following the same methodology
but starting from aldehyde ent-10 (see Supplementary data, Scheme S1). The synthesis of this
aldehyde was carried out, in this case, from commercial
reported.15
D-gulono-γ-lactone as was previously
The synthesis of 3 and 4 started from N,O-protected pyrrolidine 16 (Scheme 2) that was
synthesized from
-ribose as described by Kim and coworkers.16 Manipulation of protecting
L

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groups in 16 gave alcohol 1817 which, after oxidation followed by reaction with Bestmann-
Ohira reagent, afforded alkynyl pyrrolidine 20 in good yield. This compound was easily
transformed into pyrrolidine-triazole 3 by CuAAC with benzyl azide followed by acidic
deprotection. Attempts to synthesize the azidomethyl pyrrolidine 22 from 18 by standard
tosylation followed by nucleophilic substitution with azide anion were unsuccessful. Instead,
the carbamate 2117 was obtained after treatment with tosyl chloride. This carbamate showed
high stability and the opening reaction leading to the corresponding azidomethyl derivative
was difficult to achieve. Finally, carbamate 21 could be opened by reaction with
tetrabutylammonium azide in a sealed tube at high temperature giving the azidomethyl
pyrrolidine 22 with moderate-to-good yield, after acidic deprotection. Reaction of azide 22
with phenyl acetylene under CuAAC conditions gave pyrrolidine-triazole 4 in good yield.
Besides, azide 23, previously prepared by us,18 was transformed into triazole 7 through CuAAC
reaction and deprotection.
Scheme 2. Synthesis of pyrrolidine-triazoles 3, 4 and 7.
The analysis of the inhibition properties of compounds 1-7, ent-1 and ent-2 against a panel of
eleven commercial glycosidases was carried out (see Table S1 in Supplementary data, for
details). This initial screening showed that compound 2 was a selective inhibitor of β-
glucosidase from almond (IC50 = 8 µM) and compound 4 was a selective inhibitor of α-
galactosidase from coffee beans (IC50 = 14 µM). Triazole 3 was a moderate and selective
inhibitor of α-L-fucosidase from bovine kidney (IC50 = 168 µM). Amide 5 and thiourea 6 were
moderate-to-weak inhibitors of β-glucosidase from almond and α-mannosidase from Jack
beans. Triazoles 1, ent-1, ent-2 and 7 were weak and/or non-selective inhibitors of different
glycosidases. Thus, we envisaged triazoles 2 and 4 as interesting leads for our study and we

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chose unprotected azides 14 (Scheme 1) and 22 (Scheme 2) as the appropriate scaffolds for
the generation of the desired libraries.
Generation of two libraries of pyrrolidine-triazole hybrid molecules by parallel synthesis and
in situ biological screening.
Epimeric azidomethyl pyrrolidines 14 and 22, were reacted with 1.2 equiv.19 of a set of
commercial alkynes (a-g,j-n,p-r) and synthetic alkynes (h,i,o,s, see Supplementary data for
synthetic details) under CuAAC conditions (0.068 equiv. CuSO4/0.22 equiv. sodium ascorbate
as catalyst in ButOH-H2O as solvent) in order to generate libraries I and II, as depicted in
Scheme 3. After 24 h at room temperature, TLC and ESI-MS of the reaction mixtures showed
complete conversion and the presence of the desired pyrrolidine-triazole derivatives (see
Supplementary data for mass spectra analysis).
Scheme 3. Parallel synthesis of pyrrolidine-triazole libraries I and II.
The in situ screening of the resulting crude pyrrolidine-triazoles was carried out in a 96-well
microtiter plate. In case of library I, the % inhibition of β-glucosidase from almond in a well
containing 10 µM of each compound was measured (Figure 2). For library II, the inhibition
assay was performed against α-galactosidase from coffee beans in a well containing 50 µM of
each potential inhibitor (Figure 3). Blank experiments with CuSO4, sodium ascorbate and each
alkyne (a-s) were also carried out; none of these reagents showed any inhibition of the
corresponding enzyme at the concentration of the experiments.

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80
70
60
50
40
30
20
10
0
14 a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
Figure 2. Biological screening of library I: % inhibition of β-glucosidase from almond at 10 µM of
pyrrolidine-triazoles (pH 5, 37 ºC).
80
70
60
50
40
30
20
10
0
22 a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
Figure 3. Biological screening of library II: % inhibition of α-galactosidase from coffee beans at 50 µM of
pyrrolidine-triazoles (pH 6, 37 ºC).
The preliminary biological screening of library I showed two compounds with outstanding
inhibitory activity, 14b and 14f (72 and 65% inhibition, respectively). It must be said that, by
chance, compound 14b had been already prepared and evaluated in the scaffold selection
process (numbered as 2, Figure 1) but 14f was a new bioactive triazole derivative. The initial
azidomethyl-pyrrolidine scaffold 14 was not inhibitor at 10 µM and the other seventeen
triazoles of the library showed weak or no inhibition at this concentration, displaying the high
influence of the C-4 substituent on the triazole ring. In the case of library II, the corresponding
screening towards α-galactosidases showed four compounds with remarkable inhibition,
compounds 22b,k,p,r (62, 64, 67 and 66% inhibition, respectively). Compounds 22b,k,p,r have
in common an aromatic ring directly linked to the triazole moiety, being the only compounds
in the library with this unique structural characteristic. In this case, the azidomethyl-pyrrolidine

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scaffold 22 showed moderated-to-weak inhibition (38%) under the same conditions, being
more active than the majority of the triazoles.
Figure 4. Structure of the most potent inhibitors detected by in situ screening.
The new bioactive triazoles selected from the libraries (Figure 4) were synthesized in a higher
scale and purified by column chromatography in order to perform a more accurate inhibition
study. Their inhibitory properties were studied towards twenty commercial glycosidases and
the results are summarized in Table 1. As compounds 14b,f and 22b,k,p,r showed promising
inhibition properties towards β-glucosidase from almond and α-galactosidase from coffee
beans, respectively, human lysosomal enzymes and other mammalian enzymes were also
included in this study.
Compound 14b, that had shown good inhibition towards β-glucosidase from almonds, showed
now an unusual specificity between two different mammalian β-glucosidases; this compound
was a good inhibitor of β-glucocerebrosidase from human lysosome (IC50 = 11 µM) but did not
inhibit β-glucosidase from bovine liver. Usually, a good inhibition of bovine liver β-glucosidase
has been used as a preliminary parameter to select compounds for human lysosome β-
glucocerebrosidase inhibition.20 Significantly, this is one of the very few cases reported to date
where the β-glucosidase inhibition pattern is unusual. The result for the inhibition of the non-
mammalian enzyme (β-glucosidase from almond) correlates perfectly with those obtained in
the human version. A similar behavior was observed for compound 14f. The similarity on the
inhibition data between 14b and 14f allows us to assume that the trimethylsilyl substituent in
14f may interact with the enzyme in a similar way than the phenyl group in 14b, in spite of
their structural difference. These two compounds were specific β-glucosidase inhibitors,
presenting only weak inhibition of β-galactosidase (bovine liver) and not inhibiting any other of
the enzymes tested. Almond β-glucosidase is not fully sequenced but is deduced to belong to
CAZy21 family GH1 by limited sequence analysis.22 The X-ray structure of human lysosomal
beta-glucosidase has been reported and, on the basis of sequence similarity, this enzyme was
classified as a member of the family GH30.23 Both enzymes belong to the same clan fold,
named GH-A. Bovine liver beta-glucosidase belongs to the same CAZy family and clan fold than
almond beta-glucosidase. Thus, the three enzymes share three-dimensional structural
similarities (similar clan fold) but the human one would have only limited sequence similarity
with the others (different CAZy family). The level of selectivity detected in the inhibition of
these enzymes by our compounds could be explained by decisive non-glycone allosteric

