New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis and SPR-based binding inhibition assays

Article abstract of DOI:10.1016/j.bioorg.2020.104566

The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has been implicated in the early stages of many infection processes. DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent potentially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antagonists, mimics of the natural moiety, characterized by the presence of a real D-carbamannose unit which confers greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important information about the influence of the lipophilic substituents present in specific positions of the carba scaffold. Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a more favorable IC₅₀ value than those of the previously described real carba-analogues. This study provides valuable knowledge for the implementation of further structural modifications towards improved inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104566

Bioorganic Chemistry 107 (2021) 104566
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis
and SPR-based binding inhibition assays
,
,
,
Sebastiano Di Pietroa *, Vittorio Bordonia 1, Dalila Iacopinib, Silvia Achillic 2, Mauro Pineschia,
c
c
a
b,*
´
Michel Thepaut , Franck Fieschi , Paolo Crotti , Valeria Di Bussolo
a
`
Dipartimento di Farmacia, Universita di Pisa, Via Bonanno 33, 56126 Pisa, Italy
b
`
Dipartimento di Chimica e Chimica Industriale, Universita di Pisa, Via Moruzzi 13, 56125 Pisa, Italy
c Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has
been implicated in the early stages of many infection processes.
Anti-infective agents
Carbohydrates
DC-SIGN
DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize
mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent poten-
Glycomimetics
Lipophilic interactions
Stereoselectivity
tially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antag-
onists, mimics of the natural moiety, characterized by the presence of a real D-carbamannose unit which confers
greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present
the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with
attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were
expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands
was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important
information about the influence of the lipophilic substituents present in specific positions of the carba scaffold.
Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a
more favorable IC50 value than those of the previously described real carba-analogues. This study provides
valuable knowledge for the implementation of further structural modifications towards improved inhibitors.
1. Introduction
On mucosal surfaces, where immature dendritic cells meet patho-
gens, DC-SIGN serves as one of the very first infective agents attachment
DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule- 3
(ICAM-3)-Grabbing Non-integrin) is a member of C-type lectin receptors
(CLRs) that acts as adhesion receptor, through ICAM-3 binding, allowing
interaction between dendritic cells (DCs) and resting T cells [1–3].
Besides the ICAM-3 binding, probably the most important feature of
DC-SIGN is its ability to bind a great number of important pathogens.
This is possible due to its ability to recognize mannose- and fucose-
containing glycans, which are overexpressed on a number of bacteria,
parasites and viruses (e.g. HIV, Dengue and Ebola virus, Leishmania and
Candida species, Mycobacterium tuberculosis, Helicobater pylori, and
Schistosoma mansoni), through its carbohydrate recognition domain
(CRD) [4–6].
point and the result of this interaction is pathogen internalization,
degradation and its exposure to recruit CD4+ T-cells and to start the
humoral immune response. Moreover, DC-SIGN can also function as an
antigen receptor to allow the DC-SIGN-ligand complexes to be targeted
to lysosomal compartments and to be processed for presentation to T
cells. The dual role of DC-SIGN as adhesion receptor and antigen re-
ceptor coexists throughout all their life span [7].
Therefore, the inhibition of pathogen-DC-SIGN interaction is an
attractive concept for the development of new anti-infective agents.
Indeed, inhibition of DC-SIGN by specific antagonists, such as glyco-
mimetics or antibodies, may prevent pathogen attachment to DC and
inhibit the infection of other immune cells at its earliest steps [8–16].
* Corresponding authors.
E-mail addresses: sebastiano.dipietro@unipi.it (S. Di Pietro), valeria.dibussolo@unipi.it (V. Di Bussolo).
Present address: Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.
Present address: Univ. Grenoble Alpes, CNRS, DCM UMR 5250, F-38000 Grenoble, France.
1
2
https://doi.org/10.1016/j.bioorg.2020.104566
Received 1 July 2020; Received in revised form 9 December 2020; Accepted 14 December 2020
Available online 19 December 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
The main natural ligand recognized by DC-SIGN is the high mannose
showed a good DC-SIGN binding profile, comparable to that of the
natural disaccharide [24]. Compounds 3 are at the crossroads between 1
and 2. However, the preservation of all hydroxyl groups in the pseudo
mannose unit support the capacity to promote multiple binding sites as
in the natural disaccharide, and potentially a direct proximity to the
Phe313 hydrophobic area of the mannose unit that is not engaged in
Ca2+ chelation.
glycan (Man)9(GlcNAc)2. This branched oligosaccharide, which is pre-
sent in multiple copies in several pathogen glycoproteins, contains the
natural 1,2-mannobioside Manα(1,2)Man 1 (Fig. 1), one of the minimal
epitopes. Consequently, the individuation of glycomimetic compounds
that have good affinity for DC-SIGN, high resistance to enzymatic
degradation and low structural complexity is still a challenging research
topic. Indeed, the individuation of more effective ligands, representing
optimized lead compounds, is an essential condition also in view of a
polyvalent presentation [17,18]. In this framework, the addition of
lipophilic fragments to a sugar ‘core’ have often been applied to improve
the affinity of glycomimetics [8,19–21]. Moreover, the identification of
two areas around Phe313 in the DC-SIGN binding site that were only
partially occupied by co-crystallized tetramannoside Man4, is a pivotal
point to design more lipophilic glycomimetic DC-SIGN antagonists with
potentially increased activity [12,22].
In the search for potent monovalent DC-SIGN antagonists, in the
present work, we describe an innovative stereoselective synthesis of
pseudodisaccharides (+)-4a, (+)-4b and (+)-5a, (+)-5b mimetics of
natural disaccharide epitope 1 (Fig. 1), which are characterized by a real
carbamanno unit with an attractive lipophilic substituent, such as p-
toluensulfonyl amide (TsNH), benzyl ether (Bn) or methyl (CH3) in the C
(3), C(4) or C(6) positions. The activity of these new ligand candidates
was then evaluated by means of Surface Plasmon Resonance (SPR) in-
hibition binding assays.
The efficiency of antagonism by small molecules to inhibit viral
transfection mediated by DC-SIGN was first demonstrated by com-
pounds 2a-c (Fig. 1). These disaccharide mimetics possess a three-
dimensional structure and a conformational behaviour comparable to
2. Results and discussion
The synthesis of pseudodisaccharides (þ)-4a, (þ)-4b, (þ)-5a and
(þ)-5b, was achieved through the glycoconjugation of two different
new carbamannose glycosyl acceptors (þ)-6 and (þ)-7 with perben-
those of natural Manα(1,2)Man 1 and display a micromolar activity
against DC-SIGN mediated Ebola viral infection [10,13,17].
However, surprisingly, the binding mode of compounds 2 series
showed to be very different from those observed for the natural ligand
zoylated α-D-mannopyranosyl trichloroacetimidate (-)-8 as the typical
and shared glycosyl donor [25] (Scheme 2 and 3).
Man
α(1,2)Man disaccharide. The latter, due to the presence of equato-
Noteworthy, these two glycosyl acceptors possess a common, even if
differently protected, key carba-alcohol intermediate (Scheme 1).
Indeed, (þ)-6 and (þ)-7 are respectively synthetized starting from
known trans-amino alcohol (þ)-9 and from β-epoxide (þ)-10β, which
both derived from the key di-protected carba-alcohols (-)-11 (benzy-
lated) and (-)-12 (p-methoxybenzylated). In particular, trans amino
alcohol (þ)-9 [26] was obtained from the ring opening reaction of
β-vinyl epoxide (-)-13 [27,28] with sodium azide and its successive
reduction with PPh3, whereas the latter is synthesized from (-)-12
through a series of selective and/or orthogonal protection/deprotection
reactions. In contrast, β epoxide (þ)-10β [29] arises from carba-alcohol
(-)-11 by means of the elaboration of the primary alcohol functionality
(tosylation and its reduction) and a final epoxidation reaction.
