Trehalose-based neuroprotective autophagy inducers

Article abstract of DOI:10.1016/j.bmcl.2021.127929

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism

Full text of DOI:10.1016/j.bmcl.2021.127929

Bioorg. Med. Chem. Lett. 40 (2021) 127929
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Trehalose-based neuroprotective autophagy inducers
,
,
,f
Giulia Assonia e, Giulia Frapportia e, Eleonora Colombob, Davide Gornatib
,
,
,*
Maria Dolores Perez-Carrionc, Laura Politod, Pierfausto Senecib *, Giovanni Piccolia
,
,
*
Daniela Arosiod
a Department of Cellular, Computational And Integrative Biology, (CIBIO), Via Sommarive 9, I-38123 Povo, TN, Italy
b
`
Chemistry Department, Universita Statale di Milano, Via Golgi 19, I-20133 Milan, Italy
c
´
Unidad Asociada Neurodeath, Departamento de Ciencias Medicas, Universidad de Castilla-La Mancha, Albacete, Spain
d Istituto di Scienze e Tecnologie Chimiche (SCITEC) “Giulio Natta”, Consiglio Nazionale delle Ricerche (CNR), Via C. Golgi 19, I-20133 Milan, Italy and Via G. Fantoli
16/15, I-20138 Milan, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the
aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information
about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and
small molecule prodrugs (2–5), putative probes (6–10) and inorganic nanovectors (12a – thiol-PEG-triazole-
trehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing
properties. While small molecules 2–11 showed no pro-autophagic behavior at sub-millimolar concentrations,
Trehalose
Neurodegeneration
Autophagy
Chemical probes
Nanoparticles
Prodrugs
trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 μM trehalose
concentration without any significant toxicity at the same concentration.
α
,
α
-Trehalose 1 (trehalose from now on, Fig. 1) is a non-reducing
transformations12. Its regioselective functionalization led to biologically
active small molecules (hits, leads13–14 and chemical probes15–16), to
disaccharide formed by a 1,1 linkage (refractory to glucosidase cleav-
age) between two D-glucose molecules1. It is widely bio-synthesized in
the biological world, but absent in mammals2. In extremophile organ-
isms it stabilizes life processes in extreme conditions (freezing or
dehydration3, surviving heat and desiccation stress4. It is used in food
processing and cosmetics5, and for the reduction of misfolding of path-
ologic, aggregated proteins6.
glycoclusters17–18 and to trehalose-based nanoparticles19
.
This study aimed to develop some robust, high yield routes to key
trehalose intermediates and small molecule prodrugs (2–5, Fig. 3), pu-
tative probes (6–10) and inorganic nanovectors (12a – thiol-PEG-
triazole-trehalose constructs 11) to be tested for their autophagy-
inducing properties, bioavailability and molecular mechanism of action.
The strategy followed to prepare acetylated trehalose derivatives 2–5
– using published procedures, often optimizing them in terms of yields,
sometimes introducing major changes - is reported in Appendix A –
Supplementary Material. Acetylated trehalose derivatives 2–5 were
submitted to biology profiling as putative autophagy inducers.
Due to its high hydrophilicity, trehalose poorly permeates biological
membranes. It is not naturally occurring in higher vertebrates, which
express on their intestinal membrane trehalases7 to hydrolyze trehalose
in two molecules of glucose, preventing its oral administration. Thus,
extremely high dosages are needed for in vivo effects.
Robust in vitro evidence show that trehalose induces autophagy at
≥100 mM (Fig. 2)8. Mechanistic hypotheses include blocking glucose
transporters at the plasma membrane (GLUT) to stimulate autophagy9,
and inducing autophagy via transient transcription factor EB (TFEB)-
6-Azido trehalose 612 and 6,6′-bisazido trehalose 720 were previously
synthesized. Rather than reproducing such synthetic routes, we envis-
aged and successfully assessed a single synthetic strategy which led us to
both synthetic targets. Such strategy is shown in Scheme 1. Namely, we
submitted trehalose to bromination with 2 equivalents of NBS (step a),
then we per-acetylated the crude (step b, Scheme 1). Mono- and bis-
mediated lysosomal enlargement and membrane permeabilization10
.
Trehalose
underwent
synthetic11
and
biosynthetic
* Corresponding authors.
E-mail addresses: pierfausto.seneci@unimi.it (P. Seneci), giovanni.piccoli@unitn.it (G. Piccoli), daniela.arosio@scitec.cnr.it (D. Arosio).
These authors contributed equally to this project.
e
f
Current address: Procos, SpA, via G. Matteotti 249, I-28602 Cameri (NO), Italy.
https://doi.org/10.1016/j.bmcl.2021.127929
Received 30 October 2020; Received in revised form 25 February 2021; Accepted 27 February 2021
