molecules-30-00226

Article
A Bis-Glycosylamine Strategy for the Synthesis of Dimeric

Iminosugars Based on a DAB-1 Scaffold
Kamilia Ould Lamara 1 , Nathan Noël 1 , Fabien Massicot 1 , Jean-Luc Vasse 1 , Stéphane P. Vincent 2
and Jean-Bernard Behr 1, *
1 Université de Reims Champagne-Ardenne, CNRS, ICMR, 51097 Reims, France;

kamilia.ouldlamara@gmail.com (K.O.L.); nathan.noel@univ-reims.fr (N.N.);
fabien.massicot@univ-reims.fr (F.M.); jean-luc.vasse@univ-reims.fr (J.-L.V.)
2 NARILIS (Namur Research Institute for Life Sciences), UNamur, Rue de Bruxelles 61, 5000 Namur, Belgium;
stephane.vincent@unamur.be
* Correspondence: jb.behr@univ-reims.fr
Abstract: A straightforward synthetic route towards DAB-1 scaffolded dimeric iminosugars

is described here, starting from readily available bis-glycosylamines. The method allows
the integration of a variety of linkages (aryl, alkyl, polyethyleneglycol chains) between
both iminosugars through the choice of the bis-amine used in the first step. Moreover, an
additional substituent (allyl, ethynyl) may be inserted into the structure via nucleophilic
addition of an organometallic reagent to the starting bis-glycosylamine. A symmetrical
ethynyl-iminosugar proved susceptible to intramolecular Glaser coupling, affording the
corresponding macrocyclic structure. Dimeric iminosugars were tested towards a series of
commercial glycosidases to uncover potencies and selectivities when compared to DAB-1,
their monomeric counterpart. Whereas a significant drop in inhibition potencies was
observed towards glucosidases, some compounds displayed unexpected potent inhibition
of β-galactosidase.
Keywords: iminosugars; multivalency; glycosylamines; glycosidases; inhibition; glycophanes
Academic Editors: Francesca Cardona

and Macarena Martínez-Bailén
Received: 9 December 2024

1. Introduction
Revised: 4 January 2025 N-glycosides are biomolecules, which originate from the condensation of carbohy-
Accepted: 7 January 2025 drates with amines or related N-nucleophilic species [1]. Nucleosides or glycoproteins
Published: 8 January 2025
belong to this family, for which the newly formed anomeric bond is enzymatically pro-
Citation: Lamara, K.O.; Noël, N.; cessed in biological systems. In such cases, where the N-nucleophile is different from a
Massicot, F.; Vasse, J.-L.; Vincent, S.P.;
primary amine, the anomeric bond is rather stable and tolerates the aqueous conditions of
Behr, J.-B. A Bis-Glycosylamine
biological media. Glycosylamines resulting from reactions of sugars with primary amines,
Strategy for the Synthesis of Dimeric
Iminosugars Based on a DAB-1
however, are much more susceptible to hydrolysis or decomposition [2,3]. Conjugation
Scaffold. Molecules 2025, 30, 226. of carbohydrates with the amino function of aminoacids for instance occurs under heat-
https://doi.org/10.3390/ ing and culminates in the so-called Maillard transformation after subsequent Amadori
molecules30020226 or Heyns rearrangements [4,5]. Nevertheless, for synthetic purposes, such glycosylamines
Copyright: © 2025 by the authors. might be prepared in non-aqueous media and are conventionally used without further
Licensee MDPI, Basel, Switzerland. purification in subsequent steps, leading to highly valuable products such as iminosugars,
This article is an open access article a family of nitrogen-containing carbohydrate analogues [6]. As a relevant example, tri-O-
distributed under the terms and
benzyl-L-xylofuranose 2 might be N-glycosylated with benzylamine in dichloromethane
conditions of the Creative Commons
at room temperature in the presence of molecular sieves as a dehydrating agent to af-
Attribution (CC BY) license
(https://creativecommons.org/
ford the expected anomeric amine 3 (Scheme 1) [7]. As observed with simple reducing
licenses/by/4.0/). carbohydrates, mutarotation in glycosylamines enforces an equilibrium between closed
Molecules 2025, 30, 226 https://doi.org/10.3390/molecules30020226

30, x FOR PEER REVIEW 2 of 21
Molecules 2025, 30, 226 2 of 21
(Scheme 1) [7]. As observed with simple reducing carbohydrates, mutarotation in

glycosylamines enforces an equilibrium between closed alpha/beta isomers as well as the
alpha/beta isomers as well as the open imine form. Reduction [8–12] or nucleophilic
open imine form. Reduction
addition [13–18][8–12]
is thusor nucleophilic
possible addition
in order to [13–18]
functionalize is thus
further possible in into
the glycosylamines
order to functionalize further thederivatives.
non-hydrolizable glycosylamines into non-hydrolizable derivatives.
O OH O OH
H 2N Bn NHBn NBn
RO BnO BnO
CH 2 Cl 2, MS
RO OR BnO OBn BnO OBn
1 : R=H 3
steps 1) R-M
2 : R=Bn 2) MsCl
HO OH
H H Bn
N N N R
CF 2P(O)(OH)2 N
HO BnO *
HO HO H
HO OH HO OH BnO OBn
HO OH
5a 5b 5c 4
Scheme 1. Thestrategy
Scheme 1. The glycosylamine glycosylamine strategy
towards towards iminosugars
monomeric monomeric iminosugars
[7]. [7].
Aminosugar 3 for instance was the starting point for the synthesis of difluoromethyl-
Aminosugar 3 for instance was the starting point for the synthesis of
phosphono-iminosugar 5a [19], designed as a potential inhibitor of chitin synthase. The
difluoromethylphosphono-iminosugar
diastereoselective addition 5aof[19],
LiCF designed as a potential inhibitor of chitin
2 P(O)(OEt)2 onto 3 followed by cyclization in the
synthase. The presence
diastereoselective addition of LiCF
of methanesulfonylchloride 2P(O)(OEt)
afforded 2 onto
the expected 3 followed
iminosugar by
intermediate 4
cyclization in the(Rpresence of methanesulfonylchloride
= CF2 P(O)(OEt) afforded thechloride
2 ). Instead, the use of allylmagnesium expected iminosugar
in the key nucleophilic
intermediate 4 (R = CF2P(O)(OEt)2). Instead, the use of allylmagnesium chloride inwhich
addition on glycosylamine 3 produced an allyl-pyrrolidine 4 (R = Allyl), the key could be
transformed
nucleophilic addition in few steps into
on glycosylamine 3 6-deoxy-homo-DMDP
produced an allyl-pyrrolidine 5b (an alkaloid 4 (Rextracted
= Allyl), from the
bulbs of Hyacinthus orientalis) [20] as well as into
which could be transformed in few steps into 6-deoxy-homo-DMDP 5b (an alkaloid 7-deoxy casuarine 5c [7], a non-natural
inhibitor of amyloglucosidase. Bis-glycosylamines, molecular tools that connect two car-
extracted from the bulbs of Hyacinthus orientalis) [20] as well as into 7-deoxy casuarine 5c
bohydrate moieties via a diamino linker, are rather seldom and have been used mainly
[7], a non-natural inhibitor of amyloglucosidase. Bis-glycosylamines, molecular tools that
in the elaboration of metal-coordinating carbohydrates. The examples from the literature
connect two carbohydrate moieties via a diamino
focus on bis-glycosylamines preparedlinker, are rather seldom and
from protecting-group-free have been
reducing sugars and
used mainly in the elaboration of metal-coordinating carbohydrates. The examples
diamino-alkyl, diamino-aryl, or diethylenetriamine as the nitrogenated counterpart [21–28]. from
the literature focus
Theironcomplexing
bis-glycosylamines
ability has prepared
been studied from protecting-group-free
further in several reports but reducing
the use of bis-
glycosylaminesdiamino-aryl,
sugars and diamino-alkyl, as intermediates ortowards the synthesis of as
diethylenetriamine more theelaborated
nitrogenated structures is,
to the best of our knowledge, presently undescribed, notably
counterpart [21–28]. Their complexing ability has been studied further in several reports for the synthesis of multiva-
lent iminosugars.
but the use of bis-glycosylamines as intermediates towards the synthesis of more
The presence of multiple copies of a given ligand gathered on a unique chemical
elaborated structures is, to the best of our knowledge, presently undescribed, notably for
structure, a phenomenon found in nature to improve host-guest interactions [29,30], is
the synthesis of multivalent
a recent strategy iminosugars.
in pharmaceutical research to increase the performances of enzyme
The presenceinhibitors via the so-calledofmultivalent
of multiple copies a given ligand gathered
effect. During on decade,
the last a unique chemical
a major step forward
structure, a phenomenon
was achieved found in nature to
with multivalent improveinhibitors,
glycosidase host-guest interactions
culminating [29,30],
in binding is a
enhancement
recent strategy by in several orders of magnitude
pharmaceutical research[31–36]. Homo-dimeric
to increase compounds areof
the performances theenzyme
most simple of
the series [37,38]. Although they generally display a modest
inhibitors via the so-called multivalent effect. During the last decade, a major step forward but measurable multivalent
effect, dimeric iminosugars remain attractive for mechanistic investigations. For instance,
was achieved with multivalent glycosidase inhibitors, culminating in binding
striking results were obtained with dimer 6: kinetic analysis showed a 19-fold increase in
enhancement by several orders of magnitude [31–36]. Homo-dimeric compounds are the
the inhibition potency for 6 (IC50 = 0.108 µM) relative to the monomer towards fucosidase
most simple of(Figure
the series [37,38]. Although they generally display a modest but
1) [39]. Moreover, the use of stereoisomers of 6 allowed a detailed analysis of
measurable multivalent
the mechanismeffect,ofdimeric
inhibitioniminosugars
of such a dimericremain attractive
structure, at a for mechanistic
molecular level. In the
investigations. For
sameinstance,
manner,striking
multivalentresults were obtained
architectures with dimer
with general structure6: kinetic
7 basedanalysis
on N-tethered
showed a 19-fold increase in the inhibition potency for 6 (IC50 = 0.108 µM) relative to the
pyrrolidine 8, a natural product named DAB-1, afforded strong inhibitors of α-mannosidase,
monomer towards N-acetylgalactosamine-6-sulfatase,
fucosidase (Figure 1) [39]. Moreover, and protein tyrosine
the usephosphatase
of stereoisomers 1B [40–44]. of 6Resting
upon our experience acquired in the field of iminosugar synthesis
allowed a detailed analysis of the mechanism of inhibition of such a dimeric structure, at by using glycosylamines
a molecular level. In the same manner, multivalent architectures with general structure 7
based on N-tethered pyrrolidine 8, a natural product named DAB-1, afforded strong
inhibitors of α-mannosidase, N-acetylgalactosamine-6-sulfatase, and protein tyrosine
30, x FOR PEER REVIEW 3 of 21
Molecules 2025, 30, 226 3 of 21

synthesis by using glycosylamines as key intermediates, we aimed to come up with an
extension of the method to prepare dimeric iminosugars based on a DAB-1 scaffold
as key intermediates, we aimed to come up with an extension of the method to prepare
(Figure 1). The synthetic sequence exploits bis-glycosylamines as intermediates, which
dimeric iminosugars based on a DAB-1 scaffold (Figure 1). The synthetic sequence exploits
easily allows variation of the spacer
bis-glycosylamines (alkyl, aryl,
as intermediates, PEG)
which separating
easily both pyrrolidines
allows variation by the
of the spacer (alkyl, aryl,
crucial choice of theseparating
PEG) starting di-amine.
both pyrrolidines by the crucial choice of the starting di-amine.
Figure 1. Structures of key compounds.