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interactions close to the active site that should be more important than the glycone
interactions.
Table 1. Concentration of pyrrolidine-triazole inhibitors giving 50% inhibition (IC50) of a set of
glycosidases.
Compound
Enzymes1
14b
14f
22b
22k
22p
22r
β-glucosidase
(almond)
8 µM
NI
7 µM
NI
NI
NI
NI
NI
NI
β-glucosidase
(bovine liver)
240 µM
28 µM
14 µM
171 µM
173 µM
887 µM
888 µM
187 µM
14 µM
159 µM
268 µM
β-glucocerebrosidase
(human lysosome)
11 µM
NI
19 µM
NI
19 µM 31 µM
17 µM 13 µM
α-galactosidase
(coffee beans)
α-galactosidase A
(human lysosome)
NI
NI
NI
185 µM
β-galactosidase
(bovine liver)
458 µM
151 µM
42 µM 338 µM
NI: non inhibition (or less than 50% inhibition) at 1 mM of inhibitor.
1Non inhibition was detected towards these enzymes: α-
-fucosidase from bovine kidney, α-glucosidase from yeast
L
and rice, β-galactosidases from Escherichia coli and Aspergillus oryzae, amyloglucosidases from Aspergillus niger and
Rhizopus sp, α-mannosidase from Jack beans, β-mannosidase from snail, α,α-trehalase from porcine kidney, α-
rhamnosidase from Penicillium decumbens, β-glucuronidases from Escherichia coli and bovine liver, and β-N-
acetylglucosaminidase from Jack beans.
Compounds 22b,k,p,r were less specific than the corresponding epimers in the inhibition
assays. In general, they showed good inhibition towards α-galactosidase from coffee beans
(IC50 = 14-17 µM) but the inhibition potency decreased 10-fold towards human lysosomal α-
galactosidase A (IC50 = 159-185 µM), although both enzymes belong to the same CAZy family
(GH27).21 Surprisingly, human lysosomal β-glucocerebrosidase did not discriminate between
epimers 14b and 22b, showing both compounds the same range of activity towards this
enzyme. Moreover, 22b showed weak inhibition towards other mammalian glycosidases, such
as β-galactosidase and β-glucosidase from bovine liver. The presence of a bulkier group in the
triazole substituent of compound 22k led to an increase in the inhibition selectivity, reaching
good inhibitory activity in α-galactosidase from coffee beans (IC50 = 17 µM) and human
lysosomal β-glucocerebrosidase (IC50 = 19 µM). The inhibition pattern of compounds 22p and
22r was similar to that of analogue 22b.

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Conclusions
We have used two epimeric and structurally simple azidomethyl-pyrrolidines as scaffolds for
the generation of two libraries of pyrrolidine-triazole glycosidase inhibitors. We have
demonstrated that the biological analysis of the library can be performed in situ without
purification and isolation of the individual compounds. This rapid preliminary analysis allowed
us to select several β-glucosidase and α-galactosidase inhibitors as models for the assessment
towards human glycosidases. The derivatives that showed the best inhibition towards β-
glucosidase from almonds were also found to be inhibitors of the corresponding human
enzyme. However this correspondence was not observed between the latter and the bovine
liver enzyme. Although the new pyrrolidines described in this work do not contain a
hydroxymethyl recognition motif on the skeleton, which is present in the major part of
synthetic and natural pyrrolidine-derived glycosidase inhibitors,24,25 they showed selective
inhibition towards β-glucosidases or α-galactosidases. In this sense, compounds 14b,f are the
first examples of β-glucocerebrosidase inhibitors on the low micromolar range derived from a
3,4-dihydroxypyrrolidine. The absence of the hydroxymethyl group and the incorporation of an
important hydrophobic component in these compounds may improve their cell and ER
permeability for potential pharmacological applications. As far as we are aware, the
interaction of a hydroxymethyl group, installed on other differently configured pyrrolidine
iminosugars, with the Asp127 of the active site of human β-glucocerebrosidase has been
recently reported.26 We are currently working on the application of our method on other
scaffolds, once we have already established the appropriate tool for the rapid assessment. In
this way, we would be able to evaluate the convenience of the presence of that group in our
systems.
Experimental part.
General methods
Optical rotations were measured in a 1.0 cm or 1.0 dm tube with a Jasco P-2000
spectropolarimeter. Infrared spectra were recorded with a Jasco FTIR-410 spectrophotometer.
1H and 13C NMR spectra were recorded with a Bruker AMX300, AV300, AV500 and AVIII500 for
solutions in CDCl3, CD3OD and DMSO-d6 at room temperature except when indicated. δ are
given in ppm and J in Hz. All the assignments were confirmed by COSY and HSQC experiments.
Mass spectra (CI and ESI) were recorded on Micromass AutoSpeQ and QTRAP spectrometers.
NMR and mass spectra were registered in CITIUS (University of Seville). TLC was performed on
silica gel HF254 (Merck), with detection by UV light charring with H2SO4, p-anisaldehyde, vanillin,
ninhydrin, KMnO4 or with Pancaldi reagent [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O]. Silica gel 60
(Merck, 63–200 μm) was used for preparative chromatography.
Inhibition studies with commercial enzymes
The % of inhibition towards the corresponding glycosidase was determined in the presence of
1 mM of the inhibitor on the well. Each enzymatic assay (final volume 0.12 mL) contains 0.01
to 0.5 units/mL of the enzyme and 10 mM aqueous solution of the appropriate p-nitrophenyl
glycopyranoside (substrate) buffered to the optimal pH of the enzyme. Enzyme and inhibitor
were preincubated for 5 min at rt, and the reaction started by addition of the substrate. After

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20 min of incubation at 37 °C, the reaction was stopped by addition of 0.1 mL of sodium borate
solution (pH 9.8). The p-nitrophenolate formed was measured by visible absorption
spectroscopy at 405 nm. Under these conditions, the p-nitrophenolate released led to optical
densities linear with both reaction time and concentration of the enzyme. For the best
inhibitors (% inhibition ≥ 80), the IC50 value (concentration of inhibitor required for 50%
inhibition of enzyme activity) was determined from plots of % inhibition versus inhibitor
concentration. Each experiment (%, IC50) is performed by duplicate and the average value is
given (see Table S1 in Supplementary data).
Inhibition studies with human lysosomal enzymes
The inhibitory activity toward human lysosomal β-glucocerebrosidase and α-galactosidase A
was measured with 4-methylumbelliferyl-β-D-glucopyranoside and 4-methylumbelliferyl-α-D-
galactopyranoside as substrate. The reaction mixture consisted on 100 mM McIlvaine buffer
(pH 5.2), 0.25% sodium taurocholate and 0.1% Triton X-100, and the appropriate amount of
enzyme. The reaction mixture was pre-incubated at 0˚C for 45 min, and the reaction was
started by adding 3 mM substrate solution, followed by incubation at 37 ˚C for 30 min. The
reaction was stopped by the addition of 1.6 mL of the solution of 400 mM glycine-NaOH
solution (pH 10.6). The released 4-methylumbelliferone was measured (excitation 362 nm,
emission 450 nm) with a F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).
Generation of the libraries I and II followed by in situ biological screening.
Library I: To a 0.30 mL of a solution of azide 14 (23 mM in tBuOH–H2O (2 : 1)) in an eppendorf,
0.10 mL of a solution of the corresponding alkyne (a-s) (83 mM in tBuOH) was added followed
by 25 μL of an aqueous solution of sodium ascorbate (30 mM) and 25 μL of an aqueous
solution of CuSO4 (9.4 mM). The final concentration of the azide 14 in each eppendorf was 15
mM. The resulting mixtures were left at room temperature for 24 h and monitored for
completion by TLC and ESI-MS (see Supplementary data for mass spectra analysis). Then, the
reactions were diluted with water to the desired concentration and placed in a 96-well
microtiter plate in order to perform the enzymatic assays. In the preliminary screening of the
resulting crude (pyrrolidin-2-yl)triazoles 14a-s, % of inhibition towards β-glucosidase from
almonds was determined at 10 μM of the pyrrolidine-triazole on each well (supposing
quantitative conversion in the click reaction).
Library II: This library was generated and screened following the same procedure that for
Library I, except that azide 22, instead of 14, was used as starting material and the biological
analysis was performed towards α-galactosidases from coffee beans. In this case, the %
inhibition of crude triazoles 22a-s was determined at 50 μM on each well.
Synthesis of compounds
N-tert-Butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-talitol (9).14 To a solution
of 813 (1.05 g, 3.58 mmol) in MeOH (25 mL), Boc2O (1.59 g, 7.15 mmol) and Pd/C (10%, cat.)
were added and the reaction mixture was stirred under an atmosphere of hydrogen for 2 h.
The crude was filtered through celite and the solvent removed in vacuo. The resulting residue
was purified by chromatography column on silica gel (ether:cyclohexane 4:1) to give 9 (0.92 g,