Available online 9 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
a protecting group switch from benzyl to acetyl (steps e, f), an azide
substitution (step g), and a carefully controlled deacetylation (step h,
Scheme 3). Target 4-(5-azidopentyl) trehalose 9 was obtained in mod-
erate, unoptimized yields and was submitted to biology profiling as a
putative autophagy inducer.
Due to the easier synthetic accessibility in larger amounts of 6-azido
trehalose 6 when compared to azides 7 to 9, we used the former as a key
intermediate to be converted either into a chemical probe for mecha-
nistic studies, and into constructs for nanovector preparation. Per-
acetylated 6-azido trehalose 16 was reacted with N-(prop-2-yn-1-yl)
biotinamide 28 in a click chemistry protocol (step a, Scheme 4), yielding
a per-acetylated, triazole-connected biotin-trehalose adduct in excellent
yields. Deacetylation (step b, Scheme 4) led in excellent yields to pure
target probe 10, which was submitted to biology profiling as a putative
autophagy inducer.
Fig. 1.
α,α-Trehalose 1: chemical structure.
brominated, per-acetylated trehaloses 13 and 14 were obtained in
moderate yields, and separated by chromatography. Their substitution
with sodium azide and deacetylation in controlled conditions (steps c
and d, Scheme 1) led to 6-azido trehalose 6 and 6,6′-bis-azidotrehalose 7
in good yields. Both were submitted to biology profiling as putative
autophagy inducers.
Gold nanoparticles are reliable theranostics tools, providing benefits
in terms of drug delivery22, photothermal and microwave therapies23
.
Four trehalose monoazides were recently reported, by replacing each
hydroxy group of a glucose ring with an azide12. We did introduce the
azide group on one or both primary hydroxyls in trehalose (compounds
6 and 7), but we felt that the insertion of a small linker between
trehalose and each azide on secondary hydroxyls could preserve its
properties, while making an azide more accessible for further derivati-
zation. Thus, we targeted 2- and 4-(5-azidopentyl) trehalose 8 and 9.
As to the former, its synthesis entailed the introduction of acetals on
positions 4 and 6 and per-silylation (step a, Scheme 2), followed by a one
pot desilylation – 3-benzylation reaction21 (step b). Benzylation with
sub-stoichiometric benzyl bromide (step c) produced, after chromatog-
raphy, desired tri-benzyloxy, mono-2-hydroxy trehalose 19 in moderate
yields. Its transformation into target 2-(5-azidopentyl) trehalose 8 in
good yields entailed alkylation with a large excess of dibromopentane,
hydrogenolytic deprotection, peracetylation, azide substitution and
careful deacetylation (steps d to h, Scheme 2). Compound 8 was sub-
mitted to biology profiling as a putative autophagy inducer.
Their surface functionalization with biologically active compounds is
quite developed24
.
Our efforts towards adduct 11 are depicted in Scheme 5. t-Butyl
protected PEG alcohol 30 was converted into protected thiol 31, C-
deprotected (32) and amidated with propargylamine (33, steps a-c,
Scheme 5) in standard conditions with excellent yields. Then, linker 33
was conjugated through copper-catalyzed Huisgen cycloaddition with
acetylated 6-azido trehalose 15 to give PEG-triazole trehalose conjugate
34 in good yields (step d). Deacetylation (step e, Scheme 5) in standard
conditions led to excellent yields of sufficiently pure target adduct 11;
the presence of disulfide-connected dimer 35 does not affect AuNPs’
preparation because it should be reduced in situ by NaBH4 (vide infra).
Trehalose-bearing AuNPs 12a were prepared by reduction of tetra-
chloroauric acid by NaBH4 in presence of the thiol-PEG-triazole treha-
lose conjugate 11, using a reported procedure25. The resulting dark
suspension of AuNPs was shaken for ca. 2 h at RT, and purified by
centrifugal filtration. AuNPs 12a were water-soluble and could be re-
dissolved after lyophilization. TEM analysis showed that trehalose-
functionalized AuNPs 12a were characterized by a very small core (ca.
4.6 nm) and were almost uniformly dispersed, without signs of aggre-
gation (see Supplementary Material).
Trehalose was transformed into its mono-acetal using a 1:1 reagent
ratio followed by per-benzylation (steps a and b, Scheme 3) to yield
compound 23. Regioselective acetal opening (step c) was followed by a
nucleophilic substitution with a large excess of dibromopentane (step d),
Fig. 2. Autophagy induction and cytotoxicity assays on compound 1 upon 24 h treatment (A). Graphs report LC3BII/LC3BI ratio and LC3BII amount expressed as fold
over
α
-tubulin (B), and cell viability. Each value was normalized on not treated control, set at 1. Data are expressed as mean ± S.E.; n = 6–8, ** p < 0.01.
2