The synthesis of the carbamannose glycosyl acceptor (þ)-6 (Scheme
rial hydroxyl groups in C(3) and C(4) in both reductive and non-
reductive mannose units, can bind, alternatively, the DC-SIGN CRD by
the Ca2+ bridge. Therefore, two different binding modes (major and
minor) are possible (see Figure S1 in supporting information). Com-
pounds 2 series, due to their modified structure can bind only through
their non-reductive end mannose and thus possess a unique binding
mode [17,23]. A second difference in the binding modes of the natural
dimannose and the one of compounds 2 relies on the orientation of the
second mannose or pseudomannose unit. While the second mannose
points towards the Phe313 in both binding mode of 1, the pseudo-
mannose unit folds back on the Val351.
We previously reported that pseudo-1,2-
α-mannobioside 3a and 3b
(Fig. 1), characterized by the presence of a real carba manno unit,
Fig. 1. Minimal epitope Man
disaccharides 4a,b and 5a,b.
α
(1,2)Man 1 and chemical structure of disaccharide mimetics 2a-c, real carba analogues 3a and 3b and newly synthesized pseudo-
2

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
Scheme 1. Key intermediate compounds 11 and 12 as starting products for the synthesis of known 9 and 10β precursors.
Scheme 2. Stereoselective synthesis of carbamannose glycosyl acceptor 6 and 7: i) TsCl, Py; ii) t-BuOk, ACN; iii) NaOAc, DMF (aq); iv) MeONa, MeOH; v) MCPBA,
DCM; vi) Ac2O, Py; vii) and viii) 1.3 M solution of 2-azidoethanol, DCM, Cu(OTf)2.
2) started with the protection of amino alcohol (þ)-9 with tosyl chloride
(2.5 eq) in pyridine to give trans- N,O-ditosylate (-)-14, which was
efficiently cyclized with t-BuOK in acetonitrile to afford aziridine
requirements, was acetylated (Ac2O/Py) to afford (-)-19β as a new
carbaglycosylating agent ready for the nucleophilic ring opening reac-
tion. Subsequently, (-)-19β was subjected to reaction with a freshly
prepared 1.3 M solution of 2-azido-1-ethanol in dichloromethane [31] in
the presence of a catalytic amount of Cu(OTf)2 as the Lewis acid [32] to
afford the desired (þ)-6.
(þ)-15
α in a very good yield (75% overall yield for two steps). Subse-
quently, the acetolysis of aziridine (þ)-15
α
with sodium acetate in
aqueous DMF (80% v/v) allowed the formation of a mixture of acetate
(þ)-16 together with allylic alcohol (-)-17. A portion of the crude
mixture was purified in order to isolate and characterize both products,
while the rest of the crude mixture was directly hydrolyzed (MeONa/
MeOH) to afford only pure alcohol (-)-17.
As previously reported on the behaviour of 1,2-epoxides with
β-manno configuration [24,33], even in the case of epoxide (-)-19β, the
nucleophilic attack exclusively occurs in a trans-diaxial fashion, regio-
selectively at the favored C(1) oxirane carbon. In fact, although inter-
mediate (-)-19β possesses two electrophilic positions, C(1) and C(2),
carbon 1 is flanked by a sterically non hindered and electron-rich
methylene group, therefore it becomes the favourite reactive site. The
In this pathway aziridine (þ)-15α is a smart compound that leads
towards new carbaglycosyl donor (þ)-6, bearing a N-tosyl substituted
amino functionality at the C(4) position in a completely regio- and
stereoselective fashion. Allyl alcohol (-)-17 is then epoxidized by m-
CPBA, to afford a 40:60 mixture of the two diasteroisomeric 5a-carba
corresponding
α-O-carbamanno glycosyl acceptor (þ)-6 is then ob-
tained through a completely C(1) regio- and anti-stereoselective ring
opening process from (-)-19β.
epoxide (-)-18α and (-)-18β. Strongly supported by the completely
β-stereoselective process observed in the epoxidation of the corre-
sponding 3,4 trans diol analogue of compound 18 [24], we were sur-
prised to observe such a poorly stereoselective epoxidation process.
However, the configuration of both epoxides was exactly assigned by 1H
NMR spectra analysis and by appropriate NOE experiments [30]. After
the separation step, only (-)-18β, with the correct stereochemistry
In the same way, new glycosyl acceptor (þ)-7 was also readily
prepared by ring opening of β-6-methyl epoxide (þ)-10β [29] with 2-
azido-1-ethanol. The process turned out to be still completely C(1)
regio- and anti-stereoselective (Scheme 2).
The glycosylation reaction of glycosyl acceptors (þ)-6 and (þ)-7
with the classical manno-trichloroacetimidate glycosyl donors (-)-8 was
3

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
efficiently carried out under standard conditions [34] in dichloro-
methane as solvent and in the presence of a typical promoter (TMSOTf)
at ꢀ 20 ◦C for about 2 h, using molecular sieves AW300 to provide
strictly anhydrous reaction conditions (Scheme 3). By this way pseu-
dodisaccharides (-)-20 and (-)-21 fully-O-protected were obtained in
(þ)-4b from (-)-20 and (þ)-5b from (-)-21. Finally, catalytic hydro-
genolysis of (þ)-4b and (þ)-5b removed all the benzyl ethers and
reduced the azido functionality to afford the pseudodisaccharides
(þ)-4a and (þ)-5a, respectively, with a 2-aminoethoxy chain at the
pseudoanomeric C(1) carbon (Scheme 3).
good yields and in a completely
α
-stereoselective fashion, thanks to the
As a result, the stereoselective synthesis of (þ)-4b, (þ)-5b, (þ)-4a
anomeric effect and the anchimeric assistance of the benzoyl group on C
(2) of the glycosyl donor. The final elaboration of the two glyconjugates
(-)-20 and (-)-21 towards the target pseudodisaccharides took into ac-
count the orthogonal protecting groups previously introduced. There-
fore, deprotection of benzoyl and acetyl esters in both glycoconjugates
and (þ)-5a carba analogues of minimal natural epitope Manα1,2Man,
with appealing lipophilic features, was realized.
3. DC-SIGN binding inhibition assays
´
under Zemplen conditions affords 2-azidoethoxy pseudodisaccharide
SPR (Surface Plasmon Resonance) assays were performed in order to
Scheme 3. Stereoselective synthesis of pseudodisaccharides 4b, 5b, 4a and 5a: i) TMSOTf, DCM, MS AW 300; ii) MeONa, MeOH; iii) H2 1 atm, Pd/C (10% w/
w), MeOH.
4

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
investigate the inhibitory power of compounds 4a, 4b, 5a and 5b while
state that 3a binds as Man
α
(1,2)Man does (while no effect would be
D-mannose and compound 3b were used as controls. DC-SIGN (20
μM)
expected if 3a would bind as compound 2). Finally, in order to reach a
higher degree of hydrophobicity, benzyl groups in the C(3) and the C(4)
position of the carba unit were added to the structure of compound 5a,
leading to compound 5b. After the previous analysis on 5a, we could
have assumed that the methyl group would behave the same way as for
was injected over an SPR flow cell functionalised with mannosylated-
BSA. Serial dilution of DC-SIGN antagonists, starting from 5000 μM,
were injected as well and the IC50 values were obtained (Fig. 2 and
Tab.1).
The mere addition of a NH-toluenesulfonyl group in the C(4) position
of the carba unit in compound 4a did not help the interaction with DC-
5a, causing an IC50 of, at least, 2000 μM. Moreover, the addition of the
two benzyl groups, awaited to promote DC-SIGN interaction, seemed to
hamper the binding. Therefore, we could not measure any inhibition for
compound 5b, as shown by the sensorgram in Fig. 2D and by the Table 1.