Figure 1. Structures of key compounds.
2. Results and Discussion
2. Results and Discussion
A critical aspect in the design of divalent glycosidase inhibitors is the chemical nature
and thein
A critical aspect length of the linker
the design which separates
of divalent glycosidase the two iminosugars.
inhibitors is the A variety ofnature
chemical spacers
have been devised in literature, which comprise alkyl, aromatic, polyamino, or PEG chains,
and the length of the linker which separates the two iminosugars. A variety of spacers
each of them requiring specific chemistry for its introduction on the pre-formed pyrro-
have been devised in literature, which comprise alkyl, aromatic, polyamino, or PEG
lidine [35,45–48]. Here, the linker originates from the di-amine that is used to build the
chains, each ofbis-glycosylamine,
them requiringenabling specifica large
chemistry
variety for its introduction
of possibilities. Thus, inon the the
first pre-formed
series of experi-
pyrrolidine [35,45–48].
ments, weHere, thethe
evaluated linker originates
reactivity from
of a series the di-amine
of diamines thatfeature
9a–e, which is used to build
various chain
lengths and
the bis-glycosylamine, structures
enabling a (Scheme 2). To this
large variety aim, each amine
of possibilities. was stirred
Thus, in thewithfirsttri-O-benzyl-
series of
L -xylofuranose 2 in dichloromethane in the presence of molecular sieves, following our
experiments, we evaluated the reactivity of a series of diamines 9a–e, which feature
own methodology for the synthesis of monomeric glycosylamines [7]. However, whereas
various chain lengths and structures (Scheme 2). To this aim, each amine was stirred with
an excess of amine could be used in the latter case as no competition occurs between
tri-O-benzyl-L-xylofuranose 2 in dichloromethane
mono and bis-glycosylation, the stoichiometry inofthethe presence
reaction hasofto molecular sieves,
be carefully controlled
following our here
ownin methodology
order to secure the forformation
the synthesis of monomeric
of the symmetrical glycosylamines
expected bis-glycosylamine [7].by
However, whereas an excess ofofthe
the introduction amine could be used
sub-stoichiometric amountin the latter
of the caseFortunately,
amine. as no competition
the reaction
occurs between mono and bis-glycosylation, the stoichiometry of the reaction hasthetomajor
proved efficient, whatever the amine used, affording glycosylamines 10a–e as be
compounds. Bis-glycosylation of the amine was confirmed by HRMS with the presence of
carefully controlled here in order to secure the formation of the symmetrical expected bis-
peaks corresponding to m/z = [MH]+ and m/z = [MH2 ]2+ /2 . In some cases, a small amount
glycosylamine ofby the introduction of the sub-stoichiometric amount of the amine.
the monofunctionalized bis-amine (ie, tethering of only one sugar moiety) was identified
Fortunately, thein thereaction
Mass or NMR proved
spectra.efficient,
Nevertheless,whatever the amine
the crude product was used used,as suchaffording
in the next
glycosylaminesstep 10a–e as the major
since purification compounds.
was not possible at thisBis-glycosylation of the
stage due to the lability amine
of these was
adducts.
confirmed by HRMS with the presence of peaks corresponding to m/z = [MH] and m/zimi-
Next, we wished to use 10a–e as intermediates for the preparation of
+ dimeric =
nosugars. The first option consists of reducing the hemiaminal to the corresponding amine,
[MH2] /2. In some cases, a small amount of the monofunctionalized bis-amine (ie,
2+
which could lead, after cyclization via an SN 2 process and benzyl deprotection, to new
tethering of only one sugar moiety) was identified in the Mass or NMR spectra.
dimeric DAB-1 models in a very straightforward manner. Although mild reducing agents
Nevertheless, the
suchcrude
as NaBHproduct was used as such in the next step since purification was
4 or NaBH3 CN react readily with simple glycosylamines [8–11], we found
not possible at LiAlH
this stage due
4 as the mostto efficient
the lability
hydrideof these
donor adducts.
for our purpose. Treating bis-glycosylamines
Next, we 10a–e
wished to excess
with an use 10a–e
of LiAlHas 4 (4intermediates
equiv) in Et 2 O forfor the preparation
1 h afforded amines 11a–e ofindimeric
acceptable
iminosugars. Theyields. Alloption
first compounds proved
consists ofstable and were
reducing thepurified and fullytocharacterized
hemiaminal at this stage.
the corresponding
amine, which could lead, after cyclization via an SN2 process and benzyl deprotection, to
new dimeric DAB-1 models in a very straightforward manner. Although mild reducing
agents such as NaBH4 or NaBH3CN react readily with simple glycosylamines [8–11], we
characterized at this stage. A subsequent cyclization is required to reach the pyrrolidin
Molecules 2025, 30, 226 4 of 21
core of DAB-1. This might be performed by activation of the free hydroxyl at C-4 of bot
sugar moieties, via mesylates or triflates for instance, followed by a spontaneou
intramolecular
A subsequentattack of nitrogen
cyclization to to
is required the generated
reach electrophilic
the pyrrolidine center.This
core of DAB-1. Contrasting
might be resul
performed by activation of the free hydroxyl at C-4 of both sugar
were obtained with compounds 11a–e after treatment with methanesulfonyl chloride moieties, via mesylates or i
triflates for instance, followed by a spontaneous intramolecular
pyridine, following a protocol used for monomeric analogues [7,19,20]. Whereas 11a,b attack of nitrogen to the
generated electrophilic center. Contrasting results were obtained with compounds 11a–e
gave the expected bis-pyrrolidines 12a,b,d in a single step, a mixture of sever
after treatment with methanesulfonyl chloride in pyridine, following a protocol used for
inseparable
monomeric products
analogueswas obtained
[7,19,20]. Whereas with 11c,e,
11a,b,d gavedue to competition
the expected between
bis-pyrrolidines N- and O
12a,b,d
mesylation.
in a singleSome
step, a attempts were made
mixture of several to limit
inseparable productsthe was
reaction
obtainedatwith
nitrogen,
11c,e, duebytochangin
temperature
competition (0between
°C, 80 N-°C),
andactivator (Tf2O),
O-mesylation. Someorattempts
base (NEt ), buttoall
were3made failed.
limit The expecte
the reaction
at nitrogen, by changing temperature (0 ◦ C, 80 ◦ C), activator (Tf O), or base (NEt ), but all grou
pyrrolidines 12c,e were finally obtained by incorporating a transient Boc protecting
2 3
failed. The expected pyrrolidines 12c,e were finally obtained by incorporating a transient
at nitrogen, enabling exclusive O-mesylation and subsequent cyclization after Bo
Boc protecting group at nitrogen, enabling exclusive O-mesylation and subsequent cycliza-
deprotection with HCl and neutralization during work-up. Final deprotection of benz
tion after Boc deprotection with HCl and neutralization during work-up. Final deprotection
groups was performed
of benzyl groups was with BCl3 with
performed in CH 2Clin
BCl 3
2, to
CHafford bis-iminosugars 13a–e, which wer
2 Cl2 , to afford bis-iminosugars 13a–e,
tested against
which were glycosidases (see below).
tested against glycosidases (see below).
H2 N Linker NH 2 N N
O OH O H Linker H O
BnO 9ae BnO OBn
BnO OBn i BnO OBn BnO OBn

2 10ae (no purification)
ii
BnO
BnO OBn OH N N HO
iii H Linker H
N Linker N BnO OBn
BnO OBn
BnO OBn BnO OBn
12a,b,d OBn 11ae
(5872%)
vii
iv,v,vi
HO BnO
HO OH vii BnO OBn

N Linker N N Linker N
HO OH BnO OBn
13ae
OH 12c,e OBn
9a: H2N NH 2 9b: H2 N 9c: H2 N NH 2

NH 2
O NH 2 O
9d: H2N O 9e: H2N O O NH 2
Scheme 2. Synthesis
Scheme ofofbis-iminosugars
2. Synthesis 13a–e.
bis-iminosugars 13a–e. Reagents
Reagents and and conditions:
conditions: (i) 9a–e,(i) 9a–e, molecular
molecular sieves siev
◦ ◦
(4 Å),(4CH
Å), CH2 Cl2 , r.t., 23–48 h; (ii) LiAlH4 , Et2 O, 0 C, 1 h; (iii) MsCl, pyridine, 0 C, 2.5 h; (iv) Et3 N,
2Cl2, r.t., 23–48 h; (ii) LiAlH4, Et2O, 0 °C, 1 h; (iii) MsCl, pyridine, 0 °C, 2.5 h; (iv) Et3N
◦
DMAP, Boc2 O, MeOH, r.t., 1–12 h; (v) Et3 N, MsCl, 0 C then r.t., 40 min; (vi) conc. HCl, EtOH, 12 h at
DMAP,
60 ◦ Boc 2O, MeOH, r.t., 1–12 h; (v) Et3N, MsCl,
C (11c) or 29 h at r.t. (11e); (vii) BCl , CH Cl , 0 ◦ C,02.5
°Ch.then r.t., 40 min; (vi) conc. HCl, EtOH, 12
3 2 2
at 60 °C (11c) or 29 h at r.t. (11e); (vii) BCl3, CH2Cl2, 0 °C, 2.5 h.
The incorporation of an additional substituent onto the final pyrrolidine structur

was attempted next (Scheme 3). To this aim, bis-glycosylamine 10d was used as a mod
x FOR PEER REVIEW 5 of 21
Molecules 2025, 30, 226 5 of 21
The incorporation
described for monovalent of an additional
glycosylamines substituent
[7]. A 60:40 onto of
mixture the two
final pyrrolidine structure was
diastereoisomers
attempted next (Scheme 3). To this aim, bis-glycosylamine 10d was used as a model and was
14d was obtained (50% yield), which proved unseparable at this stage. According to their
treated with an excess (6 equiv) of allyMgCl in THF, in the same manner as that described for
NMR spectra, themonovalent
major isomer revealed[7].a Asymmetrical
glycosylamines structure
60:40 mixture of (either (R,R)
two diastereoisomers 14dor
was(S,S)
obtained
configurations at (50%
both yield), which proved unseparable at this stage. According to their NMR an
new stereocenters, see below), whereas the minor one presented spectra,
unsymmetrical structure with two opposite configurations (R,S) generated during the at
the major isomer revealed a symmetrical structure (either (R,R) or (S,S) configurations
bothselectivity
addition. Yield and new stereocenters, see below), in
were improved whereas the minorof
the presence oneLiCl,
presented an unsymmetrical
an additive that
structure with two opposite configurations (R,S) generated during the addition. Yield
proved efficient previously in nucleophilic additions to N-t-butanesulfinyl
and selectivity were improved in the presence of LiCl, an additive that proved efficient
glycosylamines [49], affording an 85:15 mixture of diastereoisomers 14d(R,R) and
previously in nucleophilic additions to N-t-butanesulfinyl glycosylamines [49], affording
14d(R,S) in 85% yield.
an 85:15 mixture of diastereoisomers 14d(R,R) and 14d(R,S) in 85% yield.
Scheme
Scheme 3. Synthesis 3. Synthesis
of dimeric of dimeric allyl-pyrrolidine
allyl-pyrrolidine 16d(R,R) and 16d(R,R) and determination
determination of the configuration
of the configuration
of 19d. Reagents and conditions: (i) AllylMgCl, THF, 0 ◦ C then r.t., 7 h; (ii) LiCl, AllylMgCl, THF,
of 19d. Reagents and conditions: (i) AllylMgCl, THF, 0 °C then r.t.,◦ 7 h; (ii) ◦LiCl, AllylMgCl, THF,
◦−78 C then r.t., 2 h 30; (iii) MsCl, pyridine, THF, −78 C then 0 C, 3 h; (iv) BCl3 , CH2 Cl2 , 0 ◦ C,
−78 °C then r.t., 2 h 230; (iii)(v)MsCl,
h 30; 9d (0.5pyridine, THF,45−78
equiv), MeOH, ◦ C,°C
4 h;then 0 °C, 3(6h;equiv),
(vi) AllylBr (iv) BCl 3, CH2Cl2, 0 °C, 2 h 30;
indium powder (3 equiv), MeOH,
(v) 9d (0.5 equiv), MeOH,
r.t., 16 h,45 °C,(two
58% 4 h;steps).
(vi) AllylBr (6 equiv), indium powder (3 equiv), MeOH, r.t., 16
h, 58% (two steps).
A cyclization protocol (MsCl in pyridine) was applied to the mixture

14d(R,R)/14d(R,S), which gave pyrrolidines 15d(R,R)/15d(R,S) in 79% yield. Both
Molecules 2025, 30, 226 6 of 21
A cyclization protocol (MsCl in pyridine) was applied to the mixture 14d(R,R)/14d(R,S),

which gave pyrrolidines 15d(R,R)/15d(R,S) in 79% yield. Both compounds were separated
at this stage and the (R) configurations at the newly formed stereogenic centers in the
structure of the major isomer were established using NOESY experiments. Deprotection
was performed next on 15d(R,R), which encompasses an all-trans configuration, the one
observed in most active glucosidase inhibitors such as the standard iminosugars DMDP or
DNJ [20]. Dimeric allyl-pyrrolidine 16d(R,R) obtained after treatment with BCl3 (56% yield)
was also tested against our panel of commercial glycosidases. An attempt was conducted
on 15d(R,R) to promote metathesis in order to generate the macrocyclic structure 17d.
Unfortunately, cyclization proved unfavorable, and only isomerization of the double bonds
was mediated by the Grubbs catalyst to give 18d. Interestingly, a more direct procedure
for allylation was conducted on unprotected D-xylose 1 by indium-mediated nucleophilic
addition on an unprotected bis-glycosylamine, as previously described with monomeric gly-
cosylamines [50]. The reaction proved successful and afforded polyhydroxy bis-allylamine
19d in 58% yield. Chemical correlation was attempted to determine the configuration of
the newly formed stereogenic centers in 19d. To this end, debenzylation of a sample of the
mixture 14d(R,R)/14d(R,S) 85/15 with BCl3 afforded samples of 19d(R,R)/19d(R,S) with
ascertained configuration, which served as reference. According to their non-equivalent
NMR spectra (see Supplementary Materials), 19d and 19d(R,R) feature distinct config-
urations, but both are symmetrical compounds in view of the simplicity of their NMR
spectra. Thus, the (S,S) configuration was assigned to the major diastereoisomer resulting
from indium-mediated allylation. The selectivity of this reaction is in full agreement with
previous reactions on monomeric glycosylamines [50].
To go even further in the synthesis of macrocyclic structures by using our method,
another strategy was designed, which would exploit alkyne/alkyne Glaser-type coupling
to form the corresponding diyne (Scheme 4). To this aim, the addition of an ethynyl nu-
cleophile was attempted on glycosylamine 10d. Whereas acetylide (via the corresponding
Grignard) afforded unsatisfactory results TMS-acetylide was added selectively to 10d
(d.e. = 90%), affording the expected bis-amine 20d in 37% yield. Here, the (S) configura-
tion was attributed to the newly formed stereocenters according to NOESY experiments
conducted on the TMS-alkyne after cyclization, which is the opposite of that observed
for the allyl-analogue. After the standard intramolecular nucleophilic substitution pro-
tocol mediated by MsCl, trimethylsilyl groups were removed (TBAF) to afford the bis-
ethynylpyrrolidine 21d. Gratifyingly, treatment of 21d with copper(II) acetate monohydrate
and nickel chloride to promote intramolecular diyne coupling afforded glycophane 22d in
82% yield [51]. At this point, no tentative was made to debenzylate further 22d.
Finally, enzymatic assays were performed with compounds 13a–e and 16d on a series
of commercial glycosidases (α-glucosidase from rice and from yeast, β-glucosidase from
almond, β-glucosidase and α-rhamnosidase from A. niger, β-galactosidase from A. orizae,
α-mannosidase and β-N-acetylglucosaminidase from Jack bean and α-galactosidase from
green coffee bean) to assess potency and selectivity of these new compounds (Table 1).
A prepared sample of DAB-1 8 was also tested under the same conditions for ease of
comparison [52]. A first set of biological assays was effected at 1 mM concentration of
inhibitor. Total annihilation (>95%) of enzyme activity at 1 mM is generally a prerequisite
for high inhibition potencies. Conversely, % inhibition below 95% at 1 mM is typical
of modest or poor inhibitors with half maximal inhibitory concentrations usually above
100 µM. Former results from our laboratories have always followed this empirical rule.
Thus, IC50 was only determined in cases where inhibition at 1 mM was greater than 95%,
by assaying decreasing concentrations of inhibitors.
afford the bis-ethynylpyrrolidine 21d. Gratifyingly, treatment of 21d with copper(II)
acetate monohydrate and nickel chloride to promote intramolecular diyne coupling
afforded
Molecules 2025, 30, 226 glycophane 22d in 82% yield [51]. At this point, no tentative was made to7 of 21
debenzylate further 22d.
Scheme 4. SynthesisScheme 4. Synthesis of