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
Fig. 3. Targeted trehalose modifications: putative prodrugs (2–5), analogues (6–9), chemical probes (10), trehalose-linker constructs (11) and trehalose-based
nanovectors (12a).
Scheme 1. Synthesis of 6-azido trehalose 6 and 6,6′-bisazido trehalose 7.
Similarly, standard PEG-AuNPs 12b were prepared by substituting
trehalose conjugate 11 with PEG methyl ether thiol (average MW 800).
TEM analysis confirmed their quality, so that both AuNPs 12a and 12b
were submitted to biology profiling as putative autophagy inducers.
We initially profiled derivatives 2–10 and nanovectors 12a in HeLa
cells for their capability to induce autophagy. We monitored autophagy
by tracking the mobility shift from LC3I to LC3II, that is a proxy for the
induction of autophagy; and the amount of LC3II, that correlates with
the number of autophagosomes8. We measured
α -tubulin levels as an
internal control in our assays. We treated HeLa cultures for 24 h at 37 ◦C
with compounds 2–6 (2.5 mM), 7 (25, 50
μM), 8 (25, 50 μM), 9 (2.5
mM), 10 (200 M, 2 mM), control vehicle (DMSO), and trehalose (100
μ
3

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
OTMS
OTMS
OH
OH
OH
O
O
O
O
OBn
O
O
O
OH
OH
a
O
b
O
O
O
O
Ph
O
O
Ph
O
OH
HO
OH
OTMS
OTMS
OBn
OH
O
Ph
O
Ph
1
OH
17
18
OBn
OBn
O
O
OBn
c
O
d
O
O
O
OBn
O
O
O
Ph
O
O
OH
O
O
Ph
O
OBn
O
Ph
OBn
Br
19
20
O
Ph
OAc
OAc
OH
O
O
O
OAc
OAc
O
O
O
OAc
OAc
O
O
O
OH
OH
OH
e,f
g
AcO
AcO
AcO
AcO
HO
HO
h
O
O
OAc
OAc
N3
OH
N3
OAc
21
OAc
22
8
Br
a) PhCH(OMe)2 2.2eq, cat PTSA, DMF, 105-100°C, 100min, then TMSCl, 1H-Imidazole, 0° to RT, 18hrs, 76%; b)
cat TMSOTf, PhCHO, Et3SiH, DCM -78°C to 0°C, 6hrs, 80%; c) BnBr 0.75eq, NaH, THF, 0°C to RT, 6hrs, 49%; d)
Br(CH2)5Br 10eq, NaH 2eq, DMF, 55°C, 4hrs, 71% e) H2, Pd-C, MeOH/THF 4:1, RT, 24hrs; f) Ac2O, cat DMAP,
DCM/Py, RT, 4hrs, 71% over two steps; g) NaN3, DMF 65°C, 16hrs, 90%; h) NaOMe, MeOH, RT, 24hrs, 96%.
Scheme 2. Synthesis of 2-(5-azidopentyl) trehalose 8.
OBn