SIGN, leading to an IC50 of 1541 ± 57.98 μM (Fig. 2B), similar to the one
found for the previously published 3a and 3b carbasugars (1453 and
1467
μM respectively). [24] On the other hand, the contemporary
presence of a hydrophobic toluenesulfonyl group in the C(4) position
and of a benzyl group in the C(6) position of the carba unit enhanced the
4. Conclusion
antagonist’s inhibitory potency (compound 4b IC50 of 1190 ± 54.44
μM,
In conclusion, we have successfully planned and implemented the
challenging stereoselective synthesis of four new pseudodisaccharides
(þ)-4b, (þ)-5b, (þ)-4a and (þ)-5a as innovative real carba-analogues
Fig. 2C). Therefore, we support the hypothesis that the single aromatic
group in C(6) is able to establish a hydrophobic interaction with DC-
SIGN Phe313.
of the minimal natural epitope Manα(1,2)Man1. Compounds (þ)-6 and
Next we wanted to test whether the addition of a different nonpolar
group in the C(6) position could be beneficial to the interaction with DC-
SIGN. This way compound 5a, which presents a methyl group in the C(6)
position of the carba unit, was tested by SPR. Unfortunately, the inhi-
bition assay revealed a worsening of the binding, with an IC50 of 2136 ±
(þ)-7 represent the key carbamannose glycosyl acceptors in the syn-
thetic pathway. The new pseudomannobiosides were designed to check
the activity of real carba-analogues characterized by the presence of
attractive lipophilic substituents to extend contacts with the protein
surface, which might improve the DC-SIGN affinity and specificity. Their
affinity was evaluated using SPR binding inhibition assays and signifi-
cant IC50 values were found. In particular, the carbadisaccharide 4b
60.81 μM comparable to the single D-Man one (Fig. 2B), showing that not
all the hydrophobic modifications are suitable for the awaited interac-
tion. However, the loss of affinity from compound 3a to 5a highlights
the existence of an interaction involving this OH group. This is fully
compatible with the binding mode of the natural compound and notably
the minor binding mode of Fig. S1 in supporting information (where an
H-bond with Asn365 is awaited, not shown). It is thus a new argument to
shows a very interesting IC50 of 1190 μM, similar to the value previously
reported for mimics 2. The presence of lipophilic groups only in C(4), or
in C4 and C6 in new real carba-analogues 4a and 4b may drastically
modify their binding mode, when compared to previously described real
carbadisaccharides 3. Here, IC50 increase of the new 5a compound when
Fig. 2. A) Inhibition curves for 4a, 4b, 5a and D-Man. Increasing concentrations of the inhibitors are plotted against DC-SIGN activity expressed as percentage. D-
Man: grey square, 4b:black round, 4a: black triangle, 5a: grey star. B) Extracted corresponding IC50 histograms, in grey colour the control compounds. The com-
pounds containing p-toluensulfonyl amide substitution (4a and 4b) are striped, while 5a is left blank. C) Sensorgram of competition of DC-SIGN ECD binding to Man-
BSA surface by 4b. D) Sensorgram of competition of DC-SIGN ECD binding to Man-BSA surface by 5b.
5

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
(δ relative to residual solvent peak for 1H and 13C). FTIR spectra were
obtained with an IR Cary 600 FTIR (Agilent Tecnologies). Yields refer to
isolated and purified products. High-resolution mass spectrometry
(HRMS)-ESI/QTOF analysis were performed using a Waters Xevo® G2-S
QTof at Mass Spectrometry facility, nanobio chemistry platform of the
Institute of Molecular Chemistry of Grenoble (PCN-ICMG). Compounds
are dissolved in 1:1 acetonitrile/water (concentration 5 × 10-5 M). The
SPR experiments were performed on a BIAcore T200 using a CM3 sensor
S series chip (see dedicated section).
Table 1
IC50 values (µM) for D-Mannose and compounds 4a, 4b, 5a and 5b.
Compound
IC50 µM
2290 ± 129.4 µM
1541 ± 57.98 µM
1190 ± 54.44 µM
2136 ± 60.81 µM
5.2. Synthetic procedures
5.2.1. 6-O-Benzyl-3-deoxy-3-(N-Tosylamino)-4-O-Tosyl-5a-carba-D-gulal.
(-)-14
A solution of trans-amino alcohol (þ)-9 [27] (0.110 g, 0.47 mmol,
1.0 eq) in anhydrous pyridine (1.0 mL) was treated with an excess of
tosyl chloride (0.224 g, 1.18 mmol, 2.5 eq). The reaction mixture was
stirred for 72 h at room temperature and then co-evaporated with
toluene. The brown solid crude residue was purified by flash chroma-
tography (SiO2, CH2Cl2/AcOEt = 9:1) to afford the pure di-N,O-tosyl
derivative (-)-14 (0.135 g, Yield = 53%) as a pale yellow solid.
n.d.
20
◦
Rf = 0.40 (1:1 Hexane/AcOEt). M.p. 136–138 C. [
α
]
= -9.3◦ (c
2.78, CHCl3). FTIR (neat)
ν
(cmꢀ 1) = 3278, 3089, 3064,D3033, 2963,
2919, 2854, 2802, 1922, 1811, 1710, 1655, 1598, 1495, 1363, 1335,
1262, 1189, 1174, 1094, 1075, 1041, 916. 1H NMR (250 MHz, CDCl3), δ
(ppm) = 7.87–7.20 (m, 4H, Ar), 7.37–7.28 (m, 7H, Ar), 7.20–7.17 (m,
2H, Ar), 5.93–5.81 (m, 1H, H2), 5.26–5.16 (m, 1H, H1), 4.81 (m, 1H,
compared to both compounds 3 and natural epitope 1 (looking at IC50
and structure of binding modes respectively) suggests a similar binding
mode, as expected, for all these compounds who share a pair of hydroxyl
group on C(3) and C(4) of both units. On the contrary, for the newly
synthetized mimics 4a, 4b and 5b where one or two OH groups in C(3)
and C(4) of the carba unit are modified, we could reasonably assume
binding poses that are more similar to those of compounds 2 (see Fig. 2
C). In fact, the binding to DC-SIGN through Ca2+ is not possible anymore
for the “reductive end“ of 4a and 4b, whereas only binding poses
mimicking the one observed with compounds 2 are still possible by
means of the two equatorial OH groups in C(3) and C(4) of the non-
reductive mannose unit. Noteworthy, the lower IC50 value obtained by
4b, when compared to that of 4a, for the first time, can be an evidence
that the lipophilic group in C(6) is probably engaged in hydrophobic
interactions with DC-SIGN key target residues. This observation is in
agreement with the recent report of the binding mode of a newly
described derivative of compound 2 where a benzylamide arm in posi-
tion C(6) is interacting with DC-SIGN surface [35]. Further investigation
to understand the role of substituent in C(6) position in compounds that
can adopt specific binding mode, as well as an amplification of the ac-
tivity by multivalent scaffolds, is currently underway.
–
H4,), 4.49 (m, 1H, N H), 4.14 (s, 2H, Ar-CH2), 3.93 (m, 1H, H3), 3.14
(dd, J = 6.72, 9.16 Hz, 1H, H6), 2.99 (dd, J = 7.49, 9.16 Hz, 1H, H6′),
2.42 (s, 3H, N-Ar-CH3), 2.35 (s, 3H, O-Ar-CH3), 2.22–2.07 (m, 1H, H5),
2.02–1.86 (m, 2H, H5a
α
-β). 13C NMR (62.5 MHz, CDCl3), δ (ppm) =
145.2, 144.2, 138.5, 137.0, 133.3, 132.4, 130.2 (C2), 130.1, 128.7,
128.6, 127.8, 127.7 (C1), 127.6, 121.8, 78.8 (C4), 73.0 (Ar-CH2), 70.3
(C6), 50.3 (C3), 33.1 (C5), 24.1 (C5a), 21.9 (O-Ar-CH3), 21.9 (N-Ar-
CH3).