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ꢀ ꢂꢄꢅ
ꢀ ꢂꢄꢆ
+ 44.8 (c 1.17, CH2Cl2) (Lit: . ꢁ
3.04 mmol, 85%) as a white solid. ꢁ
+ 54 (c 1.0, CHCl3)).
ꢃ
ꢃ
1
IR (ν cm-1) 3606-3243 (OH), 2989, 2934, 1655 (C=O). H-NMR (300 MHz, DMSO-d6, 363 K, δ
ppm, J Hz) δ 4.73-4.66 (m, 2H, H-2, H-3), 4.52 (d, 1H, JOH,5=4.8, OH-5), 4.11 (t, 1H, JOH,6a = JOH,6b
=
5.9, OH-6), 4.03 (ap. bs, 1H, H-4), 3.73-3.63 (m, 2H, H-5, H-6a), 3.44-3.25 (m, 3H, H-6b, H-1a, H-
13
1b), 1.42 (s, 9H, -C(CH3)3), 1.33 (s, 3H, -C(CH3)2), 1.26 (s, 3H, -C(CH3)2). C-NMR (75.4 MHz,
DMSO-d6, 363 K, δ ppm) δ 154.0 (C=O, Boc), 109.8 (-C(CH3)2), 82.4, 79.0 (C-2, C-3), 78.4 (-
C(CH3)3), 72.4 (C-5), 64.8 (C-4), 62.6 (C-1), 53.2 (C-6), 27.7 (-C(CH3)3), 26.5 (-C(CH3)2), 24.6 (-
C(CH3)2). HRCIMS m/z found 304.1760, calc. for C14H25NO6 [M+H]+: 304.1760.
(2R,3S,4R)-N-tert-Butoxycarbonyl-2-formyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol (10).14 To
o
a solution of 9 (353 mg, 1.17 mmol) in THF (5 mL) at 0 C, a solution of NaIO4 (502 mg, 2.34
mmol) in water (8 mL) was added. The reaction mixture was stirred at r.t. for 2 h and then THF
was removed in vacuo and the aqueous crude was extracted twice with CH2Cl2. The organic
layers were washed with sat. aq. sol. of NaHCO3 and brine, dried over Na2SO4, filtered and
ꢀ ꢂꢄꢅ
evaporated to give 10 (317 mg, 1.17 mmol, quant.) as a colorless oil. ꢁ
+ 55.9 (c 0.84,
ꢃ
-1
ꢀ ꢂꢄꢆ
CH2Cl2) (Lit: ꢁ
+ 35.5 (c 1.0, CHCl3)). IR (ν cm ) 2979, 2936, 1735 (C=O), 1692 (C=O), 1300,
ꢃ
1158, 1075, 1051. 1H-NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 9.65, 9.611 (s, 1H, CHO), 4.83 (ap. t,
1H, H-3), 4.70-4.65 (m, 2H, H-2, H-4), 3.96-3.83 (m, 1H, H-5a), 3.42-3.30 (m, 1H, H-5b), 1.48 (s,
9H, -C(CH3)3), 1.47 (s, 3H, -C(CH3)2), 1.30 (s, 3H, -C(CH3)2). 13C-NMR (75.4 MHz, CDCl3, δ ppm) δ
198.2, 198.01 (CHO), 154.2 (C=O, Boc), 112.7 (-C(CH3)2), 81.1, 81.01 (-C(CH3)3), 80.0, 79.01 (C-3),
79.6, 78.6, 71.51 (C-2, C-4), 52.4, 51.91 (C-5), 28.5, 28.4, 28.4, 25.71 (-C(CH3)3), 27.1, 27.01 (-
C(CH3)2), 25.1 (-C(CH3)2). HRCIMS m/z found 272.1491, calc. for C13H21NO5 [M+H]: 272.1498.
(2S,3S,4R)-N-tert-Butoxycarbonyl-2-ethynyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol (11). To a
o
solution of 10 (317 mg, 1.17 mmol) in anhydrous MeOH (16 mL) at 0 C, K2CO3 (327 mg, 2.34
mmol) and Bestmann-Ohira reagent (200 µL, 1.29 mmol), were slowly added. The reaction
mixture was stirred at r.t. for 4 h and then Et2O and sat. aq. sol. of NaHCO3 were added. The
aqueous phase was extracted twice with CH2Cl2. The organic layers were washed with brine,
dried over Na2SO4, filtered and evaporated. The resulting residue was purified by
chromatography column on silica gel (EtOAc:cyclohexane 1:5) to give 11 (223 mg, 0.83 mmol,
-1
ꢀ ꢂꢄꢇ
71%) as a colorless oil. ꢁ
+ 105.1 (c 0.67, CH2Cl2). IR (ν cm ) 3262 (≡CH), 2978, 2936, 1698
ꢃ
1
(C=O), 1391, 1158, 880. H-NMR (300 MHz, DMSO-d6, 363 K, δ ppm, J Hz) δ 4.81 (ap. t, 1H, H-
2
4), 4.68 (d, 1H, J3,4 = 5.7, H-3), 4.54 (ap. bs, 1H, H-2), 3.68 (d, 1H, J5a,5b = 12.9, H-5a), 3.31 (dd,
1H, J5b,4 = 4.5, H-5b), 3.20 (d, 1H, J2’,2 = 2.4, H-2’), 1.43 (s, 9H, -C(CH3)3), 1.35 (s, 3H, -C(CH3)2),
1.27 (s, 3H, -C(CH3)2). 13C-NMR (75.4 MHz, DMSO-d6, 363 K, δ ppm) δ 153.0 (C=O, Boc), 110.9 (-
C(CH3)2), 84.4 (C-3), 79.6 (-C(CH3)3), 79.1 (C-1’), 78.2 (C-4), 74.7 (C-2’), 54.1 (C-2), 50.3 (C-5),
27.6 (-C(CH3)3), 26.0 (-C(CH3)2), 24.3 (-C(CH3)2).HRESIMS m/z found 290.1364, calc. for
C14H21NO4 [M+Na]+: 290.1363.
(2S,3S,4R)-2-[(1-benzyl)-1H-1,2,3-triazol-4-yl]-pyrrolidine-3,4-diol hydrochloride (1). To a
solution of 11 (61 mg, 0.23 mmol) in toluene (2.1 mL), benzylazide (47 mg, 0.35 mmol), DIPEA
(153 µL, 0.87 mmol) and CuI (13 mg, 0.07 mmol) were added. After stirring at r.t. for 1 d., aq.
sat. sol. of NaHCO3 (5 mL) was added and the aqueous phase was extracted with EtOAc. The
organic layers were dried, filtered and evaporated. The resulting residue was purified by
1 The signals are unfolded by the presence of two rotamers.