of the glycophane theReagents
22d. glycophane 22d.
and Reagents and
conditions: conditions: (i)3HC
(i) HCC-SiMe ≡C-SiMe
, EtMgBr, 3 , EtMgBr,
THF,
THF, 0 ◦ C then r.t., 17 h; (ii) MsCl, pyridine, THF, O ◦ C then r.t., 6 h; (iii) TBAF, THF, r.t., 3 h;
0 °C then r.t., 17 h; (ii) MsCl, pyridine, THF, O °C then r.t., 6 h;
◦ (iii) TBAF, THF, r.t., 3 h; (iv)
(iv) Cu(OAc)2 ·H2 O, NiCl2 , pyridine, EtOH, CHCl3 , 60 C, 16 h.
Cu(OAc)2·H2O, NiCl2, pyridine, EtOH, CHCl3, 60 °C, 16 h.
Table 1. Glycosidase inhibition potencies of prepared compounds a,b .
Enzyme 13a 13b 13c 13d 13e 16d DAB-1

α-glucosidase >95%
40% 77% 92% 90% 72% 26%
(rice) 7.8 µM
α-glucosidase 95% >95%
88% 88% 94% 65% –20% c
(Sac. cerevisiae) 39 µM 0.69 µM
β-glucosidase 95%
13% 16% 60% 24% 23% 25%
(almond) 79 µM
β-glucosidase >95%
24% 43% 94% 93% 91% 51%
(Asp. niger) 62 µM
β-galactosidase >95% >95%
93% 92% 81% 58% 76%
(Asp. orizae) 2.8 µM 31 µM
α-mannosidase >95% >95% >95%
75% 84% 82% 32%
(Jack bean) 18 µM 34 µM 49 µM
α-rhamnosidase
67% 24% 69% 46% 18% 25% 34%
(Asp. niger)
α-galactosidase
38% NI 61% 22% 22% NI 11%
(green coffee)
β-GlcNAc-ase
NI NI NI –38% c NI –33% c 14%
(Jack bean)
a% inhibition at 1 mM concentration of inhibitor; IC50 in µM, determined when >95% inhibition. b NI means
no impact on enzyme activity (less than 5% inhibition or activation). c negative values mean activation of
enzyme activity.
DAB-1 8 was chosen as a model iminosugar for our experiments examining the effect
of dimerization. Pyrrolidine 8 entails a specific hydroxyl distribution closely related to
that of glucose. As a consequence, DAB-1 has strong affinities for enzymes operating
with glucosides or glucoconjugates, a result supported by our own experiments towards
alpha- or beta-glucosidases from different origins with IC50 ’s in the range 0.69–79 µM
(Table 1). However, structural analogies of DAB-1 with other biologically relevant car-
bohydrates such as mannose induce cross-inhibitions, as observed with α-mannosidase
(IC50 = 49 µM). Regarding dimers 13a–e, a significant decrease in glucosidase inhibition
Molecules 2025, 30, 226 8 of 21
potencies is observed here, with only 13b displaying potent alpha-glucosidase (baker’s
yeast) inhibition with IC50 = 39 µM. However, a more detailed analysis of the results
suggests that the linker separating the two pyrrolidine rings plays a role in the tightness
of the inhibitor binding to the enzyme. Compound 13d for instance, displays about 90%
inhibition towards the two alpha-glucosidases and the beta-glucosidase from Aspergillus
niger at 1 mM, whereas 13a induces much weaker enzyme inhibition. The introduction of
an additional substituent as found in 16d, was detrimental for enzyme-inhibitor recognition
regarding alpha-glucosidases. Whereas its non-substituted counterpart 13d displayed 90%
inhibition at 1 mM, only 26% inhibition was observed with allyl-pyrrolidine 16d (rice
alpha-glucosidase). An astonishing 20% enhancement of enzyme activity was observed in
the presence of the latter regarding alpha-glucosidase from yeast. An analogous activation
was observed with 16d and 13d towards beta-N-acetylglucosaminidase from Jack beans
(33% and 38% activation, respectively). Concerning the other tested enzymes, dimeriza-
tion caused a significant enhancement of alpha-mannosidase inhibition by 13c and 13e
(IC50 = 18 µM and 34 µM, respectively) when compared to DAB-1 (IC50 = 49 µM) a trend
already observed with other multimeric iminosugars towards this same enzyme [31]. Inter-
estingly, whereas monomer 8 is a poor inhibitor of beta-galactosidase, dimeric structures
13a–13d displayed >90% inhibition at 1 mM culminating to IC50 ’s = 2.8 µM and 31 µM in
the case of 13b and 13c. Some β-galactosidase-inhibiting iminosugars have been considered
for their pharmacological chaperone (PC) potency in the treatment of GM1 gangliosido-
sis, an inherited disorder caused by the disruption of human β-galactosidase. At low
concentrations, such inhibitors impede the degradation of misfolded proteins, restoring
residual activity in patients suffering from deficient enzymes. The potency and selectivity
of bis-iminosugars 13 towards β-galactosidase, as observed here, make them valuable
candidates for further studies on their use as PC in the treatment of GM1 gangliosidosis.
In conclusion, we describe here a new and straightforward synthetic route towards
dimeric iminosugars, starting from bis-glycosylamines, which are easily prepared by
the reaction of a carbohydrate with 0.5 equiv of a diamine. Reduction of such dimeric
N-glycosides with LiAlH4 followed by MsCl-induced cyclization affords the protected
bis-iminosugar, with the inverted configuration at C-4 when compared to the carbohy-
drate substrate. The method allows the integration of a variety of linkages (aryl, alkyl,
polyethyleneglycol chains) connecting both iminosugar templates by the choice of the
bis-amine used in the first step. Moreover, an additional substituent (allyl, ethynyl) may
be inserted in the structure via nucleophilic addition of an organometallic reagent to the
bis-glycosylamine in a highly diastereoselective manner. A bis-ethynyl symmetrical imi-
nosugar proved susceptible to intramolecular Glaser coupling, affording an unprecedented
macrocyclic structure. Finally, deprotected bis-iminosugars were tested towards a series of
commercial glycosidases to uncover potencies and selectivities when compared to DAB-
1, their monomeric counterpart. Whereas a significant drop in inhibition potencies was
observed towards glucosidases when compared to DAB-1, compounds 13a–d displayed
unexpected potent inhibition of β-galactosidase. Further studies might be conducted in
the future to evaluate the effect of bis-iminosugars 13a–d on human β-galactosidase and
explore their capacity to act as PC.
3. Materials and Methods

3.1. General Methods
Reactants and reagents were purchased from standard suppliers (Sigma-Aldrich
(St. Louis, MO, USA), Alfa-Aesar (Haverhill, MA, USA), Fisher Scientific (Waltham, MA, USA))
and were used without further purification. Methanol, dichloromethane, tetrahydrofuran,
and diethyl ether were dried on a Pure Solv MD system (Innovative Technology, Inc.,
Molecules 2025, 30, 226 9 of 21
Oldham, UK). Reactions were monitored on TLC plates using Silica gel F254 (0.2 mm)
(Macherey-Nagel SAS, Hoerdt, France), detection was carried out by spraying with an
aqueous solution of KMnO4 (2%)/Na2 CO3 (4%) or alcoholic solutions of phosphomolybdic
acid or p-anisaldehyde, followed by heating. Column chromatography purifications were
performed over silica gel M 9385 (40–63 µm) Kieselgel 60 (Macherey-Nagel SAS, Hoerdt,
France). NMR spectra were recorded on Bruker AC 500 (Billerica, MA, USA, 500 MHz
for 1 H, 126 MHz for 13 C) or 600 (600 MHz for 1 H and 150 MHz for 13 C) spectrometers.
Chemical shifts are expressed in parts per million (ppm) and were calibrated to deuterated
or residual non-deuterated solvent peaks for 1 H and 13 C spectra. Coupling constants are
in Hz and splitting pattern abbreviations are: br, broad; s, singlet; d, doublet; t, triplet; q,
quadruplet; m, multiplet. DEPT and JMOD 1D NMR experiments, COSY, HSQC, HMBC,
and NOESY 2D NMR experiments were used to confirm the NMR peak assignments for
all compounds. Optical rotations were determined at 25 ◦ C with an Anton Paar Model
MCP 5100 polarimeter (Graz, Austria). Mass Spectra (MS) and High Resolution Mass
Spectra (HRMS) were performed on a Waters Corp. Q-TOF Micro micromass positive ESI
(CV = 30 V) (Milford, MA, USA).
3.2. General Procedure for the Synthesis of Bis-Glycosylamines 10a–e