5.2.2. (1S, 5S,6R)-5-(Benzyloxymethyl)-7-tosyl-7-azabicyclo [4.1.0]hept-
2-ene (+)-15
α
A solution of trans- N-tosylamino-O-tosyl derivative (-)-14 (0.066 g,
0.122 mmol, 1.0 eq) in CH3CN (1.9 mL) was treated with t-BuOK
(0.0274 g, 0.244 mmol, 2.0 eq) at room temperature and the reaction
mixture was stirred for 1.5 h at room temperature. After dilution with
Et2O and filtration on celite pad, the evaporated organic solution was
characterized by the pure N-Tosyl aziridine (þ)-15α (0.0438 g, Yield =
97%) as a pale yellow liquid which was used without further
purification.
Rf = 0.66 (1:1 hexane/AcOEt). [
α
]
20 = +74.4◦ (c 0,51 CHCl3). FTIR
(cmꢀ 1) = 3062, 3033, 2919,D2853, 2191, 1642, 1598, 1495,
5. Experimental
(neat)
ν
1454, 1321, 1156, 1090, 973, 949, 843, 814, 695, 670. 1H NMR (250
MHz, CDCl3), δ (ppm) = 7.82–7.75 (m, 2H, Ar), 7.41–7.21 (m, 7H, Ar),
5.91–5.69 (m, 2H, H1 + H2), 4.48 (bs, 2H, Ar-CH2), 3.34 (dd, J = 9.07,
16.6 Hz, 1H, H6), 3.33–3.25 (m, 2H, H3 + H6′), 3.17–3.07(m, 1H, H4),
2.51–2.45 (m, 1H, H5), 2.43 (s, 3H, N-Ar-CH3), 2.22–2.10 (m, 1H,
5.1. Materials and methods
All solvents and chemicals were used as purchased without further
purification. All reactions were performed in a flame-dried modified
Schlenk (Kjeldahl shape) flask fitted with a glass stopper or rubber
septum under a positive pressure of argon. Chromatographic separations
were performed on silica gel columns by flash (Kieselgel 40,
0.040–0.063 mm; Merck) or by automated chromatography with Iso-
lera® Biotage. Reactions were followed by thin-layer chromatography
(TLC) on Merck aluminum silica gel (60 F254) sheets that were visual-
ized under a UV lamp. Evaporation was performed in vacuo (rotating
evaporator). Sodium sulfate was always used as the drying agent.
Melting points were determined with a Kofler hot-stage apparatus and
are uncorrected. Optical rotations were measured with a ATAGO AP-300
Automatic Polarimeter at 25 ◦C. 1H- and 13C NMR spectra were recorded
with a Bruker Avance 250 MHz spectrometer using the indicated
deuterated solvents. Chemical shifts are given in parts per million (ppm)
H5aα
), 2.03–1.90 (m, 1H, H5aβ). 13C NMR (62.5 MHz, CDCl3), δ (ppm)
= 144.7, 138.4, 135.9, 131.9 (C2), 130.0, 128.8, 128.1, 128.0, 127.9
(C1), 120.8, 73.5 (Ar-CH2), 70.9 (C6), 43.0 (C5), 36.2 (C4), 30.4 (C3),
24.0 (C5a), 21.9 (N-Ar-CH3).
5.2.3. 3-O-Acetyl-6-O-benzyl-4-deoxy-4-(N-tosylamino)-5a-carba-D-
glucal. (+)-16
N-Tosyl aziridine (þ)-15α (0.0438 g, 0.119 mmol, 1.0 eq) was dis-
solved in an aqueous DMF solution (80% v/v). Then, sodium acetate
(0.0389 g, 0.474 mmol, 4.0 eq) was added and the reaction was stirred
for 24 h at room temperature. The crude mixture was diluted with Et2O,
washed with brine, dried with anhydrous Na2SO4, filtered and the sol-
vent evaporated. The residual solid consisted of a 50:50 mixture of trans-
6

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
3,4-N-(tosylamino)-acetoxy derivative (þ)-16 and trans-3,4-N-(tosyla-
mino)-hydroxy derivative (-)-17. The mixture has been purified by
preparative TLC (SiO2, 0.5 cm, 1:1, Hexane/AcOEt) to afford (þ)-16 as a
white solid (0.026 g, Yield = 50%).
NMR (250 MHz, CDCl3), δ (ppm) = 7.77–7.65 (m, 2H, Ar), 7.45–7.19
(m, 7H, Ar), 6.21 (d, J = 4.9 Hz, 1H, NH), 4.36 (d, J = 11.67 Hz, 1H, Ar-
CH2), 4.34 (d, J = 2.37, 1H, OH), 4.24 (d, J = 11.67 Hz,1H, Ar-CH2),
3.89 (dd, J = 7.5, 2.1 Hz, 1H, H3), 3.26–3.11 (m, 3H, H1 + H2 + H6),
2.81–2.68 (m, 2H, H4 + H6′), 2.43 (s, 3H, Ar-CH3), 2.08–1.98 (m, 1H,
M.p. 106–108 ◦C. Rf = 0.48 (1:1, Hexane/AcOEt). [
α
]
20 = +54.1◦ (c
D
1.17, CHCl3). FTIR (neat)
ν
(cmꢀ 1) = 3727, 3628, 3315, 2963, 2364,
H5), 1.90–1.75 (m, 1H, H5a
α
), 1.52–1.39 (m, 1H, H5aβ). 13C NMR (62.5
2340, 2097, 1338, 1260, 1086, 1020, 798, 727. 1H NMR (250 MHz,
CDCl3), δ (ppm) = 7.72 (d, J = 8.3 Hz, 2H, Ar), 7.38–7.21 (m, 7H, Ar),
5.77 (m, 1H, H1), 5.39 (m, 1H, H2), 5.25 (m, 1H, H3), 5.08 (m, 1H, H4),
4.48 (d, J = 11.7, 1H, Ar-CH2), 4.35 (d, J = 11.6, 1H, Ar-CH2), 3.62–3.49
(m, 2H, H5 + H6), 3.39 (dd, J = 9.4, 5.2 Hz, 1H, H6), 2.39 (s, 3H, N-Ar-
CH3), 2.29 (m, 1H, H5a), 2.16–1.92 (m, 1H, H5a), 1.60 (s, 3H, COCH3).
13C NMR (62.5 MHz, CDCl3), δ (ppm) = 171.4 (COCH3), 143.2, 139.1,
138.2, 129.6 (C2), 128.6, 127.9, 127.9 (C1), 127.2, 125.7, 73.8 (Ar-
CH2), 73.4 (C3), 72.1 (C6), 57.3 (C4), 39.3 (C5), 29.5 (C5a), 21.6 (N-Ar-
CH3), 20.8 (COCH3).
MHz, CDCl3), δ (ppm) = 144.1, 137.0, 136.3, 129.9, 128.8, 128.2,
127.7, 127.6, 73.5 (Ar-CH2), 72.9 (C6), 72.0 (C3), 61.7 (C2), 52.3 (C1),
31.3 (C4), 29.8 (C5a), 28.0 (C5), 21.7 (Ar-CH3).
5.2.7. 6-O-Benzyl-3-O-acetyl-4-deoxy-4-(N-tosylamino)-1,2-anhydro-5a-
carba-β-D-mannopyranose. (-)-19β
Epoxide (-)-18β (0.010 g, 0.025 mmol, 1.0 eq) was dissolved in
anhydrous pyridine (0.3 mL) and treated with freshly distilled acetic
anhydride (0.16 mL) at 0 ◦C. The reaction mixture was stirred for 4 h at
room temperature. Co-evaporation of the reaction mixture with toluene
afforded the pure product (-)-19β (0.011 g, Yield = 99%), which was
used in the next step without further purification.