ACCEPTED MANUSCRIPT
chromatography column on silica gel (EtOAc:cyclohexane 1:2) to give the protected
pyrrolidine-triazole (70 mg, 0.17 mmol, 76%) as a white solid. A solution of this compound (37
mg, 0.10 mmol) in HCl (1M):THF (1:1, 2.4 mL) was stirred at r.t. for 2 d., and then evaporated
- 47.0 (c 0.60, MeOH). IR (ν cm-1) 3285
ꢀ ꢂꢄꢅ
to give 1 (32 mg, 0.11 mmol, quant) as a solid. ꢁ
ꢃ
1
(OH, NH), 1445, 1121, 814, 719. H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 8.22 (s, 1H, H-5’),
7.39-7.35 (m, 5H, H-aromat.), 5.65 (s, 2H, -CH2Ph), 4.72 (d, 1H, J2,3 = 8.1, H-2), 4.51 (dd, 1H, J3,4 =
2
3.9, H-3), 4.44-4.42 (m, 1H, H-4), 3.63 (dd, 1H, J5a,5b = 12.6, J5a,4 = 4.2, H-5a), 3.35 (dd, 1H,
J5b,4=1.5, H-5b). 13C-NMR (75.4 MHz, MeOD, δ ppm) δ 142.5 (C-4’), 136.5 (Cq aromat.), 130.1 (C
aromat.), 129.7 (C aromat.), 129.4 (C aromat.), 125.7 (C-5’), 76.8 (C-3), 70.8 (C-4), 57.1 (C-2),
55.1 (-CH2Ph), 51.2 (C-5). HRCIMS m/z found 261.1345, calc. for C13H17N4O2 [M]: 261.1352.
(2S,3S,4R)-N-tert-Butoxycarbonyl-2-hydroxymethyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol
(12).14 A solution of crude aldehyde 10 (1.26 g, 4.66 mmol) in ethanol was added to a
suspension of NaBH4 (184 mg, 4.66 mmol) in ethanol (10 mL). After stirring at r.t. for 50 min,
aq. sol. of. NH4Cl was added and the aqueous phase was extracted with EtOAc, dried over
Na2SO4, filtered and evaporated. The resulting residue was purified by chromatography
column on silica gel (EtOAc:cyclohexane 2:1) to give 12 (1.24 g, 4.54 mmol, 97%) as a colorless
1
ꢀ ꢂꢄꢆ
ꢀ ꢂꢄꢈ
oil. ꢁ + 40.7 (c 0.64, CH2Cl2) (Lit: ꢁ + 56 (c 1.0, CHCl3)). H-NMR (300 MHz, CDCl3, δ ppm,
ꢃ
ꢃ
J Hz) δ 4.72-4.54 (m, 2H), 4.14-4.02 (m, 1H), 3.88-3.63 (m, 3H), 3.49 (dd, 1H, J = 12.6, J = 5.4),
2.82 (bs, 1H, OH), 1.46 (s, 12H, -C(CH3)2, -C(CH3)3), 1.32 (s, 3H, -C(CH3)2).
(2S,3S,4R)-N-tert-Butoxycarbonyl-2-azidomethyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol
(13).14 To a solution of 12 (1.25 g, 4.57 mmol) in anhydrous pyridine (20 mL) at 0 °C, TsCl (4.40
g, 22.8 mmol) was added. After stirring overnight, water was added and the solvent was
removed in vacuo. The crude was dissolved in CH2Cl2, washed with HCl 1M, aq. sol. of. NaHCO3
and brine. The organic layer was dried over Na2SO4, filtered and evaporated. The resulting
residue was purified by chromatography column on silica gel (ether:cyclohexane 1:1) to give
the corresponding tosylate derivative (1.81 g, 4.23 mmol, 93%) as a colorless oil. To a solution
of this compound (301 mg, 0.70 mmol) in DMF (6 mL), NaN3 (114 mg, 1.75 mmol) was added.
After stirring at 70 oC for 3 h the solvent was removed in vacuo and the crude was dissolved in
CH2Cl2, washed with water and brine, dried over Na2SO4, filtered and evaporated. The resulting
residue was purified by chromatography column on silica gel (ether:cyclohexane 1:2) to give 13
ꢀ ꢂꢄꢅ
ꢀ ꢂꢄꢅ
(200.1 mg, 0.67 mmol, 96%) as a white solid. ꢁ
+ 38.0 (c 0.57, CH2Cl2) (Lit: ꢁ
+ 39.0 (c
ꢃ
ꢃ
1.0, CHCl3)). IR (ν cm-1) 2970, 2101 (N3), 1687 (C=O), 1408, 1121. 1H-NMR (300 MHz, DMSO-d6,
363 K, δ ppm, J Hz) δ 4.74 (ap. td, 1H, H-4), 4.54 (dd, 1H, J3,4 = 5.7, J3,2 = 0.3, H-3), 4.00 (ap. t,
2
1H, H-2), 3.67 (dd, 1H, J5a,5b = 12.8, J5a,4 = 0.8, H-5a), 3.50 (dd, 1H, J1’a,1’b = 12.6, J1’a,2 = 6.0, H-
1’a), 3.44 (dd, 1H, J1’b,2 = 6.0, H-1’b), 3.39 (dd, 1H, J5b,4 = 5.0, H-5b), 1.43 (s, 9H, -C(CH3)3), 1.37
13
(ap. s, 3H, -C(CH3)2), 1.27 (ap. s, 3H, -C(CH3)2). C-NMR (75.4 MHz, DMSO-d6, 363 K, δ ppm) δ
153.1 (C=O, Boc), 110.5 (-C(CH3)2), 81.9 (C-3), 78.8 (-C(CH3)3), 78.2 (C-4), 62.4 (C-2), 51.2 (C-5),
50.4 (C-1’), 27.6 (-C(CH3)3), 26.4 (-C(CH3)2), 24.5 (-C(CH3)2). HRESIMS m/z found 321.1531, calc.
for C13H22N4O4 [M+Na]+: 321.1533.
(2S,3S,4R)-2-Azidomethyl-pyrrolidine-3,4-diol (14). A solution of 13 (205 mg, 0.69 mmol) in HCl
(1M):THF 1:1 (17 mL), was stirred at r.t. overnight. Evaporation of the solvent and
chromatographic purification on Dowex 50WX8 eluting with MeOH, H2O and NH4OH 25%,

ACCEPTED MANUSCRIPT
ꢀ ꢂꢄꢆ
afforded 14 (99 mg, 0.63 mmol, 91%) as a crystalline white solid. ꢁ
- 44.1 (c 1.09, MeOH).
ꢃ
IR (ν cm-1) 3495, 3302 (OH, NH), 2101 (N3), 1263, 945. 1H-NMR (300 MHz, MeOD, δ ppm, J Hz)
δ 4.04 (td, 1H, J4,3 = J4,5a = 5.1, J4,5b = 3.3, H-4), 3.72 (dd, 1H, J3,2 = 7.5, H-3), 3.58 (dd, 1H, 2J1’a,1’b
=
2
12.5, J1’a,2 = 4.1, H-1’a), 3.40 (dd, 1H, J1’b,2 = 6.8, H-1’b), 3.14 (dd, 1H, J5a,5b = 12.0, H-5a), 3.11-
3.08 (m, 1H, H-2), 2.83 (dd, 1H, H-5b). 13C-NMR (75.4 MHz, MeOD, δ ppm) δ 75.6 (C-3), 72.5 (C-
4), 62.4 (C-2), 54.4 (C-1’), 52.4 (C-5). HRESIMS m/z found 159.0874, calc. for C5H11N4O2 [M+H]+:
159.0877.
(2S,3S,4R)-2-[(4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol (2). To a solution of
14 (66 mg, 0.42 mmol) in tBuOH:H2O 2:1 (14 mL), phenylacetylene (57 µL, 0.50 mmol), sodium
ascorbate (9 mg, 0.05 mmol) and CuSO4 (2 mg, 0.01 mmol) were added. After stirring
overnight at r.t. the solvent was evaporated and the resulting residue was purified by
chromatography column on silica gel (CH2Cl2:MeOH 10:1 → 5:1, 0.5% Et3N) to give 2 (102 mg,
-1
ꢀ ꢂꢄꢅ
0.39 mmol, 94%) as a white solid. ꢁ
- 33.9 (c 1.37, MeOH). IR (ν cm ) 3283, 3128 (OH, NH),
ꢃ
2923, 1424, 1109, 765, 692. 1H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 8.35 (s, 1H, H-5’’), 7.83-
7.80 (m, 2H, H-aromat.), 7.46-7.40 (m, 2H, H-aromat), 7.37-7.32 (m, 1H, H-aromat), 4.69 (dd,
1H, 2J1’a,1’b = 14.1, J1’a,2 = 4.2, H-1’a), 4.47 (dd, 1H, J1’b,2 = 8.1, H-1’b), 4.05 (td, 1H, J4,3 = J4,5a = 4.8,
J4,5b = 2.9, H-4), 3.79 (dd, 1H, J3,2 = 7.8, H-3), 3.53 (ap. td, 1H, H-2), 3.15 (dd, 1H, 2J5a,5b = 12.0, H-
5a), 2.88 (dd, 1H, H-5b). 13C-NMR (75.4 MHz, MeOD, δ ppm) δ 148.8 (C4’’), 131.7 (Cq aromat),
130.0 (C aromat.), 129.4 (C aromat.), 126.7 (C aromat.), 123.0 (C-5’’), 76.1 (C-3), 72.1 (C-4),
62.5 (C-2), 53.7 (C-1’), 52.3 (C-5). HRESIMS m/z found 261.1342, calc. for C13H17N4O2 [M+H]+:
261.1346.
(2S, 3S, 4R)-N-tert-Butoxycarbonyl-2-aminomethyl-3,4-O-isopropylidene-pirrolidine-3,4-diol
(15). To a solution of 13 (88 mg, 0.30 mmol) in MeOH (2.1 mL), Pd/C (10%, cat.) was added and
the reaction mixture was stirred under an atmosphere of hydrogen for 1.5 h. The crude was
filtered through celite and the solvent removed in vacuo. The resulting residue was purified by
chromatography column on silica gel (CH2Cl2:MeOH 40:1, 1% Et3N) to give 15 (77 mg, 0.28
+ 36.8 (c 0.72, CH2Cl2). IR (ν cm-1) 3352, 2977 (OH, NH),
1
ꢀ ꢂꢄꢅ
mmol, 95%) as a colorless oil. ꢁ
ꢃ
1696, 1671, 1402, 1158, 1036, 852. H-NMR (300 MHz, DMSO-d6, 363 K, δ ppm, J Hz) δ 4.70
(ap. td, 1H, H-4), 4.61 (d, 1H, J3,4 = 6.0, H-3), 3.80 (ap. t, 1H, H-2), 3.60 (dd, 1H, 2J5a,5b = 12.8, J5a,4
= 0.8, H-5a), 3.36 (dd, 1H, J5b,4 = 5.1, H-5b), 2.66 (dd, 1H, J1’a,1’b = 12.8, J1’a,2 = 5.3, H-1’a), 2.60
(dd, 1H, J1’b,2 = 6.8, H-1’b), 1.42 (s, 9H, -C(CH3)3), 1.35 (s, 3H, -C(CH3)2), 1.26 (s, 3H, -C(CH3)2). 13C-
NMR (75.4 MHz, DMSO-d6, 363 K, δ ppm) δ 153.4 (C=O, Boc), 110.0 (-C(CH3)2), 82.0 (C-3), 78.3
(C-4), 78.1 (-C(CH3)3), 65.3 (C-2), 51.5 (C-5), 41.6 (C-1’), 27.7 (-C(CH3)3), 26.4 (-C(CH3)2), 24.6 (-
C(CH3)2). HRESIMS m/z found 273.1811, calc. for C15H25N2O4 [M+H]+: 273.1809.
(2S, 3S, 4R)-2-Benzamidomethyl-pyrrolidine-3,4-diol (5). To a solution of 15 (251 mg, 0.92
o
mmol) in anhydrous CH2Cl2 (10 mL) at 0 C, benzoyl chloride (220 mL, 1.85 mmol) and Et3N
(270 mL, 1.85 mmol) were added. After stirring at r.t. for 2 h, the solution was washed with aq.
sol. of. NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and
evaported. The resulting residue was purified by chromatography column on silica gel
(EtOAc:cyclohexane 1:2 → EtOAc:cyclohexane 1:1) to give the corresponding protected
pyrrolidine-amide (356 mg, 0.94 mmol, quant.) as a white solid. A solution of this compound
(278 mg, 0.74 mmol) in HCl (4M):THF (1:1, 18 mL) was stirred at r.t. for 3 h. Evaporation of the
solvent and chromatographic purification on Dowex 50WX8 eluting with MeOH, H2O and