To a solution of 2,3,5-tri-O-benzyl-L-xylofuranose 2 (1 equiv) in anhydrous dichloromethane
was added molecular sieves (4 Å) then diamine 9a–e (0.5 equiv), and the mixture was stirred
at room temperature until total consumption of 2. The solution was filtrated and concen-
trated under reduced pressure to afford an anomeric mixture of the bis-glycosylamine
which was not purified further.
(10a) According to general procedure, the reaction of 2 (200 mg, 0.475 mmol) and 9a
(0.237 mmol, 32.3 mg) in dichloromethane (3.7 mL) in the presence of molecular sieves
(450 mg) afforded after 48 h 10a as a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.40–7.24 (m,
34 H, Ar-H), 5.03 (d, J = 3.5 Hz, 0.7 H, 2 × 1-Hmin ), 4.76 (d, J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj ),
4.64–4.45 (m, 12 H, 6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.41 (q, J = 6.0 Hz, 0.7 H, 2 × 4-Hmin ),
4.34 (q, J = 5.3 Hz, 1.3 H, 2 × 4-Hmaj ), 4.15 (d, J = 13.5 Hz, 0.7 H, 2 × CHa Hb -NHmin ), 4.07 (dd,
J = 13.4, 3.6 Hz, 1.3 H, 2 × CHa Hb -NHmaj ), 4.01–3.98 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin ),
3.95–3.93 (m, 1.3 H, 2 × 2-Hmaj ), 3.91–3.89 (m, 0.7 H, 2 × 2-Hmin ), 3.82–3.68 (m,
6 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin + 2 × 5-Hmaj + 2 × 5-Hmin ) ppm; 13 C NMR
(125 MHz, CDCl3 ): δ 140.7 (CIV ), 140.4 (CIV ), 138.5 (CIV ), 138.4 (CIV ), 138.1 (CIV ), 138.0 (CIV ),
137.9 (CIV ), 128.7, 128.6, 128.5, 128.5, 128.2, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7,
127.7, 127.1, 126.8, 126.7 (C-Ar), 94.6 (C-1maj ), 90.5 (C-1min ), 86.1 (C-2maj ), 81.6 (C-3maj ), 81.4
(C-3min ), 81.4 (C-2min ), 78.9 (C-4maj ), 77.2 (C-4min ), 73.6 (CH2 -Ph), 72.9 (CH2 -Phmin ), 72.4
(CH2 -Phmin ), 72.2 (CH2 -Phmaj ), 71.7 (CH2 -Phmaj ), 69.3 (C-5maj ), 69.0 (C-5min ), 50.2 (CH2 -
NHmin ), 49.9 (CH2 -NHmaj ) ppm; HRMS (ESI) m/z calcd for [C60 H64 N2 O8 + H]+ : 941.4741,
found 941.4745.
(10b) According to general procedure, the reaction of 2 (436 mg, 1.04 mmol) and
9b (71 mg, 0.52 mmol) in dichloromethane (7.8 mL) in the presence of molecular
sieves (950 mg) afforded after 27 h 10b as a yellow oil. 1 H NMR (500 MHz, CDCl3 ):
δ 7.40–7.27 (m, 34 H, Ar-H), 5.00–4.98 (m, 0.7 H, 2 × 1-Hmin ), 4.73–4.72 (m, 1.3 H,
2 × 1-Hmaj ), 4.68–4.47 (m, 12 H, 6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.39 (q, J = 5.7 Hz,
0.7 H, 2 × 4-Hmin ), 4.36 (q, J = 5.2 Hz, 1.3 H, 2 × 4-Hmaj ), 4.16 (d, J = 13.5 Hz, 0.7 H,
2 × CHa Hb -NHmin ), 4.08 (d, J = 13.2 Hz, 1.3 H, 2 × CHa Hb -NHmaj ), 4.03–4.00 (m,
2 H, 2 × 3-Hmaj + 2 × 3-Hmin ), 3.96–3.95 (m, 1.3 H, 2 × 2-Hmaj ), 3.92–3.71 (m, 6.7 H,
2 × 2-Hmin + 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin + 2 × 5-Hmaj + 2 × 5-Hmin ) ppm; 13 C
NMR (125 MHz, CDCl3 ): δ 139.1 (CIV ), 138.9 (CIV ), 138.5 (CIV ), 138.3 (CIV ), 138.0 (CIV ),
138.0 (CIV ), 137.9 (CIV ), 128.7, 128.7, 128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2,
Molecules 2025, 30, 226 10 of 21
128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7 (C-Ar), 94.4 (C-1maj ), 90.4 (C-1min ),
86.0 (C-2maj ), 81.6 (C-3maj ), 81.4 (C-3min ), 81.3 (C-2min ), 78.9 (C-4maj ), 77.2 (C-4min ), 73.6
(CH2 -Ph), 72.8 (CH2 -Phmin ), 72.3 (CH2 -Phmin ), 72.2 (CH2 -Phmaj ), 71.7 (CH2 -Phmaj ), 69.2
(C-5maj ), 69.0 (C-5min ), 49.9 (CH2 -NHmin ), 49.6 (CH2 -NHmaj ) ppm; HRMS (ESI) m/z calcd
for [C60 H64 N2 O8 + H]+ : 941.4741, found 941.4742.
(10c) According to general procedure, the reaction of 2 (600 mg, 1.43 mmol)
and 9c (103 mg, 0.71 mmol) in dichloromethane (11.1 mL) in the presence of molec-
ular sieves (1.35 g) afforded after 23 h 10c as a yellow oil. 1 H NMR (500 MHz,
CDCl3 ): δ 7.40–7.26 (m, 30 H, Ar-H), 5.04 (d, J = 3.4 Hz, 0.7 H, 2 × 1-Hmin ), 4.77 (d,
J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj ), 4.66–4.47 (m, 12 H, 6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.39
(q, J = 5.5 Hz, 0.7 H, 2 × 4-Hmin ), 4.34 (q, J = 5.4 Hz, 1.3 H, 2 × 4-Hmaj ), 4.01–3.99 (m,
2 H, 2 × 3-Hmaj + 2 × 3-Hmin ), 3.92–3.89 (m, 2 H, 2 × 2-Hmaj + 2 × 2-Hmin ), 3.80–3.67 (m,
4 H, 2 × 5-Hmaj + 2 × 5-Hmin ), 3.01–2.87 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ),
2.69–2.57 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ), 1.53–1.28 (m, 12 H, 6 × CH2 maj
+ 6 × CH2 min ) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.5 (CIV ), 138.4 (CIV ), 138.1 (CIV ),
138.0 (CIV ), 137.9 (CIV ), 137.9 (CIV ), 128.7, 128.6, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.1,
128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7 (C-Ar), 95.1 (C-1maj ),
91.0 (C-1min ), 86.1 (C-2maj ), 81.6 (C-3maj ), 81.5 (C-3min ), 81.2 (C-2min ), 78.8 (C-4maj ), 77.2
(C-4min ), 73.5 (CH2 -Ph), 72.8 (CH2 -Phmin ), 72.4 (CH2 -Phmin ), 72.2 (CH2 -Phmaj ), 71.7 (CH2 -
Phmaj ), 69.2 (C-5maj ), 68.9 (C-5min ), 46.6 (CH2 -NHmin ), 46.1 (CH2 -NHmaj ), 30.8 (CH2 ), 30.5
(CH2 ), 29.6 (CH2 ), 29.6 (CH2 ), 27.4 (CH2 ), 27.4 (CH2 ), 27.4 (CH2 ), 27.3 (CH2 ) ppm; HRMS
(ESI) m/z calcd for [C60 H72 N2 O8 + H]+ : 949.5372, found 949.5367.
(10d) According to general procedure, the reaction of 2 (100 mg, 0.24 mmol) and 9d
(18 mg, 0.12 mmol) in dichloromethane (2 mL) in the presence of molecular sieves (227 mg)
afforded after 24 h 10d as a brown oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.39–7.25 (m, 30 H, Ar-
H), 4.97 (d, J = 3.6 Hz, 0.6 H, 2 × 1-Hmin ), 4.72–4.71 (m, 1.4 H, 2 × 1-Hmaj ), 4.65–4.45 (m, 12 H,
6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.37 (q, J = 5.9 Hz, 0.6 H, 2 × 4-Hmin ), 4.34 (q, J = 5.4 Hz,
1.4 H, 2 × 4-Hmaj ), 3.99–3.98 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin ), 3.93 (m, 1.4 H, 2 × 2-Hmaj ),
3.90–3.89 (m, 0.6 H, 2 × 2-Hmin ), 3.80–3.68 (m, 4 H, 2 × 5-Hmaj + 2 × 5-Hmin ), 3.62–3.52 (m,
8 H, 4 × CH2 maj + 4 × CH2 min ), 3.19–3.07 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ),
2.88–2.75 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ) ppm; 13 C NMR (125 MHz,
CDCl3 ): δ 138.5 (CIV ), 138.4 (CIV ), 138.1 (CIV ), 138.0 (CIV ), 137.9 (CIV ), 137.9 (CIV ), 128.7,
128.7, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.8, 127.7 (C-Ar), 95.3 (C-1maj ), 90.9 (C-
1min ), 86.1 (C-2maj ), 81.6 (C-3min ), 81.5 (C-3maj ), 81.3 (C-2min ), 78.9 (C-4maj ), 77.2 (C-4min ), 73.5
(CH2 -Phmaj ), 73.5 (CH2 -Phmin ), 72.8 (CH2 -Phmin ), 72.4 (CH2 -Phmin ), 72.2 (CH2 -Phmaj ), 71.7
(CH2 -Phmaj ), 71.5 (CH2 min ), 71.3 (CH2 maj ), 70.4 (CH2 min ), 70.3 (CH2 min ), 70.2 (CH2 maj ), 70.2
(CH2 maj ), 69.2 (C-5maj ), 68.9 (C-5min ), 46.1 (CH2 -NHmin ), 45.7 (CH2 -NHmaj ) ppm; HRMS
(10e) According to general procedure, the reaction of 2 (157 mg, 0.37 mmol) and 9e
(41 mg, 0.18 mmol) in dichloromethane (3.1 mL) in the presence of molecular sieves (356 mg)
afforded after 27 h 10e as a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.39–7.26 (m, 30 H,
Ar-H), 4.95 (d, J = 3.6 Hz, 0.7 H, 2 × 1-Hmin ), 4.68 (d, J = 2.2 Hz, 1.3 H, 2 × 1-Hmaj ), 4.65–4.45
(m, 12 H, 6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.36 (q, J = 6.0 Hz, 0.7 H, 2 × 4-Hmin ), 4.30 (q,
J = 5.4 Hz, 1.3 H, 2 × 4-Hmaj ), 3.99–3.97 (m, 2 H, 2 × 3-Hmaj + 2 × 3-Hmin ), 3.87 (m, 2 H,
2 × 2-Hmaj + 2 × 2-Hmin ), 3.76–3.65 (m, 4 H, 2 × 5-Hmaj + 2 × 5-Hmin Hmin ), 3.62–3.48 (m,
12 H, 6 × CH2 maj + 6 × CH2 min ), 3.06–2.93 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ),
2.78–2.66 (m, 2 H, 2 × CHa Hb -NHmaj + 2 × CHa Hb -NHmin ), 1.81–1.69 (m, 4 H, 2 × CH2 -
CH2 -NHmaj + 2 × CH2 -CH2 -NHmin ) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.5 (CIV ),
138.4 (CIV ), 138.1 (CIV ), 138.0 (CIV ), 137.9 (CIV ), 128.7, 128.6, 128.5, 128.5, 128.5, 128.0, 127.9,
127.9, 127.9, 127.8, 127.8, 127.7, 127.7 (C-Ar), 95.2 (C-1maj ), 91.0 (C-1min ), 86.2 (C-2maj ),
Molecules 2025, 30, 226 11 of 21
81.6 (C-3maj ), 81.4 (C-2min ), 81.2 (C-3min ), 78.7 (C-4maj ), 77.2 (C-4min ), 73.6 (CH2 -Phj ), 72.8
(CH2 -Phmin ), 72.4 (CH2 -Phmin ), 72.2 (CH2 -Phmaj ), 71.7 (CH2 -Phmaj ), 70.7 (CH2 ), 70.3 (CH2 ),
69.8 (CH2 ), 69.7 (CH2 ), 69.6 (CH2 ), 69.2 (C-5maj ), 68.9 (C-5min ), 43.7 (CH2 -NHmin ), 43.3
(CH2 -NHmaj ), 30.7 (CH2 -CH2 -NHmin ), 30.6 (CH2 -CH2 -NHmaj ) ppm; HRMS (ESI) m/z calcd
for [C62 H76 N2 O11 + H]+ : 1025.5527, found 1025.5531.
3.3. Representative Procedure for the Reduction of Bis-Glycosylamines 10a–e