5.2.4. 6-O-Benzyl-4-deoxy-4-(N-tosylamino)-5a-carba-D-glucal. (-)-17
Sodium methoxide (0.00194 g, 0.036 mmol, 0.35 eq) was added to a
solution of (þ)-16 and (-)-17 (0.044 g, 0.102 mmol, 1.0 eq) in anhy-
drous MeOH (3.5 mL). The reaction mixture was stirred for 18 h at room
temperature. After dilution with additional MeOH, the mixture was
filtered on a celite pad and the filtrate was evaporated. The crude re-
action product has been purified by flash chromatography (SiO2, Hex/
AcOEt = 7:3) to afford the pure product (-)-17 as a pale yellow oil
(0.030 g, Yield = 65%).
Rf = 0.41 (1:1, Hexane/AcOEt). [
α
]
20 = –33.2◦ (c 0.19, CHCl3). FTIR
(neat)
ν
(cmꢀ 1) = 3280, 3063, 3029, D2958, 2922, 2856, 1729, 1234,
1157, 1092, 1026. 1H NMR (250 MHz, CDCl3), δ (ppm) = 7.76–7.65 (m,
2H, Ar), 7.43–7.11 (m, 7H, Ar), 5.02 (dd, J = 9.75, 1.3, 1H, H3), 4.87
(bs, 1H, NH), 4.45 (d, J = 11.7 Hz, 1H, Ar-CH2), 4.32 (d, J = 11.7 Hz, 1H,
Ar-CH2), 3.74–3.39 (m, 2H, H4 + H6), 3.34–3.20 (m, 3H, H1 + H2 +
H6′), 2.40 (s, 3H, Ar-CH3), 1.90–1.72 (m, 3H, H5 + H5a
α-β), 1.60 (s, 3H,
COCH3). 13C NMR (62.5 MHz, CDCl3), δ (ppm) = 171.6 (COCH3), 143.2,
139.0, 138.1, 129.7, 128.5, 127.9, 127.2, 125.4, 73.7 (C3), 73.3 (Ar-
CH2), 71.7 (C6), 54.9 (C2), 53.1 (C1), 39.8 (C4), 29.8 (C5a), 27.5 (C5),
21.6 (Ar-CH3), 20.5 (COCH3).
Rf = 0.15 (7:3, hexane/AcOEt). [
α
]
20 = -30.0◦ (c 0.42, CHCl3). FTIR
(neat)
ν
(cmꢀ 1) = 3484, 3264, 3031,D2921, 2852, 1598, 1453, 1322,
1154, 1089, 814. 1H NMR (250 MHz, CDCl3), δ (ppm) = 7.78–7.70 (m,
2H, Ar), 7.40–7.23 (m, 7H, Ar), 6.20 (d, J = 4.28 Hz, 1H, NH), 5.70–5.53
(m, 2H, H1 + H2), 4.39 (d, J = 11.7 Hz, 1H, Ar-CH2), 4.26 (d, J = 11.7
Hz, 1H, Ar-CH2), 4.24–4.18 (m, 1H, H3), 3.98 (bs, 1H, OH), 3.20 (dd, J
= 9.92, 3.09 Hz, 1H, H6), 2.98–2.75 (m, 2H, H4 + H6′), 2.43 (s, 3H, Ar-
5.2.8. (2-Azidoethyl)-3-O-acetyl-6-O-benzyl-4-deoxy-4-(N-tosylamino)-
5a-carba-α-D-mannopyranoside. (+)-6
Epoxide (-)-19β (0.029 g, 0.065 mmol, 1.0 eq) was dissolved in a 1.3
M solution of 2-azido-1-ethanol in CH2Cl2 (0.5 mL, 0.258 mmol, 4.0 eq)
and then Cu(OTf)2 (0.007 g, 0.019 mmol, 0.3 eq) was added. The re-
action was stirred for 18 h at room temperature. The mixture was diluted
with 10 mL of CH2Cl2 and washed respectively with saturated solution of
1:1 NaHCO3/NH4Cl mixture and brine. The organic phase was dried
with anhydrous Na2SO4, filtered and the solvent evaporated. The crude
was purified by flash chromatography (SiO2, Hex/AcOEt = 1:1) and
carbamannoside (þ)-6 was obtained as yellow solid (0.012 g, Yield =
34%).
CH3), 2.04–1.94 (m, 1H, H5), 1.73–1.60 (m, 2H, H5a
α
-β). 13C NMR
(62.5 MHz, CDCl3), δ (ppm) = 143.9, 137.2, 136.4, 129.8, 129.8 (C2),
129.1, 128.7, 128.1, 127.7, 127.6 (C1), 126.1, 73.5 (Ar-CH2), 73.3 (C3),
73.1 (C6), 62.1 (C4), 37.3 (C5), 28.9 (C5a), 21.7 (Ar-CH3).
5.2.5. 6-O-Benzyl-4-deoxy-4-(N-tosylamino)-1,2-anhydro-5a-carba-β-D-
mannopyranose. (-)-18β
MCPBA (70%) (0.077 g, 0.31 mmol, 1.2 eq) was added to a solution
of alcohol derivative (-)-17 (0.101 g, 0.26 mmol, 1.0 eq) in anhydrous
CH2Cl2 (5.0 mL) at 0 C. The reaction mixture was stirred for 18 h at
Rf = 0.40 (1:1, hexane/AcOEt, eluted twice). [
α
]
20 = +11.5◦ (c 0.76,
◦
room temperature. The mixture was diluted with CH2Cl2 and washed
with a saturated solution of Na2SO3, saturated solution of NaHCO3 and
brine, dried with anhydrous sodium sulfate, filtered and the solvent
evaporated. The residual solid consisted of a 60:40 mixture of diaste-
CHCl3). FTIR (neat)
ν
(cmꢀ 1) = 3289, 2962, 2925,D2107, 1730, 1454,
1413, 1327, 1260, 1090, 1019, 798. 1H NMR (250 MHz, CDCl3), δ
(ppm) = 7.76–7.63 (m, 2H, Ar), 7.41–7.16 (m, 7H, Ar), 5.03 (dd, J =
10.5, 2.8 Hz, 1H, H3), 4.57 (d, J = 9.0 Hz, 1H, H2), 4.28 (s, 2H, Ar-CH2),
4.05 (m, 1H, OH), 3.89–3.52 (m, 4H, H4 + H6 + 2H OCH2CH2N3),
3.46–3.21 (m, 4H, H1 + H6′+2H OCH2CH2N3), 2.39 (s, 3H, Ar-CH3),
reoisomeric epoxides (-)-18β and (-)-18α, which was purified by pre-
parative TLC (SiO2, 0.5 cm, Hex/AcOEt = 4:6) to afford the pure
-epoxide (-)-18 (0.035 g, Yield = 37%) and β-epoxide (-)-18β (0.055
g, Yield = 59%).
α
α
2.21–1.84 (m, 3H, H5 + H5a
α
-β), 1.77 (s, 3H, COCH3). 13C NMR (62.5
MHz, CDCl3), δ (ppm) = 171.1 (COCH3), 143.0, 139.1, 138.5, 129.6,
128.3, 127.5, 127.4, 127.3, 127.0, 95.3, 73.9 (C1), 72.8 (C3), 71.0 (C2),
70.0 (Ar-CH2), 68.3 (C6), 53.9 (OCH2CH2N3), 50.8 (OCH2CH2N3), 37.7
(C4), 29.8 (C5a), 27.4 (C5), 21.5 (Ar-CH3), 20.8 (COCH3).
