Cis-Glyco-fused benzopyran compounds as new amyloid-β peptide ligands

Article abstract of DOI:10.1039/c1cc13046c

A small library of glyco-fused benzopyran compounds has been synthesised. Their interaction features with Aβ peptides have been characterised by using STD-NMR and trNOESY experiments. The conformational analysis of the compounds has also been carried out

Full text of DOI:10.1039/c1cc13046c

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COMMUNICATION
cis-Glyco-fused benzopyran compounds as new amyloid-b peptide
ligandswz
Cristina Airoldi,a Francisco Cardona,ab Erika Sironi,a Laura Colombo,c Mario Salmona,c
Artur Silva,b Francesco Nicotraa and Barbara La Ferla*a
Received 24th May 2011, Accepted 28th July 2011
DOI: 10.1039/c1cc13046c
A small library of glyco-fused benzopyran compounds has been
synthesised. Their interaction features with Ab peptides have
been characterised by using STD-NMR and trNOESY experi-
ments. The conformational analysis of the compounds has also
been carried out through molecular mechanics (MM) and
molecular dynamics (MD) simulations.
Alzheimer’s disease (AD) is the most common cause of
dementia among neurodegenerative diseases in the elderly
population.1–5 A central pathological feature of AD is the
accumulation of misfolded amyloid-b (Ab) peptides in the
form of oligomers and amyloid fibrils and plaques in the brain.
Molecules able to stabilize the soluble Ab conformation, to
destabilize the altered amyloidogenic conformer, and to prevent
the required conformational transition could be effective
inhibitors of amyloid plaque formation and very potent drug
candidates for AD treatment.6–8 Many natural and synthetic
compounds able to interact as Ab ligands have been identified.
Among them, we have paid attention to a set of small
molecules in which aromatic moieties seem to play a key role.9
Unfortunately, many of these compounds lack solubility,
chemical stability and/or show pharmacological activities not
directly correlated to AD. Therefore, the correct therapeutic
evaluation of these molecules towards AD cannot be performed
in a straightforward manner. In this context, and in order to
overcome these chemical-based limitations, we have designed
a pool of potential Ab-ligands which display a glyco-fused
benzopyran structure (Scheme 1), therefore maintaining the
required aromatic moiety, while generating chemically stable
and water soluble compounds. Moreover, the glycidic entity
assures further possible derivatizations, such as conjugation to
Scheme 1
other molecular entities (nanoparticles, polymeric supports, etc.),
which may be employed as useful features for diagnostic
and therapeutic purposes. Notably glycomimetics inserted in
these kinds of carbohydrate–aromatic hybrids have recently
gained great interest.10,11 The employed synthetic strategy
exploits the reaction between o-hydroxybenzaldehydes and
glycals using a catalytic amount of scandium triflate in the
presence of trimethyl orthoformate (TMOF), as described by
Yadav et al.12 In order to verify the influence of the various
parts of the molecule on the interaction with the Ab peptide,
we generated a small library of glyco-fused benzopyran
compounds, using differently substituted o-hydroxybenzaldehydes
and employing both glucal (8) and galactal (9). In all cases,
we obtained cis-fused pyrano[3,2-b]benzopyran (21–91% yield),
but in contrast to previous reports,12 the reaction afforded a
variable ratio of separable mixtures of two diastereoisomers at
C5 (Table 1); the major isomer was then deprotected to afford
the final compounds 20–29.
From the molecular recognition perspective, very recently,
we have employed Saturation Transfer Difference (STD)
NMR experiments13–22 to characterize the interaction of
Ab1–42 peptide with tetracycline, thioflavin T23 and curcumin
derivatives.24 Thus, the same methodology has been applied
herein to check the effect of glyco-fused benzopyran derivatives
on the Ab1–42 oligomer recognition process.
In particular, the interaction studies with the amyloid peptide
Ab1–42 were carried out using the debenzylated compounds
20–29, and STD-NMR and trNOESY experiments.
STD-NMR experiments were performed using ligand : peptide
20 : 1 mixtures dissolved in deuterated PBS, pH 7.4, 25 1C.
Each mixture was analyzed irradiating the sample at ꢀ1.0 ppm
to achieve the selective saturation of some aliphatic resonances
of Ab oligomers. The presence of NMR signals of the test
a Dept. of Biotechnology and Biosciences, University of
Milano-Bicocca, P.zza della Scienza 2, 20126, Milan, Italy.
E-mail: barbara.laferla@unimib.it; Fax: +39 0264483565;
Tel: +39 0264483421
b Dept. of Chemistry, University of Aveiro, Campus Universitario de
Santiago, 3810-193 Aveiro, Portugal
c Dept. of Molecular Biochemistry and Pharmacology Mario Negri
Institute for Pharmacological Research, via La Masa 19, 20156,
Milan, Italy
w This article is part of the ChemComm ‘Glycochemistry and
glycobiology’ web themed issue.
z Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13046c
c
10266 Chem. Commun., 2011, 47, 10266–10268
This journal is The Royal Society of Chemistry 2011