To a solution of bis-glycosylamine 10a–e (1 equiv) in anhydrous diethyl ether at
0 ◦ C under argon atmosphere was added dropwise lithium aluminium hydride 1 M in
diethyl ether (4 equiv). The mixture was stirred for 1 h at the same temperature and then
sequentially supplemented dropwise with water (38 µL per mL of LiAlH4 1 M used), an
aqueous solution of sodium hydroxide 3 M (38 µL per mL of LiAlH4 1 M used) and water
again (114 µL per mL of LiAlH4 1 M used). The resulting mixture was filtered, the inorganic
residue was washed with ether, and the resulting ether solution was then combined with
the organic layer of the filtrate. The solution was dried with magnesium sulfate, filtered,
and concentrated under reduced pressure, and finally, the crude was purified by column
chromatography on silica gel.
(11a) According to general procedure, the reaction of 10a (232 mg, 0.246 mmol) and
LiAlH4 1 M in diethyl ether (984 µL, 0.984 mmol) in diethyl ether (2.5 mL) afforded after
purification (eluent: EtOAc/PE gradient, 80/20 to 95/5) 11a (149 mg, 0.158 mmol, 64%) as
a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.36–7.15 (m, 34 H, Ar-H), 4.56–4.42 (m, 12 H,
6 × CH2 -Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.83–3.76 (m, 4 H, 2 × 3-H + 2 × NH-CHa Hb -Ph),
3.72–3.52 (m, 8 H, 2 × 2-H + 2 × NH-CHa Hb -Ph + 2 × 5-H), 2.97 (dd, J = 12.2, 1.4 Hz, 2 H,
2 × 1-Ha Hb ), 2.83 (dd, J = 12.2, 5.1 Hz, 2 H, 2 × 1-Ha Hb ) ppm; 13 C NMR (125 MHz, CDCl3 ):
δ 138.5 (CIV ), 138.4 (CIV ), 138.1 (CIV ), 129.2, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.0,
128.0, 127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.7, 127.7 (C-Ar), 76.8 (C-3), 76.2 (C-2), 73.8
(CH2 -Ph), 73.3 (CH2 -Ph), 72.1 (CH2 -Ph), 70.9 (C-5), 66.7 (C-4), 53.6 (NH-CH2 -Ph), 46.5 (C-1)
ppm; [α]25 +
D = +6.3 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C60 H68 N2 O8 + H] : 945.5054,
found 945.5063.
(11b) According to general procedure, the reaction of 10b (583 mg, 0.619 mmol) and
LiAlH4 1 M in diethyl ether (2.48 mL, 2.476 mmol) in diethyl ether (6 mL) afforded after
purification (eluent: EtOAc/PE gradient, 80/20 to 95/5) 11b (357 mg, 0.377 mmol, 61%) as
a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.36–7.22 (m, 34 H, Ar-H), 4.56–4.41 (m, 12 H,
6 × CH2 -Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.81–3.77 (m, 4 H, 2 × 3-H + 2 × NH-CHa Hb -
Ph), 3.68–3.61 (m, 6 H, 2 × 2-H + 2 × NH-CHa Hb -Ph + 2 × 5-Ha Hb ), 3.58–3.54 (m, 2 H,
2 × 5-Ha Hb ), 2.95 (dd, J = 12.2, 1.4 Hz, 2 H, 2 × 1-Ha Hb ), 2.83 (dd, J = 12.2, 5.0 Hz, 2 H,
2 × 1-Ha Hb ) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.6 (CIV ), 138.4 (CIV ), 138.1 (CIV ), 137.9
(CIV ), 128.8, 128.7, 128.7, 128.6, 128.6, 128.5, 128.4, 128.4, 128.0, 127.9, 127.9, 127.6 (C-Ar),
76.7 (C-3), 76.2 (C-2), 73.8 (CH2 -Ph), 73.3 (CH2 -Ph), 72.1 (CH2 -Ph), 70.9 (C-5), 66.5 (C-4),
53.5 (NH-CH2 -Ph), 46.3 (C-1) ppm; [α]25 D = +14.8 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for
+
[C60 H68 N2 O8 + H] : 945.5054, found 945.5051.
(11c) According to general procedure, the reaction of 10c (597 mg, 0.629 mmol) and
purification (eluent: EtOAc/PE 70/30 then EtOAc/MeOH 95/5) 11c (388 mg, 0.407 mmol,
65%) as a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.25 (m, 30 H, Ar-H), 4.57–4.47 (m,
12 H, 6 × CH2 -Ph), 4.09–4.06 (m, 2 H, 2 × 4-H), 3.76 (d, J = 5.8 Hz, 2 H, 2 × 3-H), 3.67–3.61
(m, 4 H, 2 × 2-H + 2 × 5-Ha Hb ), 3.56 (t, J = 8.5 Hz, 2 H, 2 × 5-Ha Hb ), 2.90 (dd, J = 12.2,
1.3 Hz, 2 H, 2 × 1-Ha Hb ), 2.79 (dd, J = 12.2, 5.0 Hz, 2 H, 2 × 1-Ha Hb ), 2.55 (t, J = 7.4 Hz, 4 H,
2 × NH-CH2 -CH2 ), 1.53–1.38 (m, 4 H, 2 × NH-CH2 -CH2 ), 1.28–1.24 (m, 8 H, 4 × CH2 ) ppm;
13 C NMR (125 MHz, CDCl ): δ 138.6 (CIV ), 138.5 (CIV ), 138.2 (CIV ), 128.6, 128.6, 128.5, 128.5,
3
Molecules 2025, 30, 226 12 of 21
128.4, 128.4, 128.0, 127.9, 127.9, 127.9, 127.6 (C-Ar), 76.7 (C-3), 76.2 (C-2), 73.7 (CH2 -Ph),
73.3 (CH2 -Ph), 72.0 (CH2 -Ph), 71.0 (C-5), 66.3 (C-4), 49.6 (NH-CH2 -CH2 ), 46.5 (C-1), 29.5
(NH-CH2 -CH2 ), 29.4 (CH2 ), 27.3 (CH2 ) ppm; [α]25 D = +20.3 (c 1, CHCl3 ). HRMS (ESI) m/z
calcd for [C60 H76 N2 O8 + H]+ : 953.5680, found 953.5685.
(11d) According to general procedure, the reaction of 10d (1.07 g, 1.12 mmol) and
purification (eluent: EtOAc/MeOH gradient, 80/20 to 50/50) 11d (773 mg, 0.806 mmol,
72%) as a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.25 (m, 30 H, Ar-H), 4.57–4.45
(m, 12 H, 6 × CH2 -Ph), 4.10 (t, J = 6.7 Hz, 2 H, 2 × 4-H), 3.76 (dm, J = 5.9 Hz, 2 H, 2 × 3-H),
3.68–3.66 (m, 2 H, 2 × 2-H), 3.64–3.49 (m, 12 H, 2 × 5-H + 4 × CH2 ), 2.90 (dd, J = 12.5,
1.6 Hz, 2 H, 2 × 1-Ha Hb ), 2.83 (dd, J = 12.5, 4.8 Hz, 2 H, 2 × 1-Ha Hb ), 2.80–2.70 (m, 4 H,
2 × NH-CH2 -CH2 ) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.5 (CIV ), 138.4 (CIV ), 138.1
(CIV ), 128.5, 128.4, 128.3, 128.3, 127.8, 127.8, 127.8, 127.5 (C-Ar), 76.9 (C-3), 76.2 (C-2), 73.6
(CH2 -Ph), 73.2 (CH2 -Ph), 71.8 (CH2 -Ph), 71.0 (C-5), 70.3 (CH2 ), 69.7 (CH2 ), 66.4 (C-4), 48.7
(NH-CH2 -CH2 ), 46.2 (C-1) ppm; [α]25 D = +14.9 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for
[C58 H72 N2 O10 + H]+ : 957.5265, found 957.5270.
(11e) According to general procedure, the reaction of 10e (0.578 g, 0.564 mmol) and
purification (eluent: EtOAc/MeOH gradient, 80/20 to 50/50) 11e (336 mg, 0.327 mmol,
(m, 12 H, 6 × CH2 -Ph), 4.10–4.07 (m, 2 H, 2 × 4-H), 3.77 (dm, J = 5.8 Hz, 2 H, 2 × 3-H),
3.69–3.67 (m, 2 H, 2 × 2-H), 3.65–3.45 (m, 16 H, 2 × 5-H + 6 × CH2 ), 2.91 (dd, J = 12.2,
1.1 Hz, 2 H, 2 × 1-Ha Hb ), 2.82 (dd, J = 12.2, 4.9 Hz, 2 H, 2 × 1-Ha Hb ), 2.75–2.64 (m, 4 H,
2 × NH-CH2 -CH2 ), 1.79–1.74 (m, 4 H, 2 × NH-CH2 -CH2 ) ppm; 13 C NMR (125 MHz,
CDCl3 ): δ 138.5 (CIV ), 138.3 (CIV ), 138.1 (CIV ), 128.4, 128.4, 128.3, 128.3, 128.3, 128.0, 127.8,
127.8, 127.7, 127.7, 127.5 (C-Ar), 76.6 (C-3), 76.0 (C-2), 73.5 (CH2 -Ph), 73.1 (CH2 -Ph), 71.8
(CH2 -Ph), 70.9 (C-5), 70.5 (CH2 ), 70.1 (CH2 ), 69.5 (CH2 ), 66.3 (C-4), 46.9 (NH-CH2 -CH2 ),
46.4 (C-1), 29.2 (NH-CH2 -CH2 ) ppm; [α]25 D = +8.0 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for
[C62 H80 N2 O11 + H]+ : 1029.5840, found 1029.5833.
3.4. Representative Procedure for the Synthesis of Dimeric Iminosugars 12a,b,d

To a solution of 11a,b,d (1 equiv) in pyridine at 0 ◦ C was added dropwise methanesul-
fonyl chloride (2.4 equiv). The mixture was stirred for 2.5 h at the same temperature then the
reaction was quenched with water (10 mL) and extracted with ethyl acetate (3 × 5 mL). The
organic phase was dried with magnesium sulfate, filtered, and concentrated under reduced
pressure, and finally, the crude was purified by column chromatography on silica gel.
methanesulfonyl chloride (38 µL, 0.475 mmol) in pyridine (2.5 mL) afforded after purifica-
tion (eluent: EtOAc/PE gradient, 20/80 to 30/70) 12a (33 mg, 0.036 mmol, 19%) as a yellow
oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.33–7.27 (m, 34 H, Ar-H), 4.54–4.53 (m, 8 H, 4 × CH2 -Ph),
4.46 (d, J = 12.2 Hz, 2 H, 2 × CHa Hb Ph), 4.38 (d, J = 12.2 Hz, 2 H, 2 × CHa Hb Ph), 4.14 (d,
J = 12.2 Hz, 2 H, 2 × N-CHa Hb Ar), 3.93–3.91 (m, 4 H, 2 × 3-H + 2 × 4-H), 3.64–3.62 (m, 4 H,
2 × 6-H), 3.49 (d, J = 12.2 Hz, 2 H, 2 × N-CHa Hb Ar), 3.05 (d, J = 10.7 Hz, 2 H, 2 × 2-Ha Hb ),
2.89 (q, J = 5.2 Hz, 2 H, 2 × 5-H), 2.58 (dd, J = 10.7, 5.1 Hz, 2 H, 2 × 2-Ha Hb ) ppm; 13 C NMR
(125 MHz, CDCl3 ): δ 138.7 (CIV ), 138.6 (CIV ), 138.4 (CIV ), 129.7, 128.5, 128.4, 128.2, 127.9,
127.9, 127.8, 127.7, 127.7, 127.6 (C-Ar), 86.1 (C-4), 81.7 (C-3), 73.3 (CH2 -Ph), 71.5 (CH2 -Ph),
71.4 (C-6), 71.0 (CH2 -Ph), 68.6 (C-5), 59.2 (N-CH2 -Ar), 57.1 (C-2) ppm; [α]25 D = +22.2 (c 1,
CHCl3 ). HRMS (ESI) m/z calcd for [C60 H64 N2 O6 + H]+ : 909.4843, found 909.4849.
methanesulfonyl chloride (33 µL, 0.422 mmol) in pyridine (2 mL) afforded after purification
Molecules 2025, 30, 226 13 of 21
(eluent: EtOAc/PE gradient, 20/80 to 30/70) 12b (65 mg, 0.071 mmol, 41%) as a yellow oil.
1H NMR (500 MHz, CDCl3 ): δ 7.34–7.27 (m, 34 H, Ar-H), 4.54–4.53 (m, 8 H, 4 × CH2 -Ph),
4.47 (d, J = 12.2 Hz, 2 H, 2 × CHa Hb Ph), 4.39 (d, J = 12.2 Hz, 2 H, 2 × CHa Hb Ph), 4.13 (d,
J = 13.2 Hz, 2 H, 2 × N-CHa Hb Ar), 3.94–3.92 (m, 4 H, 2 × 3-H + 2 × 4-H), 3.65–3.61 (m, 4 H,
2 × 6-H), 3.49 (d, J = 13.2 Hz, 2 H, 2 × N-CHa Hb Ar), 3.06 (d, J = 10.7 Hz, 2 H, 2 × 2-Ha Hb ),
2.88 (q, J = 5.4 Hz, 2 H, 2 × 5-H), 2.58 (dd, J = 10.7, 5.2 Hz, 2 H, 2 × 2-Ha Hb ) ppm; 13 C
NMR (125 MHz, CDCl3 ): δ 138.6 (CIV ), 138.4 (CIV ), 137.5 (CIV ), 129.0, 128.5, 127.9, 127.8,
127.7, 127.7, 127.6 (C-Ar), 86.0 (C-4), 81.7 (C-3), 73.4 (CH2 -Ph), 71.6 (CH2 -Ph), 71.4 (C-6),
71.0 (CH2 -Ph), 68.6 (C-5), 59.0 (N-CH2 -Ar), 57.1 (C-2) ppm; [α]25 D = +18.0 (c 1, CHCl3 ).
(12d) According to general procedure, the reaction of 11d (197 mg, 0.206 mmol) and
methanesulfonyl chloride (39 µL, 0.494 mmol) in pyridine (3 mL) afforded after purification
(eluent: EtOAc/PE 70/30) 12d (66 mg, 0.072 mmol, 35%) as a yellow oil. 1 H NMR (500 MHz,
CDCl3 ): δ 7.37–7.28 (m, 30 H, Ar-H), 4.57–4.50 (m, 10 H, 4 × CH2 -Ph + 2 × CHa Hb -Ph), 4.46
(d, J = 12.3 Hz, 2 H, 2 × CHa Hb Ph), 3.94 (d, J = 5 Hz, 2 H, 2 × 3-H), 3.89 (d, J = 3.9 Hz, 2 H,
2 × 4-H), 3.64–3.53 (m, 12 H, 2 × 6-H + 4 × CH2 ), 3.27 (d, J = 10.6 Hz, 2 H, 2 × 2-Ha Hb ),
3.11 (dt, J = 12.7, 6.3 Hz, 2 H, 2 × N-CHa Hb -CH2 ), 2.82 (q, J = 5.0 Hz, 2 H, 2 × 5-H), 2.71 (dd,
J = 10.6, 5.1 Hz, 2 H, 2 × 2-Ha Hb ), 2.65 (dt, J = 12.7, 6.3 Hz, 2 H, 2 × N-CHa Hb -CH2 ) ppm;
13 C NMR (125 MHz, CDCl ): δ 138.5 (CIV ), 138.4 (CIV ), 138.3 (CIV ), 128.4, 127.9, 127.9, 127.9,
3
127.8, 127.7, 127.7, 127.7, 127.6 (C-Ar), 85.3 (C-4), 81.8 (C-3), 73.3 (CH2 -Ph), 71.4 (CH2 -Ph),
71.1 (CH2 -Ph), 71.0 (C-6), 70.4 (CH2 ), 70.3 (CH2 ), 69.5 (C-5), 58.2 (C-2), 54.5 (N-CH2 -CH2 )
ppm; [α]25 +
D = −14.1 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C58 H69 N2 O8 + H] : 921.5054,
found 921.5046.
3.5. Representative Procedure for the Synthesis of Dimeric Iminosugars 12c,e

Step 1, protection of amine functions: To a solution of 11c,e (1 equiv) in anhydrous
methanol (10 mL) at room temperature were successively added triethylamine (4 equiv), 4-
(dimethylamino)pyridine (0.2 equiv) and di-tert-butyl dicarbonate (6 equiv). The resulting
mixture was stirred until the total consumption of the starting material (typically 12 h for
11c and 1h for 11e) then the reaction was quenched with water (10 mL) and extracted with
dichloromethane (3 × 5 mL). The organic phase was dried with magnesium sulfate, filtered,
and concentrated under reduced pressure. The resulting product was used in the next step
without further purification.
Step 2, O-mesylation: To the crude obtained in step 1 was added dropwise at 0 ◦ C
triethylamine (20 equiv) then methanesulfonyl chloride (16 equiv). The mixture was
warmed to room temperature and was stirred for 40 min. The reaction was quenched
with water (10 mL) and extracted with dichloromethane (3 × 5 mL). The organic phase
was dried with MgSO4 , filtered, and concentrated under reduced pressure. The resulting
product was used in the last step without further purification.
Step 3, cyclization: To the crude obtained in step 2 and dissolved in ethanol (20 mL)
was added hydrochloric acid 37% in water (18 mL). Then, the solution was stirred for 12 h at
60 ◦ C (11c) or 29 h at room temperature (11e). Ethanol was removed under reduced pressure,
the crude was diluted with dichloromethane (40 mL) and washed with a saturated aqueous
solution of sodium hydrogen carbonate until the acid was neutralized. The organic phase
was dried with MgSO4 , filtered, and concentrated under reduced pressure, and finally, the
crude was purified by column chromatography on silica gel.
(12c) According to general procedure, the reaction of 11c (553 mg, 0.580 mmol) afforded
after the three steps and purification (eluent: EtOAc/PE 20/80) 12c (302 mg, 0.329 mmol,
(m, 10 H, 4 × CH2 -Ph + 2 × CHa Hb -Ph), 4.47 (d, J = 12.3 Hz, 2 H, 2 × CHa Hb Ph), 3.94
(d, J = 5 Hz, 2 H, 2 × 3-H), 3.92 (d, J = 3.6 Hz, 2 H, 2 × 4-H), 3.62 (dd, J = 9.5, 4.8 Hz,
Molecules 2025, 30, 226 14 of 21
2 H, 2 × 6-Ha Hb ), 3.55 (dd, J = 9.5, 6.9 Hz, 2 H, 2 × 6-Ha Hb ), 3.25 (d, J = 10.3 Hz, 2 H,
2 × 2-Ha Hb ), 2.88–2.82 (m, 2 H, 2 × N-CHa Hb -CH2 ), 2.75–2.72 (m, 2 H, 2 × 5-H), 2.58
(dd, J = 10.4, 5.0 Hz, 2 H, 2 × 2-Ha Hb ), 2.37–2.31 (m, 2 H, 2 × N-CHa Hb -CH2 ), 1.54–1.48
(m, 4H, 2 × N-CH2 -CH2 ), 1.31–1.28 (m, 8H, 4 × CH2 ) ppm; 13 C NMR (125 MHz, CDCl3 ):
δ 138.6 (CIV ), 138.4 (CIV ), 138.4 (CIV ), 128.5, 128.5, 128.4, 128.4, 128.4, 127.9, 127.9, 127.9,
127.7, 127.6 (C-Ar), 85.6 (C-4), 81.7 (C-3), 73.3 (CH2 -Ph), 71.4 (CH2 -Ph), 71.1 (C-6), 71.0
(CH2 -Ph), 69.5 (C-5), 57.4 (C-2), 55.8 (N-CH2 -CH2 ), 29.7 (CH2 ), 28.3 (N-CH2 -CH2 ), 27.7
(CH2 ) ppm; [α]25D = −26.6 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C60 H72 N2 O6 + H] :
+
917.5469, found 917.5472.