Rf = 0.10 (1:1, hexane/AcOEt). [
α
]
20 = -107.8◦ (c 0.25, CHCl3). FTIR
(neat)
ν
(cmꢀ 1) = 3528, 3233, 3002,D2962, 2927, 2890, 2860, 1153,
1091, 1030. 1H NMR (250 MHz, CDCl3), δ (ppm) = 7.78–7.66 (m, 2H,
Ar), 7.42–7.22 (m, 7H, Ar), 5.83 (d, J = 5.6 Hz, 1H, NH), 4.32 (d, J =
11.8 Hz, 1H, Ar-CH2), 4.17 (d, J = 11.8 Hz, 1H, Ar-CH2), 3.84 (d, J = 8.8
Hz, 1H, H3), 3.72 (bs, 1H, OH), 3.38 (dd, J = 4.0, 1.6 Hz, 1H, H2), 3.24
(dd, J = 4.88, 4.0 Hz, 1H, H1), 3.12–3.07 (m, 2H, H4 + H6), 2.79 (dd, J
= 9.5, 7.5 Hz, 1H, H6′), 2.42 (s, 3H, Ar-CH3), 2.07–1.50 (m, 3H, H5 +
5.2.9. 2,3,4,6-tetra-O-benzoyl-
trichloroacetimidate. (-)-8 [25]
α
-D-mannopyranosyl
A solution of 2,3,4,6-tetra-O-benzoyl-
α-D-mannose (1.42 g, 2.38
mmol, 1.0 eq) in anhydrous CH2Cl2 (8.0 mL) was treated respectively
H5a
α
-β). 13C NMR (62.5 MHz, CDCl3), δ (ppm) = 144.0, 137.2, 136.6,
with Cl3CCN (1.0 mL, 12.0 mmol, 5.0 eq) and DBU (18.0 μL, 0.12 mmol,
129.9, 128.7, 128.2, 127.7, 127.6, 73.5 (Ar-CH2), 72.9 (C6), 72.0 (C3),
57.9 (C2), 56.8 (C1), 52.8 (C4), 37.9 (C5a), 27.2 (C5), 21.7 (Ar-CH3).
0.05 eq) and stirred for 3 h at room temperature. Then, the solvent was
evaporated and the residual solid was purified by flash chromatography
(SiO2, Hex/AcOEt = 8:2) to afford (-)-8 as a white solid (1.11 g, Yield =
63%).
5.2.6. 6-O-Benzyl-4-deoxy-4-(N-tosylamino)-1,2-anhydro-5a-carba-α-D-
glucopyranose. (-)-18
α
20
D
Rf = 0.31 (1:1, hexane/AcOEt). [
α]
= -11.7◦ (c 0.82, CHCl3). 1H
7

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
5.2.10. (2-azidoethyl)-2-O-[2′,3′,4′,6′-tetra-O-benzoyl-
α-D-
under 1 atm of hydrogen at room temperature until fully reduction of the
azide group and deprotection of the benzyl group (TLC monitored (8:2
CH2Cl2/MeOH)). After 18 h the mixture was diluted with MeOH and
filtered through a celite pad to remove the catalyst. Evaporation of the
solvent afforded a white solid, which was recrystallized from (i-Pr)2O to
afford the pure 1,2-pseudomannobioside (þ)-4a (0.0036 g, Yield =
77%), as a white solid.
mannopyranosyl]-3-O-acetyl-6-O-benzyl-4-deoxy-4-(N-tosylamino)-5a-
carba-α-D-mannopyranoside. (-)-20
The glycosyl-donor (-)-8 (0.022 g, 0.029 mmol, 1.3 eq) and the
glycosyl-acceptor (þ)-6 (0.012 g, 0.023 mmol, 1.0 eq) were dissolved in
anhydrous toluene and dried overnight under vacuum to remove any
trace of water. Acid washed AW 300 molecular sieves were heated under
vacuum and added to the reaction mixture. Anhydrous CH2Cl2 (0.50
mL) and a catalytic amount of TMSOTf in CH2Cl2 (0.1 mL, 0.004 mmol,
0.2 eq) was added under argon at ꢀ 20 ◦C and the reaction mixture was
Rf = 0.04 (8:2 AcOEt/Acetone). [
α]
20 = +8.6◦ (c 0.18, CH3OH). FTIR
D
(neat)
ν
(cmꢀ 1) = 3332, 2945, 2833, 1658, 1450, 1415, 1020, 671. 1H
NMR (250 MHz, MeOD), δ (ppm) = 7.78–7.69 (m, 2H, Ar), 7.40–7.30
(m, 2H, Ar), 4.96 (s, 1H, Anomeric), 4.09–3.13 (m, 16H, H1 + H2 + H3
+ H4 + H6 + H2′+H3′+H4′+H5′+H6, OCH2CH2N3), 2.41 (s, 3H, Ar-
stirred at the same temperature for 3 h. Afterwards, Et3N (20.0 μL) was
added and the mixture was diluted with CH2Cl2 and filtered through a
Celite pad. Evaporation of the solvent afforded a crude product, which
was purified by preparative TLC (SiO2, 0.5 cm, Hex/AcOEt = 7:3). The
pure O- and N-protected pseudomannobioside (-)-20 (0.013 g, Yield =
CH3), 2.14–1.46 (m, 3H, H5a
α
-β + H5). 13C NMR (62.5 MHz, MeOD), δ
(ppm) = 144.4, 139.9, 130.6, 129.4, 128.4, 126.2, 103.7 (Anomeric),
78.5, 77.4 (C3′), 75.6 (C2′), 73.8 (C1), 72.8 (C4′), 72.6 (OCH2CH2N3),
68.8 (C3), 63.8 (C6), 64.4 (C6′), 56.5 (C4), 43.9 (OCH2CH2N3), 38.0
(C5a), 30.8 (C5), 21.5 (Ar-CH3). HRMS, (m/z) [M + H]+, calculated for
52%), was obtained as a white solid.
20
Rf = 0.22 (7:3, hexane/AcOEt, three developments). [
α
]
= -50.7◦
(c 1.07, CHCl3). FTIR (neat)
ν
(cmꢀ 1) = 3307, 2961, 2106, 1D726, 1601,
C22H37N2O11S 537.2118, found 537.2118.
1452, 1261, 1159, 1092, 1026, 708. 1H NMR (250 MHz, CDCl3), δ
(ppm) = 8.20–7.70 (m, 9H, Ar), 7.69–7.09 (m, 20H, Ar), 6.17–6.04 (t, J
= 10.2 Hz, 1H, H4′), 5.86 (dd, J = 10.2, 3.2 Hz, 1H, H2′), 5.79–5.63 (m,
1H, H3′), 5.19–5.16 (m, 1H, Anomeric), 5.02 (dd, J = 10 Hz, 2.8 Hz, 1H,
H3), 4.75–4.71 (d, J = 8.3 Hz, 1H, H6′b), 4.68–4.64 (dd, J = 12 Hz, 2.3
Hz, 1H, H6′a), 4.52 (s, 2H, Ar-CH2), 4.50–4.40 (m, 2H, H5′+H2), 4.08
(bs, 1H, H6b), 3.94–3.17 (m, 9H, H1 + H4 + H5 + H6 + H6a + 4H
5.2.13. (2-Azidoethyl)-3,4-di-O-benzyl-6-deoxy-5a-carba-α-D-
mannopyranoside. (+)-7
Epoxide (þ)-10β [29] (0.0692 g, 0.213 mmol, 1.0 eq) was solubi-
lized in a 1.3 M solution of 2-azido-1-ethanol in CH2Cl2 (0.66 mL, 0.854
mmol, 4.0 eq) and Cu(OTf)2 (0.0232 g, 0.064 mmol, 0.3 eq) was added.
The reaction was stirred for 18 h at room temperature. The mixture was
diluted with 10 mL CH2Cl2 and washed respectively with a saturated
solution of NaHCO3, saturated solution of NH4Cl and brine. The organic
phase was dried with anhydrous Na2SO4, filtered and the solvent
evaporated to afford (þ)-7 (0.079 g, Yield = 90%) as a yellow syrup,
which was used without further purification.