ACCEPTED MANUSCRIPT
ꢀ ꢂꢄꢆ
NH4OH 25%, afforded 5 (171 mg, 0.72 mmol, 98%) as a whitish solid. ꢁ
- 37.8 (c 0.65,
ꢃ
MeOH). IR (ν cm-1) 3290, 3061 (OH, NH), 1635 (C=O), 1541, 1400, 1099, 807, 690. 1H-NMR (300
MHz, MeOD, δ ppm, J Hz) δ 7.86-7.82 (m, 2H, H-aromat.), 7.54 (ap. tt, 1H, H-aromat), 7.49-7.43
(m, 2H, H-aromat), 4.08 (ap. td, 1H, H-4), 3.77 (dd, 1H, J3,2 = 6.9, J3,4 = 5.1, H-3), 3.61 (dd, 1H,
2J1’a,1’b = 13.7, J1’a,2 = 5.3, H-1’a), 3.49 (dd, 1H, J1’b,2 = 7.2, H-1’b), 3.24-3.20 (m, 1H, H-2), 3.17 (dd,
1H, 2J5a,5b = 11.9, J5a,4 = 5.3, H-5a), 2.84 (dd, 1H, J5b,4 = 3.6, H-5b). 13C-NMR (75.4 MHz, MeOD, δ
ppm) δ 170.8 (C=O), 135.4 (Cq aromat), 132.8 (C aromat.), 129.6 (C aromat.), 128.3 (C
aromat.), 76.2 (C-3), 72.3 (C-4), 63.1 (C-2), 52.0 (C-5), 43.2 (C-1’). HRESIMS m/z obsd. 237.1231,
calc. for C12H17N2O3 [M+H]+: 237.1234.
(2S, 3S, 4R)-2-Phenylthioureidomethyl-pyrrolidine-3,4-diol hydrochloride (6). To a solution of 15
(76 mg, 0.28 mmol) in anhydrous CH2Cl2 (5 mL), phenyl isothiocyanate (44 mL, 0.36 mmol) was
added. After stirring at r.t. for 6 h, the solvent was removed under vacuo and the resulting
residue was purified by chromatography column on silica gel (EtOAc:cyclohexane 1:2) to give
the corresponding protected pyrrolidine-thiourea (120 mg, 0.29 mmol, quant.) as a colorless
oil. A solution of this compound (63 mg, 0.15 mmol) in HCl (4M):THF (1:1, 3.8 mL) was stirred
ꢀ ꢂꢄꢆ
at r.t. for 3 h, and evaporated to give 6 (45 mg, 0.15 mmol, quant.) as a colorless oil. ꢁ
+
ꢃ
1
2.1 (c 0.61, MeOH). IR (ν cm-1) 3240, 3045 (OH, NH), 2359, 1539, 1313, 1136, 1040, 696. H-
NMR (300 MHz, MeOD, δ ppm, J Hz) δ 7.50-7.39 (m, 2H, H-aromat), 7.35-7.33 (m, 2H, H-
aromat), 7.29-7.23 (m, 1H, H-aromat.), 4.31-4.25 (m, 1H, H-4), 4.14-4.06 (m, 2H, H-3, H-1’a),
2
2
3.98 (dd, 1H, J1’b,1’a = 15.0, J1’b,2 = 3.9, H-1’b), 3.73 (ap. td, 1H, H-2), 3.50 (dd, 1H, J5a,5b = 12.5,
13
J5a,4 = 3.8, H-5a), 3.25 (dd, 1H, J5b,4 = 1.5, H-5b). C-NMR (75.4 MHz, MeOD, δ ppm) δ 183.6
(C=S), 139.0 (Cq aromat), 130.4 (C aromat.), 127.4 (C aromat.), 125.9 (C aromat.), 74.4 (C-3),
70.8 (C-4), 63.7 (C-2), 50.7 (C-5), 44.5 (C-1’). HRESIMS m/z found 268.1117, calc. for
C12H18N3O2S [M]+: 268.1114.
(2R,3S,4R)-N-Benzyl-2-hydroxymethyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol (17).27 To a
solution of 1628 (13.44 g, 26.8 mmol) in THF (185 mL), TBAF (30 mL, 29.5 mmol) was added.
After stirring at r.t. for 2.5 d, the solvent was removed and the resulting residue was purified
by chromatography column on silica gel (ether:cyclohexane 1:1→2:1) to give 17 (6.46 g, 24.5
1
ꢀ ꢂꢄꢆ
ꢀ ꢂꢄꢉ
mmol, 92%) as a colorless oil. ꢁ
- 81.8 (c 0.57, CH2Cl2) (Lit: ꢁ
– 80.3 (c 1.3, CHCl3)). H-
ꢃ
ꢃ
NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 7.29-7.18 (m, 5H, H-aromat.), 4.67 (dd, 1H, J = 6.3, J =
4.8), 4.55 (dd, 1H, J = 4.7, J = 6.5), 4.01 (d, 1H, J = 13.2), 3.91 (d, 1H, J = 1.8), 3.90 (s, 1H), 3.20
(d, 1H, J = 13.2), 3.04 (d, 1H, J = 11.1), 2.56 (bs, 1H, OH), 2.34 (ap. q, 1H), 2.09 (dd, 1H, J = 11.1,
J = 4.5), 1.51 (s, 3H, -C(CH3)2), 1.28 (s, 3H, -C(CH3)2).
(2R,3S,4R)-N-tert-Butoxycarbonyl-2-hydroxymethyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol
(18).17 To a solution of 17 (6.43 g, 24.4 mmol) in MeOH (180 mL), Boc2O (10.88 g, 48.8 mmol)
and Pd/C (10%, cat.) were added and the reaction mixture was stirred under hydrogen for 4 h.
The crude was filtered through celite and the solvent removed in vacuo. The resulting residue
was purified by chromatography column on silica gel (EtOAc:cyclohexane 1:2 → 1:1) to give 18
ꢀ ꢂꢄꢆ
ꢀ ꢂꢄꢆ
(5.73 g, 21.0 mmol, 86%) as a colorless oil. ꢁ
- 40.1 (c 0.58, CH2Cl2) (Lit: ꢁ
– 33.8 (c 2.1,
ꢃ
ꢃ
1
CHCl3)). H-NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 4.74 (ap. d, 3H), 3.85-3.79 (m, 3H), 3.52 (bs,
2H), 1.48 (s, 3H, -C(CH3)2), 1.44 (s, 9H, -C(CH3)3), 1.31 (s, 3H, -C(CH3)2).