OBn
OBn
OH
OH
O
O
OBn
OBn
O
O
OBn
OBn
O
O
OH
OH
c
a,b
Ph
O
O
O
O
O
OBn
HO
OBn
HO
OH
OBn
OBn
OH
23
24
OBn
OBn
OH
OAc
OBn
OBn
O
O
OAc
OAc
OAc
O
O
OBn
OBn
OBn
e,f AcO
d
O
O
O
OAc
O
OBn
OAc
OBn
26
Br
Br
25
OAc
OH
O
O
OAc
OAc
O
O
OH
OH
AcO
h
g
HO
O
O
O
OAc
O
OH
OAc
OH
27
N3
OAc
N3
OH
9
a) PhCH(OMe)2 1eq, PTSA 0.05eq, DMF, 105-100°C, 100min; b) BnBr 9eq, NaH 7.5eq, dry DMF, RT, 24hrs,
33% over two steps; c) Et3SiH 7.5eq, TFA 7.5eq, dry DCM, 0°C to RT, 4hrs, 78%; d) Br(CH2)5Br 10eq, NaH
2eq, DMF, 55°C, 4hrs, 70%; e) H2, Pd/C 10%, MeOH/THF 4:1, RT, 16hrs; f) Ac2O, cat DMAP, DCM/Py, RT,
16hrs; g) NaN3, DMF 65°C, 16hrs, 60% over three steps; h) NaOMe, MeOH, RT, 8hrs, 98%.
Scheme 3. Synthesis of 4(5-azidopentyl) trehalose 9.
mM). Unfortunately, we did not appreciate any autophagic activation at
the given experimental conditions for compounds 2–10 (Fig. 4).
We next evaluated the biological activity of PEG-AuNPs 12a (diluted
1:400, 1:200, and 1:100 for an estimated, adjusted free-trehalose con-
release of free trehalose from PEG-AuNPs 12a; in these conditions,
trehalose-bearing AuNPs 12a efficiently induced autophagy and
increased the number of autophagosome at the higher estimated con-
centration of free trehalose (40 μM, Fig. 6).
centration around 10, 20, and 40
μ
M), of their non-assembled PEG-
We verified the in vitro safety of trehalose-bearing AuNPs 12a after
24 and 48 h incubation by a standard MTT cytotoxicity assay. They
induced only limited cytotoxicity at the estimated free trehalose con-
trehalose precursor 11 (10, 20, and 40
μ
M), of the PEG-linker (5, 10, 20
μ
M) and of the standard PEG-AuNPs 12b (diluted 1:400, 1:200, and
1:100). Upon a 24 h long treatment, autophagy was not elicited by any
sample at any concentration (Fig. 5).
centration of 40 μM upon 24 h treatment, but were well tolerated upon
48 h incubation (Fig. 7).
Next, we repeated the assays after 48 h incubation to ensure the
Bafilomycin A1 is an inhibitor of autophagosome-lysosome fusion in
4