Table 1 cis-Fused glyco benzopyrans
o-Hydroxy benzaldehyde
D-Glycal
Protected products
C5 R/S (yield%)
Deprotected compounds C5 R (yield%)
1: R1,R2 = H,H
2: R1,R2 = NO2,H
3: R1,R2 = OBn,H
4: R1,R2 = OMe,H
5: R1,R2 = CH3,H
6: R1,R2 = H, OMe
7: R1,R2 = H,CH3
5
8
8
8
8
8
8
8
9
9
9
10: R1,R2,R3,R4 = H,H,H,OBn
11: R1,R2,R3,R4 = NO2,H,H,OBn
12: R1,R2,R3,R4 = OBn,H,H,OBn
13: R1,R2,R3,R4 = OMe,H,H,OBn
14: R1,R2,R3,R4 = CH3,H,H,OBn
15: R1,R2,R3,R4 = H,OMe,H,OBn
16: R1,R2,R3,R4 = H,CH3,H,Obn
17: R1,R2,R3,R4 = CH3,H,OBn,H
18: R1,R2,R3,R4 = H,OMe,OBn,H
19: R1,R2,R3,R4 = H, CH3, OBn,H
92/8 (59%)
100/0 (40%)
100/0 (35%)
85/15 (73%)
95/5 (91%)
53/47 (64%)
100/0 (45%)
100/0 (66%)
100/0 (37%)
100/0 (21%)
20: R1,R2,R3,R4 = H,H,H,OH (97%)
21: R1,R2,R3,R4 = NH2,H,H,OH (94%)
22: R1,R2,R3,R4 = OH,H,H,OH (100%)
23: R1,R2,R3,R4 = OMe,H,H,OH (96%)
24: R1,R2,R3,R4 = CH3,H,H,OH (95%)
25: R1,R2,R3,R4 = H,OMe,H,OH (97%)
26: R1,R2,R3,R4 = H,CH3,H,OH (97%)
27: R1,R2,R3,R4 = CH3,H,OH,H (98%)
28: R1,R2,R3,R4 = H,OMe,OH,H (97%)
29: R1,R2,R3,R4 = H, CH3, OH,H (98%)
6
7
Since the STD intensity is proportional to the ligand binding
affinity for the molecular target,13,14 we exploited competitive
STD experiments to rank the affinity25 of compounds 20,
22–29 for the peptide. Due to extensive resonance overlapping,
the acquisition of competitive STD spectra on a unique
mixture containing all the molecules was not feasible. Hence,
we performed three different competitive experiments of the
different molecules in the presence of Ab1–42 oligomers.
Separate experiments for mixtures containing the D-galactose
derivatives (20–26), the D-glucose analogues (27–29), or ‘‘the
best ligands’’ identified from the two previous screenings
(24 and 29) were performed. In the first and second competi-
tive experiments, we measured the STD effect on H6, and in
the third experiment on H10a (see Scheme 1). For each
molecule, the fractional STD effect was calculated as (I ꢀ I0)/I0,
where I is the intensity of the monitored signal in the
STD spectrum and I0 is the intensity of the same signal in a
reference spectrum. Compounds 24 and 29 showed the same
affinity for Ab1–42, as their H10a signals presented equal
intensities. Hence, to compare the data obtained in the differ-
ent competitive experiments, the fractional STD effects of 24
and 29 were set equal to 1 and, therefore, the relative inten-
sities for the other molecules were calculated. The results are
summarized in Fig. 2.
Fig. 1 Comparison between STD experiments acquired in the pre-
sence of compounds 21 and 24. 1H NMR (A and C) and 1D-STD
NMR (B and D) spectra recorded on Ab : ligand mixtures dissolved in
deuterated PBS at 25 1C and containing Ab1–42 (80 mM) and a test
molecule (1.6 mM) (A and B, compound 21; C and D, compound 24).
1H spectra were acquired with 64 scans, 1D-STD spectra with 512
scans and 2 s of saturation time.
molecule in the STD spectra is a non-ambiguous demonstra-
tion of the existence of interaction. Conversely, the absence of
NMR resonances in the STD spectra indicates that the
employed molecule is not an Ab ligand. In all cases, several
NMR resonances of 20–29 appeared in the corresponding
STD spectra recorded in the presence of Ab oligomers
(Fig. 1C and D and Fig. S1 (ESIz)), thus showing their ability