ACCEPTED MANUSCRIPT
(2S,3S,4R)-N-tert-Butoxycarbonyl-2-formyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol (19).17 To a
o
solution of oxalyl chloride (510 µL, 5.86 mmol) in anhydrous CH2Cl2 (6 mL) at -78 C, DMSO
o
(840 µL, 11.7 mmol) in CH2Cl2 (3 mL) was added and the solution was stirred at -78 C for 45
min. A solution of 18 (1.07 g, 3.91 mmol) in CH2Cl2 (6 mL) was then added and the reaction
o
mixture was stirred at -78 C for 2.5 h. Finally, Et3N (3.0 mL, 21 mmol) was added and the
o
mixture stirred at -78 C for 30 min. and allowed to reach r.t. The reaction was diluted with
CH2Cl2 and washed with HCl 1M, sat. aq. sol. of NaHCO3 and brine. The organic layer was dried
over Na2SO4, filtered and evaporated. The resulting residue was purified by chromatography
column on silica gel (EtOAc:cyclohexane 1:2) to give 19 (948 mg, 3.49 mmol, 89%) as a white
1
ꢀ ꢂꢄꢆ
ꢀ ꢂꢄꢇ
solid. ꢁ
- 94.6 (c 0.68, CH2Cl2) (Lit: ꢁ
- 51.6 (c 3.06, CHCl3)). H-NMR (300 MHz, CDCl3, δ
ꢃ
ꢃ
ppm, J Hz) δ 9.42 (bs, 1H, CHO), 4.97 (t, 1H, J = 6.3), 4.81 (td, 1H, J = 5.7, J = 1.8), 4.08-4.04 (m,
1H), 3.80-3.76 (m, 1H), 3.63-3.60 (m, 1H), 1.46 (s, 3H, -C(CH3)2), 1.41 (s, 9H, -C(CH3)3), 1.29 (s,
3H, -C(CH3)2).
(2R,3S,4R)-N-tert-Butoxycarbonyl-2-ethynyl-3,4-O-isopropylidene-pyrrolidine-3,4-diol (20). To a
suspension of 4-acetamidobenzenosulfonyl azide (587 mg, 2.37 mmol) and K2CO3 (1.16 g, 8.29
o
mmol) in anhydrous CH3CN (18 mL) at 0 C, dimethyl 2-oxopropylphosphonate (324 µL, 2.22
mmol) was added. After stirring at r.t. for 6 h, a solution of 19 (402 mg, 1.48 mmol) in
anhydrous MeOH (18 mL) was added and the mixture was stirred at r.t. overnight. The mixture
was filtered through celite and the solvent removed in vacuo. The resulting residue was
purified by chromatography column on silica gel (EtOAc:cyclohexane 1:5) to give 20 (214 mg,
-1
ꢀ ꢂꢄꢆ
0.80 mmol, 54%) as a colored liquid. ꢁ
+ 104.3 (c 0.68, CH2Cl2). IR (ν cm ) 3220 (
),
ꢃ
1
2978, 2938, 2115 (
), 1696 (C=O), 1392, 1157, 881. H-NMR (300 MHz, CDCl3, δ ppm, J
Hz) δ 4.78-4.60 (m, 3H, H-4, H-3, H-2), 3.86-3.79 (m, 1H, H-5a), 3.39 (dd, 1H, 2J5b,5a = 12.8, J5b,4
=
4.7, H-5b), 2.29 (d, 1H, J2’,2 = 2.4, H-2’), 1.47 (s, 9H, -C(CH3)3), 1. 42 (s, 3H, -C(CH3)2), 1.29 (s, 3H, -
C(CH3)2). 13C-NMR (75.4 MHz, CDCl3, δ ppm) δ 154.4 (C=O, Boc), 112.2 (-C(CH3)2), 85.6, 85.11 (C-
3), 80.6 (-C(CH3)3), 80.1 (C-1’), 79.7, 78.81 (C-4), 72.8 (C-2’), 55.0, 54.51 (C-2), 51.4, 50.91 (C-5),
28.5 (-C(CH3)3), 26.7 (-C(CH3)2), 25.0 (-C(CH3)2). HRESIMS m/z found 290.1364, calc. for
C14H21NO4 [M+Na]+: 290.1363.
(2R,3S,4R)-2-[(1-Benzyl)-1H-1,2,3-triazol-4-yl]-pyrrolidine-3,4-diol (3). To a solution of 20 (81
mg, 0.30 mmol) in toluene (2.8 mL), benzyl azide (80 mg, 0.60 mmol), DIPEA (200 µL, 1.15
mmol) and CuI (18 mg, 0.09 mmol) were added. After stirring at r.t. for 6 h, aq. sat. sol. of
NaHCO3 was added and the aqueous phase was extracted with EtOAc. The organic layers were
dried over Na2SO4, filtered and evaporated. The resulting residue was purified by
chromatography column on silica gel (EtOAc:cyclohexane 1:2) to give the corresponding
protected pyrrolidine-triazole (116 mg, 0.29 mmol, 96%) as a white solid. A solution of this
compound (72 mg, 0.18 mmol) in HCl (4M):THF (1:1, 4.4 mL) was stirred at r.t. for 3 h.
Evaporation of the solvent and chromatographic purification on Dowex 50WX8 eluting with
MeOH, H2O and NH4OH 25%, afforded 3 (43 mg, 0.16 mmol, 91%) as a crystalline white solid.
1
- 61.2 (c 0.62, MeOH). IR (ν cm-1) 3280, 3133 (OH, NH), 1441, 1128, 917, 720. H-NMR
ꢀ ꢂꢄꢅ
ꢁ
ꢃ
(300 MHz, MeOD, δ ppm, J Hz) δ 7.91 (s, 1H, H-5’), 7.40-7.30 (m, 5H, H-aromat.), 5.58 (s, 2H, H-
1’’), 4.19 (ap. td, 1H, H-4), 4.15 (d, 1H, J2,3 = 7.8, H-2), 4.09 (dd, 1H, J3,4 = 5.0, H-3), 3.37 (dd, 1H,
13
2J5a,5b = 12.2, J5a,4 = 5.3, H-5a), 2.91 (dd, 1H, J5b,4 = 3.2, H-5b). C-NMR (75.4 MHz, MeOD, δ
ppm) δ 149.0 (C-4’), 136.8 (Cq aromat), 130.0 (C aromat.), 129.6 (C aromat.), 129.2 (C aromat.),

