O-GlcNAcylation of truncated NAC segment alters peptide-dependent effects on α-synuclein aggregation

Article abstract of DOI:10.1016/j.bioorg.2019.103389

Numerous post-translational modifications (PTMs) of the Parkinson's disease (PD) associated α-synuclein (α-syn) protein have been recognised to play critical roles in disease aetiology. Indeed, dysregulated phosphorylation and proteolysis are thought to modulate α-syn aggregation and disease progression. Among the PTMs, enzymatic glycosylation with N-acetylglucosamine (GlcNAc) onto the protein's hydroxylated amino acid residues is reported to deliver protective effects against its pathogenic processing. This modification has been reported to alter its pathogenic self-assembly. As such, manipulation of the protein's O-GlcNAcylation status has been proposed to offer a PD therapeutic route. However, targeting upstream cellular processes can lead to mechanism-based toxicity as the enzymes governing O-GlcNAc cycling modify thousands of acceptor substrates. Small glycopeptides that couple the protective effects of O-GlcNAc with the selectivity of recognition sequences may prove useful tools to modulate protein aggregation. Here we discuss efforts to probe the effects of various O-GlcNAc modified peptides on wild-type α-synuclein aggregation.

Full text of DOI:10.1016/j.bioorg.2019.103389

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
O-GlcNAcylation of truncated NAC segment alters peptide-dependent effects
on α-synuclein aggregation
⁎
Philip Ryana,b,c, Ming-ming Xud, Andrew K. Daveya,b,c, Michael Kassioue, George D. Mellickd,
,
⁎
Santosh Rudrawara,b,c,
a School of Pharmacy and Pharmacology, Griffith University, Gold Coast, QLD 4222, Australia
b Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD 4222, Australia
c Quality Use of Medicines Network, Griffith University, Gold Coast, QLD 4222, Australia
d Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia
e School of Chemistry, The University of Sydney, NSW 2006, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Numerous post-translational modifications (PTMs) of the Parkinson’s disease (PD) associated α-synuclein (α-
syn) protein have been recognised to play critical roles in disease aetiology. Indeed, dysregulated phosphor-
ylation and proteolysis are thought to modulate α-syn aggregation and disease progression. Among the PTMs,
enzymatic glycosylation with N-acetylglucosamine (GlcNAc) onto the protein’s hydroxylated amino acid re-
sidues is reported to deliver protective effects against its pathogenic processing. This modification has been
reported to alter its pathogenic self-assembly. As such, manipulation of the protein’s O-GlcNAcylation status has
been proposed to offer a PD therapeutic route. However, targeting upstream cellular processes can lead to
mechanism-based toxicity as the enzymes governing O-GlcNAc cycling modify thousands of acceptor substrates.
Small glycopeptides that couple the protective effects of O-GlcNAc with the selectivity of recognition sequences
may prove useful tools to modulate protein aggregation. Here we discuss efforts to probe the effects of various O-
GlcNAc modified peptides on wild-type α-synuclein aggregation.
α-Synuclein aggregation
Parkinson’s disease
O-GlcNAcylation
Neurodegenerative diseases
1. Introduction
upstream, along the pathogenic cascade, the self-assembly of α-syn sits
is still undetermined. It is also unclear whether other specific molecular
There is no cure for the neurodegeneration that results in
Parkinson’s disease (PD). Specifically, understanding what causes PD
and the mechanisms leading to the neurodegenerative process is being
pursued to advance disease modifying therapies and clinically relevant
diagnostics. The main pathological hallmarks of PD arise from the
misprocessing, misfolding and self-assembly of a 140 amino-acid pro-
tein, α-synuclein (α-syn). α-Syn is highly abundant in the brain and,
when not degraded, forms beta pleated sheets and fibrillar aggregates
which eventually form insoluble Lewy bodies and Lewy neurites. These
aggregates have been used to characterize PD at autopsy [1].
events are required to trigger the process. The pathogenic process in PD
is associated with α-syn self-assembly, although the exact mechanism
responsible for inducing neurotoxicity is poorly defined. It is suggested
to be either characteristic of α-syn oligomers, or somehow triggered by
supraphysiological levels of α-syn monomers [2–4]. Aggregation prone
species may also be able to propagate between neurons [5,6], initiating
secondary nucleation events with endogenous α-syn upon arrival, ac-
celerating disease spread and progress [7].
In addition to PD, there are an array of neurodegenerative diseases
(NDs) characterized by the misfolding and self-assembly of neurotoxic
proteins. These proteins include amyloid-β and tau associated with
Alzheimer’s disease (AD); Huntingtin in Huntingtons disease (HD); and
various prion-related disorders. Chemical agents that can bind these
proteins in their monomeric or oligomeric forms selectively and with
high-affinity may be useful as early-stage diagnostic tools if detected ex
vivo. Compounds able to effectively inhibit the aggregation process are
speculated to be useful as therapeutic tools. Small peptides are able to
The predominant α-syn isoform is a 140 amino-acid sequence con-
sisting of three distinct domains: an N-terminal repeat domain spanning
residues 1–60; a central, hydrophobic region spanning residues 61–95;
and the proline-rich, highly acidic C-terminal region that spans residues
95–140. The central domain hosts the non-amyloid-β component
(NAC), a stretch of residues implicated in the pathogenic self-recogni-
tion interactions that lead to pathological aggregation. Exactly how far
⁎ Corresponding authors at: School of Pharmacy and Pharmacology, Griffith University, Gold Coast, QLD 4222, Australia.
E-mail addresses: g.mellick@griffith.edu.au (G.D. Mellick), s.rudrawar@griffith.edu.au (S. Rudrawar).
https://doi.org/10.1016/j.bioorg.2019.103389
Received 13 August 2019; Received in revised form 15 October 2019; Accepted 21 October 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:PhilipRyan,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103389