(12e) According to general procedure, the reaction of 11e (1.7 g, 1.672 mmol) afforded
after the three steps and purification (eluent: EtOAc/PE gradient, 50/50 to 100/00) 12e
(349 mg, 0.352 mmol, 21%) as a yellow oil. 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.27 (m,
30 H, Ar-H), 4.57–4.49 (m, 10 H, 4 × CH2 -Ph + 2 × CHa Hb -Ph), 4.45 (d, J = 12.3 Hz, 2 H,
2 × CHa Hb Ph), 3.94 (d, J = 5.1 Hz, 2 H, 2 × 3-H), 3.91 (d, J = 3.9 Hz, 2 H, 2 × 4-H), 3.65–3.47
(m, 16 H, 6 × CH2 + 2 × 6-H), 3.23 (d, J = 10.3 Hz, 2 H, 2 × 2-Ha Hb ), 2.95 (dt, J = 11.9,
8.0 Hz, 2 H, 2 × N-CHa Hb -CH2 ), 2.76–2.73 (m, 2 H, 2 × 5-H), 2.59 (dd, J = 10.4, 5.1 Hz,
2 H, 2 × 2-Ha Hb ), 2.43 (dt, J = 12.4, 6.7 Hz, 2 H, 2 × N-CHa Hb -CH2 ), 1.80 (dq, J = 14.4,
7.0 Hz, 4 H, 2 × N-CH2 -CH2 ) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.6 (CIV ), 138.4
(CIV ), 138.4 (CIV ), 128.5, 128.5, 128.4, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7,
127.6 (C-Ar), 85.6 (C-4), 81.7 (C-3), 73.3 (CH2 -Ph), 71.4 (CH2 -Ph), 71.1 (C-6 + CH2 -Ph), 70.7
(CH2 ), 70.3 (CH2 ), 69.8 (CH2 ), 69.5 (C-5), 57.3 (C-2), 52.4 (N-CH2 -CH2 ), 28.4 (N-CH2 -CH2 )
ppm; [α]25 +
D = −29.3 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C62 H76 N2 O9 + H] : 993.5629,
found 993.5627.
3.6. Representative Procedure for the Synthesis of Deprotected Bis-Iminosugars 13a–e

To a solution of dimeric iminosugars 12a–e (1 equiv) in anhydrous dichloromethane at
0 ◦ C and under argon atmosphere was added dropwise boron trichloride solution 1 M in
dichloromethane (10 equiv). The mixture was stirred for 2 h 30 at the same temperature
and then was quenched with methanol (3 mL). The solvent was removed under reduced
pressure and the residue was washed with diethyl ether (3 × 1 mL) and then chloro-
form (1.5 mL). Finally, the crude was purified by column chromatography on silica gel
(13a–c,e) or passed through an ion exchange resin (DOWEX 50WX8 (NH4 + ) column (Sigma-
Aldrich: St. Louis, MO, USA), elution with a solution of 0.2% ammonium bicarbonate) and
lyophilized (13d).
boron trichloride solution 1 M in dichloromethane (630 µL, 0.630 mmol) in dichloromethane
(0.5 mL) afforded after purification (eluent: CHCl3 /MeOH/NH4 OH 0.8 M 60/40/10) 13a
(8.3 mg, 0.023 mmol, 36%) as a yellow oil. 1 H NMR (500 MHz, D2 O): δ 7.55–7.54 (m, 4 H,
Ar-H), 4.38 (d, J = 12.7 Hz, 2 H, 2 × N-CHa Hb Ar), 4.21–4.20 (m, 2 H, 3-H), 4.10–4.05 (m,
4 H, N-CHa Hb Ar + 4-H), 3.83 (dd, J = 12.1, 5.4 Hz, 2 H, 2 × 6-Ha Hb ), 3.77 (dd, J = 12.1,
6.1 Hz, 2 H, 2 × 6-Ha Hb ), 3.30–3.15 (m, 6 H, 2 × 2-H + 2 × 5-H) ppm; 13 C NMR (125 MHz,
D2 O): δ 133.3 (CIV ), 132.6, 131.3, 129.5 (C-Ar), 77.6 (C-4), 74.3 (C-3), 72.8 (C-5), 59.6 (C-6),
58.9 (N-CH2 -Ar), 58.3 (C-2) ppm; [α]25 D = +3.5 (c 1, MeOH). HRMS (ESI) m/z calcd for
[C18 H28 N2 O6 + H]+ : 369.2026, found 369.2025.
boron trichloride solution 1 M in dichloromethane (1.27 mL, 1.27 mmol) in dichloromethane
(2 mL) afforded after purification (eluent: CHCl3 /MeOH gradient, 75/25 to 60/40) 13b
(18 mg, 0.049 mmol, 39%) as a yellow oil. 1 H NMR (500 MHz, D2 O): δ 7.61 (s, 4 H, Ar-H),
4.57 (d, J = 12.8 Hz, 2 H, 2 × N-CHa Hb Ar), 4.34 (d, J = 12.8 Hz, 2 H, 2 × N-CHa Hb Ar),
4.29–4.28 (m, 2 H, 3-H), 4.11–4.10 (m, 2 H, 4-H), 3.89 (dd, J = 12.3, 5.2 Hz, 2 H, 2 × 6-Ha Hb ),
Molecules 2025, 30, 226 15 of 21
3.83 (dd, J = 12.3, 6.9 Hz, 2 H, 2 × 6-Ha Hb ), 3.54–3.49 (m, 4 H, 2 × 2-Ha Hb + 2 × 5-H),
3.37–3.34 (m, 2 H, 2 × 2-Ha Hb ) ppm; 13 C NMR (125 MHz, D2 O): δ 132.2 (CIV ), 131.6 (C-Ar),
76.7 (C-4), 73.8 (C-3), 73.7 (C-5), 59.0 (C-6), 58.9 (N-CH2 -Ar), 58.5 (C-2) ppm; [α]25 D = −1.4
(c 1, MeOH). HRMS (ESI) m/z calcd for [C18 H28 N2 O6 + H]+ : 369.2026, found 369.2023.
(13c) According to general procedure, the reaction of 12c (117 mg, 0.127 mmol) and
(2 mL) afforded after purification (eluent: CHCl3 /MeOH gradient, 70/30 to 50/50) 13c
(42 mg, 0.110 mmol, 87%) as a yellow oil. 1 H NMR (500 MHz, D2 O): δ 4.36–4.35 (m,
2 H, 3-H), 4.12–4.11 (m, 2 H, 4-H), 4.01 (dd, J = 12.5, 4.9 Hz, 2 H, 2 × 6-Ha Hb ), 3.94 (dd,
J = 12.5, 7.6 Hz, 2 H, 2 × 6-Ha Hb ), 3.68 (d, J = 12.4 Hz, 2 H, 2 × 2-Ha Hb ), 3.55–3.44 (m,
6 H, 2 × 2-Ha Hb + 2 × 5-H + 2 × N-CHa Hb -CH2 ), 3.24–3.17 (m, 2 H, 2 × N-CHa Hb -CH2 ),
1.78–1.72 (m, 4 H, 2 × N-CH2 -CH2 ), 1.40–1.37 (m, 8 H, 4 × CH2 ) ppm; 13 C NMR (125 MHz,
D2 O): δ 78.4 (C-4), 77.7 (C-5), 76.3 (C-3), 61.1 (C-2), 61.0 (C-6), 59.4 (N-CH2 -CH2 ), 30.4 (CH2 ),
28.1 (CH2 ), 27.1 (N-CH2 -CH2 ) ppm; [α]25 D = −18.9 (c 0.45, H2 O). HRMS (ESI) m/z calcd for
+
[C18 H36 N2 O6 + H] : 377.2652, found 377.2650.
(13d) According to general procedure, the reaction of 12d (90 mg, 0.098 mmol) and
boron trichloride solution 1 M in dichloromethane (980 µL, 0.98 mmol) in dichloromethane
(1 mL) afforded after purification 13d (13 mg, 0.034 mmol, 35%) as a yellow oil. 1 H
NMR (500 MHz, D2 O): δ 4.11 (dt, J = 5.4, 2.5 Hz, 2 H, 2 × 3-H), 3.92 (dd, J = 5.0,
2.8 Hz, 2 H, 2 × 4-H), 3.72–3.63 (m, 12 H, 2 × 6-H + 4 × CH2 ), 3.11–3.04 (m, 4 H,
2 × 2-Ha Hb + 2 × N-CHa Hb -CH2 ), 2.83–2.79 (m, 2 H, 2 × 2-Ha Hb ), 2.64–2.61 (m, 2 H,
2 × N-CHa Hb -CH2 ), 2.58 (q, J = 5.0 Hz, 2 H, 2 × 5-H) ppm; 13 C NMR (125 MHz, D2 O):
δ 78.8 (C-4), 75.4 (C-3), 72.0 (C-5), 69.4 (CH2 ), 68.6 (CH2 ), 60.9 (C-6), 58.8 (C-2), 53.8 (N-
CH2 -CH2 ) ppm; [α]25 D = −14.6 (c 1, MeOH). HRMS (ESI) m/z calcd for [C16 H32 N2 O8 + H] :
+
381.2237, found 381.2236.

(13e) According to general procedure, the reaction of 12e (277 mg, 0.279 mmol) and
(3 mL) afforded after purification (eluent: EtOAc/MeOH/NH4 OH 0.8 M 50/50/5) 13e
(78 mg, 0.173 mmol, 62%) as a yellow oil. 1 H NMR (500 MHz, D2 O): δ 4.33–4.32 (m, 2 H,
2 × 3-H), 4.10–4.09 (m, 2 H, 2 × 4-H), 3.98 (dd, J = 12.4, 4.9 Hz, 2 H, 2 × 6-Ha Hb ), 3.91 (dd,
J = 12.4, 6.8 Hz, 2 H, 2 × 6-Ha Hb ), 3.70–3.61 (m, 14 H, 2 × 2-Ha Hb + 6 × CH2 ), 3.52–3.56
(m, 2 H, 2 × N-CHa Hb -CH2 ), 3.41 (dd, J = 12.1, 4.4 Hz, 2 H, 2 × 2-Ha Hb ), 3.36–3.35 (m,
2 H, 2 × 5-H), 3.21–3.17 (m, 2 H, 2 × N-CHa Hb -CH2 ), 2.10–1.95 (m, 4 H, 2 × N-CH2 -CH2 )
ppm; 13 C NMR (125 MHz, D2 O): δ 76.5 (C-4), 74.7 (C-5), 74.0 (C-3), 69.5 (CH2 ), 69.5 (CH2 ),
68.4 (CH2 ), 58.7 (C-6), 58.6 (C-2), 54.2 (N-CH2 -CH2 ), 25.1 (N-CH2 -CH2 ) ppm; [α]25 D = –5.7 (c
+
0.37, H2 O). HRMS (ESI) m/z calcd for [C20 H40 N2 O9 + H] : 453.2812, found 453.2814.
3.7. Procedure for Allylation of Bis-Glycosylamine 10d