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
Scheme 4. Synthesis of biotin-based trehalose chemical probe 10.
Scheme 5. Synthesis of thiol-PEG-triazole-trehalose adduct 11 for functionalization of inorganic nanovectors.
vitro and it enables to determine the activity of the autophagic flux.
as such, and the thiol-PEG-triazole-trehalose construct 11 used to build
them) should have been enough to achieve our goals.
Thus, we treated cells with compound 1 (100 mM) and 12a (40 μM)
alone or in presence of bafilomycin 1A (10 nM) for 48 h, and we
monitored the amount of LC3II (Fig. 8A). Remarkably, we noticed that
bafilomycin 1A had a synergistic effect on both compound 1 and 12a
(Fig. 8B).
Acetylated trehalose derivatives 2–5 were earlier studied as intra-
cellular delivery trehalose vehicles26, with hexa-acetylated 4 showing
increased membrane permeability. Unfortunately, we could not show an
autophagy-inducing effect up to low mM concentrations for 2–5, thus
disproving their potential usefulness as bioavailable trehalose prodrugs.
Trehalose azides 6–9 and biotinylated trehalose 10 were designed
respectively as putative photoactivatable probes27 and as an affinity
chromatography reagent28. If one among them would have shown sig-
nificant activation of the autophagic flux, we would have either
Our goal was to identify trehalose-based novel chemical entities with
measurable autophagy-inducing properties, and to synthesize biologi-
cally active trehalose-based probes to gather information about its mo-
lecular mechanism of action. We reasoned that a small array made of
prodrugs (2–5), putative probes (6–10) and inorganic nanovectors (12a
5

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
Fig. 4. Autophagy induction assay on compounds 2–10 upon 24 h treatment (A). Graphs report LC3BII/LC3BI ratio (B) and LC3BII amount expressed as fold over
-tubulin (C). Each value was normalized on proper control. Data are expressed as mean ± S.E.; n = 6–8, ** p < 0.01.
α
photoactivated it in cells showing autophagy stimulation, covalently
binding to their unknown target either before or after cell lysis (6–9) or
we would have incubated it in the same cells, whose lysates then were to
be eluted on a streptavidin-coated support to identify the bound target
(10). The lack of autophagy stimulation by trehalose analogues 6–10
prevented any mechanism of action-targeted experiment.
the pharmacodynamic properties of trehalose. Rather, we are confident
that the improved activity observed for compound 12a stems from its
improved pharmacokinetics properties, in particular stability and
membrane permeability. Indeed, further studies will be performed to
determine the bioavailability of compound 12a.
Such results could have major therapeutic consequences and will be
thoroughly exploited in the near future, up to in vivo testing in models of
diseases showing autophagy disturbance.
Such negative results suggest that any modification on trehalose
hampers its biological activity. Instead, we considered that the identi-
fication of nanocarriers to improve trehalose pharmacokinetics proper-
ties may prove to be a convenient strategy. In a previous work we
described trehalose-containing self-assembling NPs based on squa-
lene29, which did not influence the autophagic flux in our assays.
Conversely, we report here that trehalose-bearing AuNPs 12a effi-
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.
ciently induce autophagy in cellular assays at a 40 μM estimated con-
centration, i.e. at a > 1000 lower concentration than the one needed for
free trehalose to be active. We did not expect that AuNPs should modify
6

G. Assoni et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
Fig. 5. Autophagy induction assay on conjugate 11, PEG-linker, trehalose-bearing PEG-AuNPs 12a and standard PEG-AuNPs 12b upon 24 h treatment (A). Graphs
report LC3BII/LC3BI ratio (B) and LC3BII amount expressed as fold over α-tubulin (C). Each value was normalized on untreated control. Data are expressed as mean
± S.E.; n = 6–8,** p < 0.01.
Fig. 6. Autophagy induction assay on conjugate 11, PEG-linker, trehalose-bearing PEG-AuNPs 12a and standard PEG-AuNPs 12b upon 48 h treatment (A). Graphs
report LC3BII/LC3BI ratio (B) and LC3BII amount expressed as fold over
α
-tubulin (C). Each value was normalized on untreated control. Data are expressed as mean
± S.E.; n = 6–8, * p < 0.05, ** p < 0.01.
7

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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127929
Fig. 7. MTT cytotoxicity assay on compound 11, PEG linker, trehalose-bearing PEG-AuNPs 12a and standard PEG-AuNPs 12b upon 24 (left) and 48 h (right)
treatments. Graphs report viability expressed as fold over control, set at 1. Data are expressed as mean ± S.E.; n = 6–8, * p < 0.05.
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