OCH2CH2N3), 2.37 (s, 3H, Ar-CH3), 2.20 (m, 1H, H5aα-β), 1.90–1.74 (m,
1H, H5a
α
-β), 1.51 (s, 3H, COCH3). 13C NMR (62.5 MHz, CDCl3), δ (ppm)
= 171.7 (COCH3), 166.2 (Ar-COO), 165.8 (Ar-COO), 165.5 (Ar-COO),
165.2 (Ar-COO), 143.2, 138.9, 133.7, 133.4, 130.0, 130.0, 129.9, 129.8,
129.8, 129.4, 129.2, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 127.7,
127.5, 127.4, 99.8 (Anomeric), 78.1, 77.4 (C2), 76.3 (C5′), 73.2 (C1),
72.8 (Ar-CH2), 71.5 (C6), 70.4 (C3), 70.0 (C2′), 68.2 (C3′), 67.0 (C4′),
63.3 (OCH2CH2N3), 54.5 (C6′), 50.7 (OCH2CH2N3), 38.0 (C4), 29.8
(C5a), 28.3 (C5), 21.5 (Ar-CH3), 20.4 (COCH3).
Rf = 0.29 (7:3 hexane/AcOEt); [
α
]
20 = +2.0◦ (c 0.98, CHCl3); FTIR
D
(neat)
ν
(cmꢀ 1) = 3461, 2924, 2870, 2104, 1101, 1053. 1H NMR (250
MHz, CDCl3), δ (ppm) = 7.40–7.21 (m, 10H, Ar), 4.88 (d, 1H, J = 10.9
Hz, Ar-CH2), 4.69 (m, 2H, J = 11.4 Hz, Ar-CH2), 4.60 (d, 1H, J = 10.9
Hz, Ar-CH2), 4.15–4.11 (m, 1H, H1), 3.77 (dd, 1H, J = 9.1, 3.2 Hz, H2),
3.74–3.20 (m, 6H, H3 + H4 + 4H OCH2CH2N3), 2.51 (bs, 1H, OH),
5.2.11. (2-Azidoethyl)-2-O-(
α
-D-mannopyranosyl)-3-hydroxy-6-O-benzyl-
4-deoxy-4-(N-tosylamino)-5a-carba-
α
-D-mannopyranoside. (+)-4b
1.99–1.83 (m, 1H, H5), 1.76 (dt, 1H, J = 14.3, 2.8 Hz, H5aα-β),
A fleshly prepared 0.5 M solution of sodium methoxide (0.3 mL,
0.0066 mmol, 5.0 eq) in MeOH was added to a solution of 1,2-pseudo-
mannobioside (-)-20 (0.013 g, 0.012 mmol, 1.0 eq) in MeOH (0.3 mL)
and the reaction mixture was stirred for 12 h at room temperature. Then
the solution was diluted with MeOH and acidic Amberlite IRC-50 resin
was added until pH = 7 was reached. Afterwards, the solution was
filtered from the resin and the filtrate evaporated. The crude compound
was purified by crystallization from (i-Pr)2O to afford derivative (þ)-4b
1.65–1.45 (m, 1H, H5a
α
-β), 1.04 (d, 3H, J = 6.5 Hz, CH3). 13C NMR
(62.5 MHz, CDCl3), δ (ppm) = 139.1, 138.5, 128.6, 128.5, 128.1, 128.0,
128.0, 127.6, 83.1 (C4), 82.1 (C3), 77.0 (C1), 75.3 (C2), 72.7 (Ar-CH2),
69.6 (Ar-CH2), 68.2 (OCH2CH2N3), 51.0 (OCH2CH2N3), 31.6 (C5), 31.5
(C5a), 18.3 (CH3).
5.2.14. (2-Azidoethyl)-2-O-[2′,3′,4′,6′-tetra-O-benzoyl-
α-D-
mannopyranosyl]-3,4,-di-O-benzyl-6-deoxy-5a-carba-
mannopyranoside. (-)-21
α-D-
as a white solid (0.008 g, Yield = 99%).
20
Rf = 0.24 (8:2 AcOEt/Acetone). [
α
]
= +55.9◦ (c 0.86, CH3OH).
The glycosyl-donor trichloroacetimidate (-)-8 (0.178 g, 0.240 mmol,
1.3 eq) and the glycosyl-acceptor carbamannoside (þ)-7 (0.076 g,
0.185 mmol, 1.0 eq) were dissolved in anhydrous toluene and dried
overnight under vacuum to remove any trace of water. The mixture was
solubilized in anhydrous CH2Cl2 (1.52 mL) and acid washed AW 300
molecular sieves were heated under vacuum and added to the reaction
mixture. A 0.1 M solution of TMSOTf in CH2Cl2 (0.037 mL, 0.037 mmol,
0.2 eq) was added at ꢀ 20 ◦C under argon atmosphere, and then the
mixture was stirred at the same temperature for 1 h. The mixture was
then diluted with 20 mL CH2Cl2 and Et3N was added until pH = 7 was
reached. After filtration on a celite pad and evaporation of the solvent,
the crude product was purified by flash chromatography (SiO2, Hex/
AcOEt = 7:3), to afford the O-protected pseudomannobioside (-)-21
D
FTIR (neat)
ν
(cmꢀ 1) = 3399, 2923, 2107, 1711, 1598, 1453, 1304,
1153, 1073, 1057, 1019, 814. 1H NMR (250 MHz, MeOD), δ (ppm) =
7.70 (d, J = 8.2 Hz, 2H, Ar), 7.28 (m, 7H, Ar), 5.05 (s, 1H, Anomeric),
4.17 (d, J = 12.1 Hz, 1H, H2), 4.08 (d, J = 12.2 Hz, 1H, H3), 3.98 (m,
1H, H3′), 3.84 (m, 3H, H1 + H2′+H4), 3.65 (m, 8H, Ar-CH2
+
H4′+H5′+H6′+ OCH2CH2N3), 3.26 (m, 2H, H6), 3.13–3.02 (m, 1H, H5),
2.37 (s, 3H, Ar-CH3), 1.93 (m, 2H, OCH2CH2N3), 1.71 (m, 2H, H5aα-β).
13C NMR (62.5 MHz, MeOD), δ (ppm) = 144.3, 140.15, 140.0, 130.5,
129.32, 128.35, 128.20, 128.16, 103.8 (Anomeric), 79.1 (C5′), 77.5
(C2), 75.5 (C3′), 73.6 (C2′), 73.0 (C1), 72.7 (C6), 70.1 (Ar-CH2), 69.3
(C4′), 68.7 (C3), 63.1(OCH2CH2N3), 56.5 (C6′), 52.0 (OCH2CH2N3),
37.8 (C4), 29.4 (C5a), 23.1 (C5), 21.5 (Ar-CH3). HRMS, (m/z) [M +
Na]+, calculated C29H40N4O11NaS 675.2312, found 675.2324.
(0.152 g, Yield = 83%), as a white solid.