ACCEPTED MANUSCRIPT
123.9 (C-5’), 78.8 (C-3), 72.3 (C-4), 59.0 (C-2), 54.9 (C-1’’), 52.7 (C-5). HRESIMS m/z found
261.1347, calc. for C13H17N4O2 [M+H]+: 261.1346.
(5S,6R,7S,7aR)-6,7-O-isopropylidene-tetrahydropirrolo[1,2-c]-oxazol-3-one-6,7-diol (21).17 To a
solution of 18 (704 mg, 2.58 mmol) in anhydrous piridine (10 mL) at 0 °C, TsCl (1.24 g, 6.44
mmol) was added. After stirring at 0 oC to r.t. overnight, the solvent was removed in vacuo and
the resulting residue was purified by chromatography column on silica gel (EtOAc:cyclohexane
ꢀ ꢂꢄꢅ
1:1 → 2:1) to give 21 (473 mg, 2.37 mmol, 92%) as a white solid. ꢁ
- 19.8 (c 0.79, CH2Cl2)
ꢃ
1
ꢀ ꢂꢄꢇ
(Lit: ꢁ
– 4.92 (c 2.40, CHCl3)). H-NMR (300 MHz, CDCl3, δ ppm, J Hz) δ 4.81 (dd, 1H, J = 5.1,
ꢃ
J = 4.5), 4.59 (ap. t, 1H), 4.54 (dd, 1H, J = 8.7, J = 2.4), 4.41 (ap. t, 1H), 3.98 (d, 1H J = 13.5), 3.86
(ap. ddd, 1H), 3.09 (dd, 1H, J = 13.5, J = 4.2), 1.44 (s, 3H, -C(CH3)2), 1.31 (s, 3H, -C(CH3)2).
(2R,3S,4R)-2-Azidomethyl-pyrrolidine-3,4-diol (22). A solution of 21 (71 mg, 0.36 mmol) and
Bu4NN3 (708 mg, 2.49 mmol) in THF (7 mL) was heated at 100 oC in a sealed tube for 14 d. The
solvent was evaporated and the residue was purified by chromatography column on silica gel
(ether→ether:MeOH 30:1, 0.5% Et3N) to give the (2R,3S,4R)-2-azidomethyl-3,4-O-
isopropylidene-pyrrolidine-3,4-diol (42 mg, 0.21 mmol, 60%) as a yellowish oil. A solution of
this compound (101 mg, 0.51 mmol) in HCl (1M):THF 1:1 (12 mL), was stirred at r.t. overnight.
Evaporation of the solvent and chromatographic purification on Dowex 50WX8 eluting with
ꢀ ꢂꢄꢅ
MeOH, H2O and NH4OH 25%, afforded 22 (64 mg, 0.40 mmol, 80%) as a yellowish oil. ꢁ
+
ꢃ
5.6 (c 1.33, MeOH). IR (ν cm-1) 3323 (OH, NH), 2098 (N3), 1267, 1121. H-NMR (300 MHz,
1
MeOD, δ ppm, J Hz) δ 4.18 (td, 1H, J4,5a = J4,5b = 6.8, J4,3 = 4.6, H-4), 4.03 (t, 1H, J3,2 = 4.6, H-3),
2
3.62 (dd, 1H, J1’a,1’b = 12.2, J1’a,2 = 6.2, H-1’a), 3.36 (dd, 1H, J1’b,2 = 8.1, H-1’b), 3.19 (ddd, 1H, H-
2), 3.03 (dd, 1H, 2J5a,5b = 11.1, H-5a), 2.81 (dd, 1H, H-5b). 13C-NMR (75.4 MHz, MeOD, δ ppm) δ
73.7 (C-4), 72.9 (C-3), 61.2 (C-2), 52.4 (C-1’), 51.2 (C-5). HRESIMS m/z found 159.0875, calc. for
C5H11N4O2 [M+H]+: 159.0877.
(2R,3S,4R)-2-[(4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol (4). To a solution of
t
22 (32 mg, 0.20 mmol) in BuOH:H2O 2:1 (8 mL), phenylacetylenene (27 µL, 0.24 mmol),
sodium ascorbate (9 mg, 0.04 mmol) and CuSO4 (2 mg, 0.01 mmol) were added and the
solution was stirred at r.t. for 1 d. The solvent was evaporated and the resulting residue was
purified by chromatography column on silica gel (CH2Cl2:MeOH:NH4OH 6:1:0.1) to give 4 (45
ꢀ ꢂꢄꢅ
mg, 0.17 mmol, 85%) as a whitish solid. ꢁ
+ 15.5 (c 0.68, MeOH). IR (ν cm-1) 3254, 3130
(OH, NH), 1467, 1318, 1224, 1091, 766, 689. 1H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 8.33 (s,
ꢃ
1H, H-5’’), 7.83-7.79 (m, 2H, H-aromat.), 7.46-7.40 (m, 2H, H-aromat), 7.33 (ap. tt, H-aromat.),
4.75 (dd, 1H, 2J1’a,1’b = 13.8, J1’a,2 = 5.7, H-1’a), 4.50 (dd, 1H, J1’b,2 = 8.1, H-1’b), 4.21 (td, 1H, J4,5a
=
J4,5b = 6.6, J4,3 = 4.6, H-4), 4.08 (t, 1H, J3,2 = 4.7, H-3), 3.68-3.62 (m, 1H, H-2), 3.08 (dd, 1H, 2J5a,5b
= 11.3, H-5a), 2.90 (dd, 1H, H-5b). 13C-NMR (75.4 MHz, MeOD, δ ppm) δ 148.7 (C-4’’), 131.7 (Cq
aromat), 130.0 (C aromat.), 129.4 (C aromat.), 126.7 (C aromat.), 123.1 (C-5’’), 73.4 (C-4), 72.9
(C-3), 61.7 (C-2), 51.7 (C-1’), 51.0 (C-5). HRESIMS m/z found 261.1349, calc. for C13H17N4O2
[M+H]+: 261.1346.
(2R,3S,4R)-2-[(4-Phenyl)-1H-1,2,3-triazol-1-yl)ethyl)]-pyrrolidine-3,4-diol (7). To a solution of
2318 (94 mg, 0.27 mmol) in toluene (2.5 mL), phenylacetylene (122 µL, 1.08 mmol), DIPEA (180
o
µL, 1.03 mmol) and CuI (17 mg, 0.08 mmol) were added. After stirring at 50 C for 3.5 d., aq.
sat. sol. of NaHCO3 (15 mL) was added and the aqueous phase was extracted with EtOAc. The

ACCEPTED MANUSCRIPT
organic layers were dried, filtered and evaporated. The resulting residue was purified by
chromatography column on silica gel (AcOEt:cyclohexane 1:2) to give the corresponding
protected pyrrolidine-triazol (111 mg, 0.25 mmol, 91%) as a white solid. A solution of this
compound (75 mg, 0.17 mmol) in HCl (1M):THF (1:1, 6.8 mL) was stirred at r.t. for 2.5 h and
then evaporated. Chromatographic purification on silica gel (CH2Cl2:MeOH, 50:1 → 20:1)
afforded the 3,4-O-unprotected derivative (43 mg, 0.10 mmol). A solution of this compound in
MeOH (2 mL) was hydrogenated in the presence of Pd/C (10%) and HCl (5M) (73 μL) for 2 h.
Filtration through celite, evaporation and chromatographic purification on Dowex 50WX8
eluting with MeOH, H2O and NH4OH 25%, afforded 7 (7 mg, 0.03 mmol, 30%) as a white solid.
-1
1
ꢀ ꢂꢄꢈ
ꢁ
+ 66.9 (c 0.65, MeOH). IR (ν cm ) 3268 (OH, NH), 1426, 1219, 1044, 762, 690. H-NMR
ꢃ
(300 MHz, MeOD, δ ppm, J Hz) δ 8.35 (s, 1H, H-5’’), 7.81 (ap. d, 2H, H-aromat.), 7.43 (ap. t, 2H,
H-aromat.), 7.38-7.30 (m, 1H, H-aromat.), 4.63-4.49 (m, 2H, H-2’), 4.22 (ap. td, 1H, H-4), 3.99
2
(t, 1H, J3,4 = J3,2 = 4.4, H-3), 3.05 (dd, 1H, J5a,5b = 11.4, J5a,4 = 7.2, H-5a), 3.00-2.95 (m, 1H, H-2),
2.86 (dd, 1H, J5b,4 = 6.5, H-5b), 2.41-2.29 (m, 1H, H-1’a), 2.21-2.10 (m, 1H, H-1’b). 13C-NMR (75.4
MHz, MeOD, δ ppm) δ 148.9 (C-4’’), 131.7 (Cq aromat.), 130.0 (C aromat.), 129.4 (C aromat.),
126.7 (C aromat.), 122.3 (C-5’’), 73.8 (C-4), 73.1 (C-3), 59.6 (C-2), 51.4 (C-5), 49.2 (C-2’), 31.4 (C-
1’). HRESIMS m/z found 275.1498, calc. for C14H19N4O2 [M+H]+: 275.1503.
(2S,3S,4R)-2-[(4-((Trimethylsilyl)methyl)-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol (14f).
t
To a solution of 14 (21 mg, 0.13 mmol) in BuOH:H2O 2:1 (4.5 mL), trimethyl(propargyl)silane
(54 µL, 0.33 mmol), sodium ascorbate (6 mg, 0.03 mmol) and CuSO4 (1.4 mg, 0.01 mmol) were
added. After stirring 40 h. at r.t. the solvent was evaporated and the resulting residue was
purified by chromatography column on silica gel (CH2Cl2:MeOH 5:1, 0.5% Et3N) to give 14f (24
-1
ꢀ ꢂꢄꢆ
mg, 0.09 mmol, 67%) as a yellowish oil. ꢁ
- 35.9 (c 0.86, MeOH). IR (ν cm ) 3248 (OH, NH),
ꢃ
1
2954, 1542, 1428, 1248, 1047, 840. H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 7.63 (s, 1H, H-
2
5’’), 4.57 (dd, 1H, J1’a,1’b = 14.0, J1’a,2 = 4.1, H-1’a), 4.38 (dd, 1H, J1’b,2 = 7.8, H-1’b), 4.01 (td, 1H,
J4,5a = J4,3 = 4.8, J4,5b = 3.0, H-4), 3.72 (dd, 1H, J3,2 = 7.5, H-3), 3.45 (td, 1H, H-2), 3.11 (dd, 1H,
2J5a,5b = 12.0, H-5a), 2.86 (dd, 1H, H-5b), 2.11 (ap. s, 2H, -CH2Si(CH3)3), 0.04 (ap. s, 9H, (-Si(CH3)3).
13C-NMR (75.4 MHz, MeOD, δ ppm) δ 146.6 (C-4’’), 123.0 (C-5’’), 76.1 (C-3), 72.3 (C-4), 62.7 (C-
2), 53.7 (C-1’), 52.2 (C-5), 15.7 (-CH2Si(CH3)3), -1.8 (-Si(CH3)3). HRESIMS m/z found 271.1579,
calc. for C11H23N4O2Si [M+H]+: 271.1585.
(2R,3S,4R)-2-[(4-(6-Methoxynaphthalen-2-yl)-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol
t
(22k). To a solution of 22 (28 mg, 0.18 mmol) in BuOH:H2O 2:1 (7.2 mL), 2-ethynyl-6-
methoxynaphthalene (41 mg, 0.22 mmol), sodium ascorbate (8 mg, 0.04 mmol) and CuSO4 (2
mg, 0.01 mmol) were added and the solution was stirred at r.t. for 1 d. The solvent was
evaporated and the resulting residue was purified by chromatography column on silica gel
(CH2Cl2:MeOH:NH4OH 7:1:0.1 → 6:1:0.1) to give 22k (46 mg, 0.14 mmol, 76%) as a whitish
-1
ꢀ ꢂꢄꢆ
solid. ꢁ
+ 28.6 (c 0.60, MeOH). IR (ν cm ) 3253, 2919, (OH, NH), 1614, 1482, 1210, 1023,
ꢃ
1
853, 819. H-NMR (300 MHz, DMSO-d6, δ ppm, J Hz) δ 8.56 (s, 1H, H-5’’), 8.31 (bs, 1H, H-
aromat.), 7.95 (dd, 1H, J = 8.6, J = 1.7, H-aromat.), 7.88 (ap. d, 2H, H-aromat.), 7.33 (d, 1H, J =
2.3, H-aromat.), 7.18 (dd, 1H, J = 9.0, J = 2.4, H-aromat), 4.81 (bs, 1H, OH), 4.75 (bs, 1H, OH),
4.56 (dd, 1H, 2J1’a,1’b = 13.8, J1’a,2 = 4.8, H-1’a), 4.32 (dd, 1H, J1’b,2 = 8.9, H-1’b), 4.03-3.93 (m, 2H,
2
H-4, H-3), 3.88 (s, 3H, -OCH3), 3.54-3.47 (m, 1H, H-2), 3.32 (bs, 1H, NH), 2.96 (dd, 1H, J5a,5b
=
13
10.8, J5a,4 = 6.6, H-5a), 2.70 (dd, 1H, J5b,4 = 6.3, H-5b). C-NMR (75.4 MHz, DMSO-d6, δ ppm) δ