To a solution of dry lithium chloride (650 mg, 15.24 mmol, 6 equiv) in anhydrous
tetrahydrofuran (10 mL) at −78 ◦ C and under argon atmosphere was added dropwise
a solution of allyl magnesium chloride 2 M in tetrahydrofuran (7.62 mL, 15.24 mmol,
6 equiv). The mixture was stirred for 20 min then a solution of the bis-glycosylamine 10d
(2.42 g, 2.54 mmol, 1 equiv) in anhydrous tetrahydrofuran (20 mL) was added dropwise.
The mixture was stirred at −78 ◦ C for 30 min then was warmed at room temperature
and stirred until total consumption of starting material (typically 2 h). The reaction was
quenched with a saturated aqueous solution of ammonium chloride and extracted with
ethyl acetate (3 × 30 mL). The organic phase was dried with magnesium sulfate, filtered, and
concentrated under reduced pressure. The crude was purified by column chromatography
on silica gel (eluent: EtOAC/MeOH 97:3) to afford a mixture of 14d(R,R) and 14d(R,S)
(85/15) (2.24 g, 2.16 mmol, 85%) as a yellow oil.
Molecules 2025, 30, 226 16 of 21
(14d) 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.22 (m, 30 H, Ar-H), 5.80–5.72 (m, 1.7 H,
2 × 2-Hmaj ), 5.60–5.51 (m, 0.3 H, 2 × 2-Hmin ), 5.14–5.09 (m, 3.4 H, 2 × 1-Hmaj ), 5.02 (d,
J = 10.2 Hz, 0.3 H, 2 × 1-Ha Hb min ), 4.90 (d, J = 17 Hz, 0.3 H, 2 × 1-Ha Hb min ), 4.68–4.32
(m, 12 H, 6 × CH2 -Phmaj + 6 × CH2 -Phmin ), 4.16–4.13 (m, 0.3 H, 2 × 6-Hmin ), 4.08 (d,
J = 6.7 Hz, 1.7 H, 2 × 6-Hmaj ), 3.83–3.81 (m, 2 H, 2 × 7-Hmaj +2 × 7-Hmin ), 3.65–3.39
(m, 14 H, 2 × 5-Hmaj + 2 × 5-Hmin + 2 × 8-Hmaj + 2 × 8-Hmin + 4 × CH2 maj + 4 × CH2 min ),
3.06–2.93 (m, 4 H, 2 × 4-Hmaj + 2 × 4-Hmin + 2 × NH-CHa Hb maj + 2 × NH-CHa Hb min ), 2.81–
2.77 (m, 1.7 H, 2 × NH-CHa Hb maj ), 2.56–2.45 (m, 4 H, 2 × 3-Hmaj + 2 × 3-Ha Hb min + 2 × NH-
CHa Hb min ), 2.15–2.07 (m, 0.3 H, 2 × 3-Ha Hb min ) ppm; 13 C NMR (125 MHz, D2 O): δ 138.7
(CIV ), 138.6 (CIV ), 138.6 (CIV ), 138.5 (CIV ), 138.4 (CIV ), 138.3 (CIV ), 138.3 (CIV ), 138.3 (CIV ),
138.1 (CIV ), 138.0 (CIV ), 137.9 (CIV ), 135.7 (C-2min ), 135.3 (C-2maj ), 128.8, 128.7, 128.5, 128.4,
128.4, 128.3, 128.0, 127.9, 127.9, 127.8, 127.6, 127.6 (C-Ar), 118.2 (C-1maj ), 117.7 (C-1min ),
78.4 (C-7maj ), 77.1 (C-5min ), 75.1 (C-5maj + C-7min ), 73.9 (CH2 -Phmaj ), 73.6 (CH2 -Phmin ),
73.2 (CH2 -Phmin ), 73.2 (CH2 -Phmaj ), 73.0 (CH2 -Phmaj ), 72.8 (CH2 -Phmin ), 71.1 (C-8maj ), 70.8
(C-8min ), 70.3 (CH2 ), 70.2 (CH2 ), 67.2 (C-6maj ), 66.3 (C-6min ), 58.9 (C-4min ), 54.5 (C-4maj ), 46.8
(NH-CH2 maj ), 45.6 (NH-CH2 min ), 34.6 (C-3min ), 34.2 (C-3maj ) ppm; HRMS (ESI) m/z calcd
for [C64 H80 N2 O10 + H]+ : 1037.5891, found 1037.5898.
3.8. Procedure for the Synthesis of Bis-Allylpyrrolidines 15d(R,R) and 15d(R,S)

To a solution of the bis-glycosylamines 14d(R,R) and 14d(R,S) (2.24 g, 2.16 mmol,
1 equiv) in anhydrous tetrahydrofuran (10 mL) under argon atmosphere was added pyri-
dine (10 mL). The mixture was cooled to −78 ◦ C then methanesulfonyl chloride (0.84 mL,
10.81 mmol, 5 equiv) was added dropwise. The mixture was stirred for 1 h at the same
temperature, and then was warmed to 0 ◦ C and stirred for another 2 h. The reaction was
quenched with water (10 mL) and extracted with ethyl acetate (4 × 40 mL). The organic
phase was dried with magnesium sulfate, filtered, and concentrated under reduced pres-
sure, and finally, the crude was purified by column chromatography on silica gel (eluent:
PE/EtOAc 70/30) to afford 15d(R,R) (731 mg, 0.731 mmol), 15d(R,S) (100 mg, 0.1 mmol)
and a mixture of the two diastereomers (869 mg, 0.869 mmol, 15d(R,R)/15d(R,S): 83/17)).
Global yield 79% yellow oils.
(15d(R,R)) 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.28 (m, 30 H, Ar-H), 5.83–5.74 (m,
2 H, 2 × 2a-H), 5.08–5.05 (m, 4 H, 2 × 3a-H), 4.57–4.41 (m, 12 H, 6 × CH2 -Ph), 3.90 (s, 2 H,
2 × 4-H), 3.80 (s, 2 H, 2 × 3-H), 3.70–3.52 (m, 12 H, 2 × 6-H + 4 × CH2 ), 3.29–3.26 (m,
2 H, 2 × 5-H), 3.19–3.17 (m, 2 H, 2 × 2-H), 3.01–2.91 (m, 4 H, 2 × N-CH2 ), 2.54–2.49 (m,
2 H, 2 × 1a-Ha Hb ), 2.23–2.17 (m, 2 H, 2 × 1a-Ha Hb ) ppm; 13 C NMR (125 MHz, D2 O): δ
138.5 (CIV ), 135.6 (C-2a), 128.4, 128.4, 128.4, 128.0, 127.9, 127.9, 127.8, 127.6, (C-Ar), 117.2
(C-3a), 85.7 (C-3), 85.6 (C-4), 73.4 (CH2 -Ph), 71.4 (CH2 -Ph), 71.3 (CH2 -Ph), 70.6 (CH2 ), 70.5
(CH2 ), 69.5 (C-6), 66.1 (C-5), 65.7 (C-2), 46.7 (N-CH2 ), 32.1 (C-1a) ppm; [α]25 D = −8.4 (c 1,
+
MeOH). HRMS (ESI) m/z calcd for [C64 H76 N2 O8 + H] : 1001.5680, found 1001.5682.
(15d(R,S)) 1 H NMR (500 MHz, CDCl3 ): δ 7.38–7.25 (m, 30 H, Ar-H), 5.82–5.68 (m,
2 H, 2a-H + 2a′ -H), 5.10–5.04 (m, 3 H, 3a-H + 3a′ -Ha Hb ), 4.99 (dd, J = 10.2, 1.5 Hz, 1 H,
3a′ -Ha Hb ), 4.62–4.41 (m, 11 H, 5 × CH2 -Ph + CHa Hb -Ph), 4.32 (d, J = 11.7 Hz, 1 H, CHa Hb -
Ph), 3.89–3.88 (m, 2 H, 4-H + 4′ -H), 3.79–3.77 (m, 2 H, 3-H + 3′ -H), 3.69–3.51 (m, 11 H,
6-H + 6′ -Ha Hb + 4 × CH2 ), 3.43 (t, J = 9.3 Hz, 1 H, 6′ -Ha Hb ), 3.28–3.25 (m, 1 H, 5-H),
3.18–3.16 (m, 1 H, 2-H), 3.14–3.10 (m, 1 H, 5′ -H), 3.09–3.05 (m, 1 H, 2′ -H), 3.04–2.87 (m,
4 H, 2 × N-CH2 ), 2.53–2.43 (m, 2 H, 1a-Ha Hb + 1a′ -Ha Hb ), 2.34–2.29 (m, 1 H, 1a′ -Ha Hb ),
2.23–2.16 (m, 1 H, 1a-Ha Hb ) ppm; 13 C NMR (125 MHz, D2 O): δ 138.6 (CIV ), 138.5 (CIV ),
138.65 (CIV ), 138.2 (CIV ), 136.1 (C-2a′ ), 135.6 (C-2a), 128.5, 128.5, 128.4, 128.4, 128.4, 128.3,
128.3, 128.3, 128.2, 128.2, 128.2, 128.2, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7,
127.7, 127.6, (C-Ar), 117.2 (C-3a), 116.5 (C-3a′ ), 85.7 (C-3), 85.6 (C-4), 82.3 (C-4′ ), 82.3 (C-3′ ),
Molecules 2025, 30, 226 17 of 21
73.4 (CH2 -Ph), 73.2 (CH2 ′ -Ph), 72.4 (C-6′ ), 71.7 (CH2 ′ -Ph), 71.4 (CH2 -Ph), 71.3 (CH2 -Ph), 70.7
(CH2 ′ -Ph), 70.5 (CH2 ), 70.2 (CH2 ), 69.7 (C-5′ ), 69.5 (C-6), 66.9 (C-2′ ), 66.1 (C-5), 65.7 (C-2),
53.1 (N-CH2 ′ ), 46.7 (N-CH2 ), 32.8 (C-1a′ ), 32.1 (C-1a) ppm; [α]25 D = +1.9 (c 1, MeOH). HRMS
3.9. Procedure for the Synthesis of Deprotected Bis-Allylpyrrolidine 16d(R,R)

According to general procedure described in 3.6, the reaction of 15d(R,R) (27 mg,
0.027 mmol, 1 equiv) and boron trichloride solution (270 µL, 0.27 mmol) in anhydrous
dichloromethane (3 mL) afforded after purification (eluent: AcOEt/MeOH/NH4 OH 0.8 M
40/60/5) 16d(R,R) (7 mg, 0.015 mmol, 56%) as a yellow oil.
(16d(R,R)) 1 H NMR (600 MHz, D2 O): δ 5.91–5.84 (m, 2 H, 2 × 2a-H), 5.19 (dd, J = 19.9,
13.8 Hz, 4 H, 2 × 3a-H), 3.96–3.94 (s, 2 H, 2 × 4-H), 3.91–3.89 (s, 2 H, 2 × 3-H), 3.80–3.74 (m,
4 H, 2 × 6-H), 3.70–3.63 (m, 8 H, 4 × CH2 ), 3.05 (dt, J = 8.7, 4.0 Hz, 2 H, 2 × 2-H), 2.99–2.91
(m, 6 H, 2 × 5-H + 2 × N-CH2 ), 2.43 (dt, J = 14.0, 4.9 Hz, 2 H, 2 × 1a-Ha Hb ), 2.12 (dt, J = 14.0,
8.8 Hz, 2 × 1a-Ha Hb ) ppm; 13 C NMR (151 MHz, D2 O): δ 135.1 (C-2a), 117.6 (C-3a), 79.7
(C-3), 79.1 (C-4), 69.6 (CH2 ), 68.7 (CH2 ), 68.2 (C-5), 66.2 (C-2), 59.7 (C-6), 45.7 (N-CH2 ), 31.4
(C-1a) ppm; [α]25D = −17.6 (c 0.10, H2 O). HRMS (ESI) m/z calcd for [C22 H40 N2 O8 + Na] :
+
483.2682, found 483.2691.
3.10. Procedure for the Synthesis of 19d(R,R) and 19d(R,S)

To a solution of 14d(R,R) and 14d(R,S) (85/15) (150 mg, 0,145 mmol, 1 equiv) in
anhydrous dichloromethane (10 mL) at 0 ◦ C and under argon atmosphere was added
dropwise boron trichloride solution 1 M in dichloromethane (1.45 mL, 1.45 mmol, 10 equiv).
The mixture was stirred for 2 h 30 at the same temperature and then was quenched with
methanol (2 mL). The solvent was removed under reduced pressure and the residue was
washed with chloroform (3 × 1 mL) then dichloromethane (3 × 1 mL) to afford a mixture
of 19d(R,R) and 19(R,S) (85/15) (55 mg, 0.112 mmol, 77%) as a yellow oil.
(19d(R,R)) 1 H NMR (500 MHz, MeOD): δ 5.92–5.77 (m, 2 H, 2 × 2-H), 5.29 (d,
J = 17.1 Hz, 2 H, 2 × 1-Ha Hb ), 5.19 (d, J = 10.2 Hz, 2 H, 2 × 1-Ha Hb ), 3.98 (dd,
J = 5.3, 3.1 Hz, 2 H, 2 × 5-H), 3.87 (t, J = 3.3 Hz, 2 H, 2 × 6-H), 3.82–3.55 (m, 14 H,
2 × 7-H + 2 × 8-H + 4 × CH2 ), 3.52 (q, J = 6.5 Hz, 2 H, 2 × 4-H), 3.42–3.34 (m, 2 H,
2 × NH-CHa Hb ), 3.30–3.25 (m, 2 H, 2 × NH-CHa Hb ), 2.68–2.54 (m, 4 H, 2 × 3-H) ppm; 13 C
NMR (125 MHz, MeOD): δ 133.8 (C-2), 120.3 (C-1), 72.9 (C-7), 71.8 (C-6), 71.4 (CH2 ), 70.1
(C-5), 66.8 (CH2 ), 64.0 (C-8), 62.8 (C-4), 47.7 (NH-CH2 ), 33.3 (C-3) ppm; HRMS (ESI) m/z
calcd for [C22 H44 N2 O10 + H]+ : 497.3074, found 497.3070.
3.11. Procedure for Ethynylation of Bis-Glycosylamine 10d