to recognize and bind Ab1–42, with the notable exception of
compound 21, whose signals are absent (Fig. 1B).
Thus, 24, 26, 27 and 29, whose aromatic rings are substi-
tuted with a methyl group, are the ligands with the highest
affinities for Ab oligomers, followed by 23, 25 and 28, which
present a O-methyl group as a substituent, and by 20, with no
aromatic substituent. These molecules display fractional STD
effects higher than 70%. Finally, 22, with a hydroxyl group at
position 7, has the lowest affinity. These data, together with
the absence of binding of 21 (with an amine substituent) to Ab
oligomers, clearly indicate that the lower the polarity of the
substituents on the aromatic ring, the greater the compound
affinity for Ab1–42. Moreover, the position of substituents on
the aromatic ring (position 7 or 8), as well as the nature of the
saccharide entity are not relevant, as supported by the evi-
dence that compounds 24, 26, 27 and 29 showed equal binding
affinity, and the same applies for compounds 23, 25 and 28.
These findings are in full agreement with the STD-based
epitope-mapping, recorded with five different saturation times
(0.5, 1.2, 2.0, 3.0, 5.0 s) (Fig. S3, ESIz). According to the STD
relative intensities, the region of the ligand mainly involved in
the interaction with Ab (the binding epitope) is the aromatic
ring, while protons of the saccharide portion showed the least
intense STD signals. These evidences explain why the stereo-
chemistry of sugar carbons does not influence the binding
Additional trNOESY experiments acquired on the same
ligand:peptide mixtures supported these results. The change
in the sign of the cross-peaks of the test molecule, from
positive (blue color), in the absence of Ab1–42 peptide, to
negative (red color), in the presence of Ab1–42 peptide, reflects
an increase of its effective rotational motion correlation time,
and supports its binding to a large molecular entity,14 here
represented by the Ab oligomers. In agreement with the STD
results, all trNOESY spectra of 20–29 acquired in the presence
of Ab1–42 showed the key change, from positive to negative,
of the corresponding cross-peak signs, except for compound
21. In this case, the NOE-cross peaks remained positive,
indicating that this molecule does not bind to Ab1–42 in a
significant manner (Fig. S2, ESIz).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10266–10268 10267

of Health (533F/Q/1), Cariplo Foundation (Project NOBEL-
GUARD), Banca IntesaSanPaolo and PRIN (PROT.2007T7M-
SAJ) for financial support, and Flamma (Italy) for the kind gift
of Fmoc amino acids. Francisco Cardona kindly acknowledges
Fundac¸ ao para a Ciencia e Tecnologia (FCT, Portugal) for the
PhD grant (SFRH/44888/2008).
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The research leading to these results has received funding
from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no.
212043 and from Regione Lombardia, Fondo per la promozione
di accordi istituzionali, Progetto no. 4779 ‘‘Network Enabled
Drug Design (NEDD)’’. We acknowledge the Italian Ministry
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c
10268 Chem. Commun., 2011, 47, 10266–10268
This journal is The Royal Society of Chemistry 2011