ACCEPTED MANUSCRIPT
157.4 (Cq aromat.), 146.1 (C-4’’), 133.8 (Cq aromat), 129.5 (C aromat.), 128.6 (Cq aromat.),
127.3 (C aromat.), 126.3 (Cq aromat.), 124.1 (C aromat.), 123.3 (C aromat.), 121.8 (C-5’’), 119.1
(C aromat.), 106.0 (C aromat.), 72.2 (C-4), 71.8 (C-3), 59.9 (C-2), 55.2 (-OCH3), 51.3 (C-1’), 50.4
(C-5). HRESIMS m/z found 341.1603, calc. for C18H21N4O3 [M+H]+: 341.1608.
(2R, 3S, 4R)-2-[(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol (22p).
The synthesis of this compound was carried out following the procedure of 22k, except that 4-
methoxyphenylacetylene was used as alkyne. The resulting residue was purified by
chromatography column on silica gel (CH2Cl2:MeOH:NH4OH 6:1:0.1) to give 22p (50 mg, 0.17
-1
ꢀ ꢂꢄꢅ
mmol, 89%) as a whitish solid. ꢁ
+ 11.8 (c 0.82, MeOH). IR (ν cm ) 3254, 2924, (OH, NH),
ꢃ
1558, 1496, 1245, 1028, 835, 799. 1H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 8.22 (s, 1H, H-5’’),
2
7.75-7.70 (m, 2H, H-aromat.), 7.01-6.96 (m, 2H, H-aromat.), 4.73 (dd, 1H, J1’a,1’b = 13.8, J1’a,2
=
5.7, H-1’a), 4.49 (dd, 1H, J1’b,2 = 8.3, H-1’b), 4.21 (td, 1H, J4,5a = J4,5b = 6.7, J4,3 = 4.5, H-4), 4.08 (t,
1H, J4,3 = 4.6, H-3), 3.83 (s, 3H, -OCH3), 3.69-3.62 (m, 1H, H-2), 3.08 (dd, 1H, 2J5a,5b = 11.3, H-5a),
13
2.90 (dd, 1H, H-5b). C-NMR (75.4 MHz, MeOD, δ ppm) δ 161.3 (Cq aromat.), 148.7 (C-4’’),
128.0 (C aromat.), 124.3 (Cq aromat.), 122.3 (C-5’’), 115.4 (C aromat.), 73.6 (C-4), 73.0 (C-3),
61.7 (C-2), 55.8 (-OCH3), 51.9 (C-1’), 51.2 (C-5). HRESIMS m/z found 291.1453, calc. for
C14H19N4O3 [M+H]+: 291.1452.
(2R, 3S, 4R)-2-[(4-(4-Hydroxymethylphenyl)-1H-1,2,3-triazol-1-yl)methyl)]-pyrrolidine-3,4-diol
(22r). The synthesis of this compound was carried out following the procedure of 22k, except
that 4-hydroxymethylphenylacetylene was used as alkyne. The resulting residue was purified
by chromatography column on silica gel (CH2Cl2:MeOH:NH4OH 5:1:0.1 → 4:1:0.1) to give 22r
-1
ꢀ ꢂꢄꢆ
(42 mg, 0.14 mmol, 76%) as a whitish solid. ꢁ + 17.8 (c 0.82, MeOH). IR (ν cm ) 3408, 3245,
ꢃ
1
2923 (OH, NH), 1457, 1110, 1016, 972, 797. H-NMR (300 MHz, MeOD, δ ppm, J Hz) δ 8.33 (s,
2
1H, H-5’’), 7.80 (d, 2H, J = 8.4, H-aromat.), 7.43 (d, 2H, H-aromat.), 4.78 (dd, 1H, J1’a,1’b = 14.0,
J1’a,2 = 5.3, H-1’a), 4.63 (s, 2H, -CH2OH), 4.55 (dd, 1H, J1’b,2 = 8.6, H-1’b), 4.24 (td, 1H, J4,5a = J4,5b
=
=
6.7, J4,3 = 4.4, H-4), 4.11 (t, 1H, J3,4 = J3,2 = 4.5, H-3), 3.75-3.70 (m, 1H, H-2), 3.13 (dd, 1H, 2J5a,5b
13
11.4, H-5a), 2.94 (dd, 1H, H-5b). C-NMR (75.4 MHz, MeOD, δ ppm) δ 148.6 (C-4’’), 143.0 (Cq
aromat.), 130.7 (Cq aromat.), 128.5 (C aromat.), 126.7 (C aromat.), 123.0 (C-5’’), 73.5 (C-4),
73.0 (C-3), 64.9 (-CH2OH), 61.7 (C-2), 51.7 (C-1’), 51.1 (C-5). HRESIMS m/z found 291.1454, calc.
for C14H19N4O3 [M+H]+: 291.1452.
Acknowledgments
This work was supported by the Ministerio de Economía y Competitividad of Spain (CTQ2012-
31247 and CTQ2016-77270-R) and the Junta de Andalucía (FQM-345). M. M. B. acknowledges
the Spanish government for a FPU fellowship.
Appendix A. Supplementary data
Supplementary data related to this article can be found at…
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ACCEPTED MANUSCRIPT
Tuning of β-glucosidase and α-galactosidase inhibition by generation and
in situ screening of a library of pyrrolidine-triazole hybrid molecules
Macarena Martínez-Bailén,a Ana T. Carmona,a Elena Moreno-Clavijo,a Inmaculada Robina,a,*
Daisuke Ide,b Atsushi Katob and Antonio J. Moreno-Vargasa,*
aDepartment of Organic Chemistry, Faculty of Chemistry, University of Seville, C/Prof. García González, 1,
b
41012-Seville, Spain. Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194,
Japan. e-Mail: robina@us.es, ajmoreno@us.es
Graphical Abstract
Highlights
Epimeric azidomethyl pyrrolidines were used as scaffolds for glycosidase inhibitors.
Click reaction/in situ screening were combined for the fast discovery of inhibitors.
β-Glucosidase (almond) inhibition correlates with β-glucocerebrosidase inhibition.
No correlation was observed between plant and human α-galactosidase inhibitors.
The new inhibitors incorporate a lipophilic group that could help to cross membranes.