To a solution of trimethylsilylacetylene (1.65 mL, 11.6 mmol, 8 equiv) in anhydrous
tetrahydrofuran (10 mL) at 0 ◦ C and under argon atmosphere was added dropwise ethyl
magnesium bromide 3 M in diethylether (3.9 mL, 11.6 mmol, 8 equiv). The mixture was
stirred for 30 min at the same temperature then a solution of the bis-glycosylamine 10d
(1.38 g, 1.45 mmol, 1 equiv) in anhydrous tetrahydrofuran (20 mL) was added dropwise.
The mixture was stirred at 0 ◦ C for 30 min then was finally warmed at room temperature
and stirred for 16 h. The reaction was quenched with a saturated aqueous solution of
ammonium chloride and extracted with ethyl acetate (2 × 40 mL). The organic phase was
dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The
crude was purified by column chromatography on silica gel (eluent: PE/EtOAc 70/30)
to afford 20d (605 mg, 0.53 mmol, 37%) as a mixture of two diastereoisomers (95:5) and a
yellow oil.
(20d) 1 H NMR (500 MHz, CDCl3 ) major diastereomer: δ 7.36–7.27 (m, 28 H, Ar-H),
7.18–7.16 (m, 2 H, Ar-H), 4.73 (d, J = 11.8 Hz, 2 H, 2 × CHa Hb -Ph), 4.64 (d, J = 11.8 Hz,
Molecules 2025, 30, 226 18 of 21
2 H, 2 × CHa Hb -Ph), 4.51 (d, J = 12.0 Hz, 2 H, 2 × CHa Hb -Ph), 4.46 (d, J = 12.0 Hz,
2 H, 2 × CHa Hb -Ph), 4.42 (d, J = 12.1 Hz, 2 H, 2 × CHa Hb -Ph), 4.38 (d, J = 12.1 Hz, 2 H,
2 × CHa Hb -Ph), 4.09 (dd, J = 8.1, 5.8 Hz, 2 H, 2 × 6-H), 3.81–3.79 (m, 4 H, 2 × 3-H + 2 × 4-H),
3.63–3.44 (m, 14 H, 2 × 5-H + 2 × 7-H + 4 × CH2 ), 3.22–3.17 (m, 2 H, 2 × NH-CHa Hb ), 2.62
(dt, J = 12.1, 4.2 Hz, 2 H, 2 × NH-CHa Hb ), 0.2 (s, 18 H, 2 × Si(CH3 )3 ) ppm; 13 C NMR (125
MHz, CDCl3 ) major diastereomer: δ 138.7 (CIV ), 138.3 (CIV ), 138.0 (CIV ), 128.6, 128.5, 128.5,
128.4, 128.4, 128.3, 128.2, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.5 (C-Ar), 106.5 (CIV ,
C≡C-Si), 89.0 (CIV , C≡C-Si), 79.7 (C-4), 76.4 (C-5), 74.2 (CH2 -Ph), 73.7 (CH2 -Ph), 73.1 (CH2 -
Ph), 70.6 (C-7), 70.2 (CH2 ), 70.0 (CH2 ), 65.9 (C-6), 48.3 (C-3), 46.4 (NH-CH2 ), 0.1 (Si(CH3 )3 )
ppm; HRMS (ESI) m/z calcd for [C68 H88 N2 O10 Si2 + H]+ : 1149.6056, found 1149.6057.
3.12. Procedure for the Synthesis of Bis-Ethynylpyrrolidine 21d

Step 1, O-mesylation and cyclization: according to the procedure described in 3.8, the
reaction of 20d (550 mg, 0.48 mmol) and methanesulfonyl chloride (0.19 mL, 2.4 mmol) in
pyridine (3 mL) and THF (3 mL) afforded the cyclized intermediate (235 mg, 0.21 mmol,
44%) as a yellow oil after purification (eluent: PE/EtOAc 90/10).
Step 2, TMS removing: To a solution of the pyrrolidine obtained in step 1 (200 mg,
0.18 mmol, 1 equiv) in anhydrous tetrahydrofuran (10 mL) was added a solution of tetra-
butylammonium fluoride 1 M in tetrahydrofuran (0.45 mL, 0.447 mmol, 2.5 equiv). The
mixture was stirred for 3 h at room temperature and then was concentrated under reduced
pressure. The crude product was purified by column chromatography on silica gel (eluent:
EP/EtOAc 70:30) to afford 21d (140 mg, 0.144 mmol, 69%) as a colorless oil.
(21d) 1 H NMR (500 MHz, CDCl3 ): δ 7.39–7.26 (m, 30 H, Ar-H), 4.64–4.58 (m, 6 H,
2 × CH2 Ph + 2 × CHa Hb -Ph), 4.54–4.45 (m, 6 H, 2 × CH2 Ph + 2 × CHa Hb -Ph), 3.90–3.89
(m, 4 H, 2 × 2-H + 2 × 3-H), 3.87–3.86 (m, 2 H, 2 × 4-H), 3.74–3.67 (m, 4 H, 2 × CH2 ),
3.63–3.53 (m, 8 H, 2 × CH2 + 6-H), 3.17–3.03 (m, 6 H, 2 × N-CH2 + 2 × 5-H), 2.39 (s, 2 H,
2 × C≡CH) ppm; 13 C NMR (125 MHz, CDCl3 ): δ 138.5 (CIV ), 138.1 (CIV ), 138.0 (CIV ), 128.4,
128.3, 128.3, 128.3, 128.0, 127.8, 127.8, 127.7, 127.6, 127.6, 127.6, 127.5 (C-Ar), 83.6 (C-4),
82.3 (C-3), 81.2 (CIV , C≡CH), 73.7 (C≡CH), 73.1 (CH2 -Ph), 72.1 (CH2 -Ph), 71.5 (C-6), 70.9
(CH2 -Ph), 70.3 (CH2 ), 69.5 (CH2 ), 67.6 (C-5), 58.5 (C-2), 52.1 (N-CH2 ) ppm; [α]25
D = −13.6
(c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C62 H68 N2 O8 + H]+ : 969.5054, found 969.5056.
3.13. Procedure for the Synthesis of Glycophane 22d

To a solution of the bis-ethynylpyrrolidine 21d (80 mg, 0.083 mmol, 1 equiv) in a
mixture of ethanol (20 mL) and chloroform (5 mL) were successively added pyridine
(340 µL, 0.42 mmol, 5 equiv), triethylamine (350 µL, 0.25 mmol, 3 equiv), copper(II) acetate
monohydrate (17 mg, 0.083 mmol, 1 equiv) and nickel chloride (11 mg, 0.083 mmol, 1 equiv).
The mixture was heated to 60 ◦ C and stirred for 16 h. The solvents were removed under
reduced pressure and the crude was purified by column chromatography on silica gel
(eluent: EP/EtOAc 70/30) to afford 22d (67 mg, 0.068 mmol, 82%) as a colorless oil.
(22d) 1 H NMR (500 MHz, CDCl3 ): δ 7.37–7.27 (m, 26 H, Ar-H), 7.21–7.19 (m, 4 H,
Ar-H), 4.65 (d, J = 12.5 Hz, 2 H, 2 × CHa Hb -Ph), 4.57 (d, J = 12.5 Hz, 2 H, 2 × CHa Hb -Ph),
4.54 (d, J = 12.0 Hz, 2 H, 2 × CHa Hb -Ph), 4.47 (d, J = 12.0 Hz, 2 H, 2 × CHa Hb -Ph), 4.41
(d, J = 12.1 Hz, 2 H, 2 × CHa Hb -Ph), 4.38 (d, J = 12.1 Hz, 2 H, 2 × CHa Hb -Ph), 3.85–3.82
(m, 4 H, 2 × 2-H + 2 × 3-H), 3.74 (dd, J = 3.3, 1.2 Hz, 2 H, 2 × 4-H), 3.60–3.48 (m, 6 H,
2 × CH2 + 2 × CHa Hb ), 3.60–3.50 (m, 6 H, 2 × 6-H + 2 × CHa Hb ), 3.09–3.04 (m, 2 H,
2 × N-CHa Hb ), 2.91–2.83 (m, 4 H, 2 × 5-H + 2 × N-CHa Hb ) ppm; 13 C NMR (125 MHz,
CDCl3 ): δ 138.4 (CIV ), 138.0 (CIV ), 138.0 (CIV ), 128.5, 128.5, 128.5, 128.3, 127.9, 127.8, 127.8
(C-Ar), 83.9 (C-4), 82.5 (C-3), 78.0 (CIV , C≡C-C≡C), 73.3 (CH2 -Ph), 72.3 (CH2 -Ph), 71.7 (C-6),
71.4 (CH2 -Ph), 71.0 (CIV , C≡C-C≡C), 70.9 (CH2 ), 70.0 (C-5), 69.7 (CH2 ), 60.6 (C-2), 54.5
Molecules 2025, 30, 226 19 of 21
(N-CH2 ) ppm; [α]25 +

D = −84.2 (c 1, CHCl3 ). HRMS (ESI) m/z calcd for [C62 H67 N2 O8 + H] :
967.4897, found 967.4896.
3.14. Biological Assays Towards Glycosidases

All the enzymes were purchased from Sigma Chemical Co. In a typical experiment,
the glycosidase (0.013 U/mL) was pre-incubated at 33 ◦ C for 5 min in the presence of
the inhibitor in 50 mM acetate buffer (pH 5.6, except for rice α-glucosidase pH 5.1 and
yeast α-glucosidase pH 6.2). The reaction was started by the addition of the appropriate
substrate (p-nitrophenyl glycoside, 1 mM concentration) to a final volume of 250 µL. The
reaction was stopped after 10–15 min (depending on the enzyme) by the addition of 350 µL
of 0.4 M Na2 CO3 . The released p-nitrophenolate was quantified spectrophotometrically
at 415 nm with a microplate reader (300 µL of the reaction mixture per well). In cases
where inhibition was greater than 95%, IC50 values were determined further after assaying
decreasing concentrations of inhibitor. All the assays were performed in duplicate (less
than 10% variability in each case).
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules30020226/s1, analytical data of isomerized compound
18d, 1 H NMR and 13 C NMR spectra of compounds 10a–e, 11a–e, 12a–e, 13a–e, 14d, 15d(R,R) (addi-
tional NOESY), 15d(R,S), 16d(R,R), 18d, 19d, 20d, 21d (additional NOESY), 22d are available.
Author Contributions: Conceptualization, J.-B.B. and S.P.V.; syntheses, K.O.L. and N.N.; biological
assays, J.-B.B.; analyses, F.M. and J.-L.V.; writing—original draft preparation, J.-B.B. and F.M.; writing—
review and editing, F.M., J.-B.B., J.-L.V. and S.P.V.; project administration, funding acquisition, S.P.V.
and J.-B.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available in the
Supplementary Materials of this article.
Acknowledgments: The authors gratefully acknowledge support from the Univ. Reims Champagne
Ardenne (post-doctoral allocation to K.O.L.) and from CNRS. N.N. is grateful to URCA and Ecole
Doctorale ABIES for a doctoral allocation. We also thank Anthony Robert for his help in the structural
determination (NOESY experiments) of new compounds.
Conflicts of Interest: The authors declare no conflicts of interest.
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Article

A Bis-Glycosylamine Strategy for the Synthesis of Dimeric

1 Université de Reims Champagne-Ardenne, CNRS, ICMR, 51097 Reims, France;

Abstract: A straightforward synthetic route towards DAB-1 scaffolded dimeric iminosugars

Keywords: iminosugars; multivalency; glycosylamines; glycosidases; inhibition; glycophanes

Academic Editors: Francesca Cardona

Received: 9 December 2024

Molecules 2025, 30, 226 https://doi.org/10.3390/molecules30020226

Molecules 2025, 30, 226 2 of 21

(Scheme 1) [7]. As observed with simple reducing carbohydrates, mutarotation in

Molecules 2025, 30, 226 3 of 21

Figure 1. Structures of key compounds.

BnO OBn i BnO OBn BnO OBn

HO OH vii BnO OBn

9a: H2N NH 2 9b: H2 N 9c: H2 N NH 2

The incorporation of an additional substituent onto the final pyrrolidine structur

A cyclization protocol (MsCl in pyridine) was applied to the mixture

A cyclization protocol (MsCl in pyridine) was applied to the mixture 14d(R,R)/14d(R,S),

Scheme 4. SynthesisScheme 4. Synthesis of

Table 1. Glycosidase inhibition potencies of prepared compounds a,b .

Enzyme 13a 13b 13c 13d 13e 16d DAB-1

3. Materials and Methods

3.2. General Procedure for the Synthesis of Bis-Glycosylamines 10a–e

3.3. Representative Procedure for the Reduction of Bis-Glycosylamines 10a–e

3.4. Representative Procedure for the Synthesis of Dimeric Iminosugars 12a,b,d

3.5. Representative Procedure for the Synthesis of Dimeric Iminosugars 12c,e

917.5469, found 917.5472.

3.6. Representative Procedure for the Synthesis of Deprotected Bis-Iminosugars 13a–e

381.2237, found 381.2236.

3.7. Procedure for Allylation of Bis-Glycosylamine 10d

3.8. Procedure for the Synthesis of Bis-Allylpyrrolidines 15d(R,R) and 15d(R,S)

3.9. Procedure for the Synthesis of Deprotected Bis-Allylpyrrolidine 16d(R,R)

483.2682, found 483.2691.

3.10. Procedure for the Synthesis of 19d(R,R) and 19d(R,S)

3.11. Procedure for Ethynylation of Bis-Glycosylamine 10d

3.12. Procedure for the Synthesis of Bis-Ethynylpyrrolidine 21d

3.13. Procedure for the Synthesis of Glycophane 22d

(N-CH2 ) ppm; [α]25 +

3.14. Biological Assays Towards Glycosidases

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Conflicts of Interest: The authors declare no conflicts of interest.

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