20
Rf = 0.44 (8:2 Hexane/AcOEt four times eluted). [
α
]
= -80.0◦ (c
5.2.12. (2-Aminoethyl)-2-O-(
tosylamino)-5a-carba-
α
-D-mannopyranosyl)-4-deoxy-4-(N-
0.25, CHCl3). FTIR (neat)
ν
(cmꢀ 1) = 3372, 3243, 2928D, 2872, 2360,
α
-D-mannopyranoside. (+)-4a
2339, 2106, 1727, 1695, 1262. 1H NMR (250 MHz, CDCl3), δ (ppm) =
8.11–8.01 (m, 6H, Ar), 7.99–7.92 (m, 3H, Ar), 7.86–7.79 (m, 3H, Ar),
7.65–7.20 (m, 14H, Ar), 7.19–7.01 (m, 4H, Ar), 6.01 (t, 1H, J = 9.8 Hz,
H4′), 5.92–5.83 (m, 2H, H2′+H3′), 5.49 (d, 1H, J = 1.5 Hz, Anomeric),
Pseudomannobioside (þ)-4b (0.0057 g, 0.0087 mmol, 1.0 eq) was
dissolved in a 4:1 MeOH/anhydrous DMF mixture (2.0 mL) and 10 wt%
Pd/C (0.010 g) was added. The reaction mixture was constantly kept
8

S. Di Pietro et al.
Bioorganic Chemistry 107 (2021) 104566
4.96–4.35 (m, 7H, Ar-CH2 + H5′+H6′a-b), 4.22 (s, 1H, H2), 3.79 (dd,
1H, J = 9.2, 2.6 Hz, H3), 3.64 (s, 1H, H1), 3.49 (m, 2H, 2H HCH2CH2N3),
3.37–3.22 (m, 2H, 2H HCH2CH2N3), 3.19–3.10 (m, 1H, H4), 1.96–1.65
experiments were performed on a BIAcore T200 using a CM3 sensor S
series chip. Flow cells were activated as described [35]. Flow cell one
was functionalised with BSA and blocked with ethanolamine and sub-
sequently used as a control surface. Flow cells 2 and 3 were treated with
(m, 3H, H5 + H5a
α
-β), 1.10 (d, 3H, J = 6.5 Hz, CH3). 13C NMR (62.5
MHz, CDCl3), δ (ppm) = 166.3 (Ar-COO), 165.7 (Ar-COO), 165.7 (Ar-
COO), 165.2 (Ar-COO), 139.1, 138.6, 133.6, 133.5, 133.3, 130.0, 129.9,
129.8, 129.6, 129.3, 129.1, 128.7, 128.6, 128.6, 128.5, 128.4, 127.8,
127.6, 127.5, 127.4, 99.0 (Anomeric), 82.9 (C4), 81.6 (C3), 76.1 (C2),
75.5 (C5′), 73.1 (C1), 70.3 (Ar-CH2), 70.1 (C2′), 69.5 (C3′), 67.9 (C4′),
67.3 (OCH2CH2N3), 63.4 (C6′), 50.9 (OCH2CH2N3), 32.5 (C5), 31.9
(C5a), 18.2 (CH3).
BSA-Manα1-3[Manα1-6]Man (Dextra) (60 μg/ml) in 10 mM NaOAc pH
4 to reach different binding densities and blocked with ethanolamine.
The final densities on flow cells 2 and 3 were 2062 and 2183 RU,
respectively. The affinity of the various compounds for DC-SIGN ECD
were evaluated via an established inhibition assay [35] in which DC-
SIGN ECD was injected at 20
μM alone or in the presence of
increasing concentration of inhibitors (ranging from 0 to 5 mM). In-
jections were performed at 5 μL/min using 25 mM Tris-HCl pH 8, 150
5.2.15. (2-Azidoethyl)-2-O-(
α
-D-mannopyranosyl)-3,4-di-O-benzyl-6-
mM NaCl, 4 mM CaCl2, 0.05% P20 surfactant as running buffer. The
surface was regenerated by the injection of 50 mM EDTA.
deoxy-5a-carba- -D-mannopyranoside. (+)-5b
α
A freshly prepared solution 0.5 M of sodium methoxide (0.5 mL,
0.018 mmol, 0.6 eq) in anhydrous MeOH was added to a solution of 1,2-
pseudomannobioside (-)-21 (0.029 g, 0.029 mmol, 1.0 eq) in MeOH
(1.0 mL) and the reaction mixture was stirred for 12 h at room tem-
perature. Then, the solution was diluted with additional MeOH and
Amberlite resin IRC-50 was added until pH = 7. Afterwards, the solution
was filtered from the resin and the filtrate evaporated. The residual solid
was purified by preparative TLC (SiO2, 0.5 cm, AcOEt/acetone = 8:2) to
afford (þ)-5b (0.0152 g, Yield = 45%) as a yellow solid.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Rf = 0.31 (8:2 AcOEt/Acetone). [
α
]
20 = +8.5◦ (c 0.76, CHCl3). FTIR
(neat)
ν
(cmꢀ 1) = 3433, 2964, 2930, D2106, 1262, 1451, 1415, 1096,
Funding: S.A. was supported by the European Union’s Horizon 2020
research and innovation program under the Marie Sklodowska-Curie
grant agreement No. 642870 (ITN-Immunoshape). This work used the
Multistep Protein Purification Platform and the SPR platform of the ISBG
(UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for
Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02)
and GRAL, financed within the University Grenoble Alpes graduate
school CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integra-
tion into the Interdisciplinary Research Institute of Grenoble (IRIG,
CEA). F.F. also acknowledges the French Agence Nationale de la
Recherche (ANR) PIA for Glyco@Alps (ANR-15-IDEX-02). V.D.B. ac-
knowledges the University of Pisa for intramural funding.
1023, 801. 1H NMR (250 MHz, CDCl3), δ (ppm) = 7.40–7.20 (m, 10H),
5.22 (s, 1H, Anomeric), 4.82 (d, 1H, J = 10.8 Hz, H2), 4.69 (d, 1H, J =
11.8 Hz, H2′), 4.61 (d, 1H, J = 11.8 Hz, H3′), 4.53 (d, 1H, J = 10.8 Hz,
H1), 4.18–3.92 (m, 4H, Ar-CH2), 3.92–3.78 (m, 2H, H3 + H4), 3.74 (dd,
1H, J = 9.1, 2.5 Hz, H5′), 3.61–3.44 (m, 3H, H6′+H4′), 3.42–3.14 (m,
2H, OCH2CH2N3), 1.78–1.66 (m, 3H, H5 + OCH2CH2N3), 1.48–1.36 (m,
2H, H5a
α
-β), 1.01 (d, 3H, J = 6.5 Hz, CH3). 13C NMR (62.5 MHz, CDCl3),
δ (ppm) = 139.0, 138.7, 128.6, 128.5, 128.2, 128.1, 127.8, 127.7, 101.6
(Anomeric), 83.2 (C4), 81.2 (C5′), 77.0 (C3), 75.8 (C2), 75.0 (C3′), 73.0
(C1), 71.8 (C2′), 71.0 (Ar-CH2), 67.9 (C4′), 66.6 (OCH2CH2N3), 61.2
(C6′), 50.9 (OCH2CH2N3), 31.7 (C5), 29.9 (C5a), 18.2 (CH3). HRMS, (m/
z) [M + Na]+, calculated for C29H39N3O9Na 596.2584, found 596.2590.
The authors thank Dr. S. Fortunato and Mr. A. Marsico for the pre-
liminary work.
The authors declare no competing financial interest.
5.2.16. (2-Aminoethyl)-2-O-(
-D-mannopyranoside. (+)-5a
Pseudomannobioside (þ)-5b (0.0152 g, 0.0265 mmol, 1.0 eq) was
α-D-mannopyranosyl)-6-deoxy-5a-carba-
α
Appendix A. Supplementary material
dissolved in a 4:1 MeOH/anhydrous DMF mixture (2.0 mL) and 10 wt%
Pd/C (0.003 g) was added. The reaction mixture was constantly kept
under 1 atm of hydrogen at room temperature until fully reduction of the
azide group and deprotection of the benzyl groups (monitoring by TLC
(8:2 CH2Cl2/MeOH)). After 18 h the mixture was diluted with MeOH
and filtered through a celite pad. The evaporation of the solvent afforded
a crude reaction product which was re-crystallized from (i-Pr)2O to
afford pure 1,2-pseudomannobioside (þ)-5a (0.0079 g, Yield = 81%),
as a yellow solid.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104566.
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