P. Ryan, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 1. Literature examples of peptidomimetic inhibitors of Aβ aggregation. (a) The sequence KLVFFKKKKKK (I) possesses a chain of cationic kosmotropic lysine units
as disrupting domain [12]; (b) Compound II bears dehydroalanine (ΔAla), a residue which induces specific structural conformations [10]; (c) a novel bis(thiourea)
hydrazide pseudopeptide scaffold (III) consists of a blocking face resulting from H-bond-incompetent sulfur atoms and N-methylamides, and a complementarity face
resulting from the increased acidity of the thiourea protons [11].
bind amyloidogenic proteins selectively, competitively and, when ap-
propriately functionalised, are able to disrupt amyloid growth [8].
Murphy and co-workers reported a ‘hybrid peptide’ (I, Fig. 1a) which
possesses a high-affinity for binding Aβ, supplied by the well-known
Aβ-selective CHD (KLVFF) recognition sequence, coupled to a dis-
ruptive lysine hexamer sequence that interferes with Aβ self-assembly
[9].The peptide worked to accelerate aggregation of the soluble Aβ
oligomers into fibrils, reducing the concentrations of toxic putative
intermediates. Rangachari and co-workers’ dehydroalanine-bearing
peptide (II, Fig. 1b) is able to efficiently reduce Aβ-induced cell toxicity
[10], and Hecht and Klein’s novel series of amphiphilic bis(thiourea)-
hydrazide containing pseudopeptides (III, Fig. 1c) adhere to Aβ and
inhibit its self-association [11]. Each of these peptidomimetics consist
Fig. 2. (a) Reversible PTM O-GlcNAcylation of the side chain of serine; (b) α-
Synuclein O-GlcNAcylation.
of a protein-recognition component and disruptive component. The
disruption results from intermolecular interactions that work overall to
disfavour amyloid formation, altering the physicochemical properties
of the resultant protein-inhibitor complex, or by other chemical me-
chanisms.
manner [17,18]. Results suggest that O-GlcNAcylated α-syn is in-
corporated into the aggregation reaction less efficiently than un-
modified α-syn [17]. Interestingly, O-GlcNAcylation (O-GlcNAc addi-
tion) is reported to affect the proteins implicated in AD, ALS and HD in
similar ways [19]. These findings support a model where O-GlcNAcy-
lation prevents indiscriminate protein aggregation in general.
Post-translational modifications (PTM) of α-syn, (proteolysis,
phosphorylation, acetylation, glycosylation etc.) alter the chemical
properties of the protein and are proposed to modulate Lewy pathology.
The O-GlcNAc transferase catalyzed O-GlcNAcylation is a reversible
PTM and several proteins are glycosylated including α-syn (see Fig. 2).
Calpain-mediated C-terminal truncation is reported to yield aggrega-
tion-prone α-syn fragments which may promote seeded aggregation
[13]. The C-terminal domain of α-syn is reported to stabilize the dis-
ordered conformation of the α-syn monomer via interactions with the
N-terminal or NAC domains [14]. Calpain activity is reported to cor-
relate with disease progression in PD mouse models [15,16]. Phos-
phorylation is suggested to play an important role in the regulation of
α-syn aggregation, Lewy body formation and neuronal degeneration.
Glycosylation of hydroxyl-amino acids with N-acetylglucosamine (O-
GlcNAc) works competitively against phosphorylation, and it is
speculated that this may constitute a protective mechanism against PD
pathogenesis [17]. O-GlcNAc functionalization, at various native
modification sites along the NAC domain of α-syn, reportedly alters the
peptides self-assembling propensity and toxicity in a site-specific
A truncated analogue of the NAC domain of α-syn has been reported
previously to accelerate wild-type (wt) α-syn aggregation in a con-
centration-dependent manner upon co-incubation in vitro [20]. When
modified with O-GlcNAc at a site analogous to Thr72, the truncated
NAC segment (α-syn68-77, Ac-68GAVVT(O-GlcNAc)GVTAV77-NH2) did
not increase, nor accelerate wt α-syn aggregation upon co-incubation. It
was concluded that the glycopeptide was not participating in ag-
gregation under the assay conditions. Glycoside addition can influence
the capacity of a peptide to establish binding interactions with known
substrates; it also affects its hydrophilic profile, steric bulk, and its re-
sistance to metabolic processing [21].
Following from this, we sought to investigate whether PD-relevant
NAC segments, known to bind α-syn selectively, would, when deco-
rated with the disruptive O-GlcNAc unit, act as inhibitors of the pa-
thogenic associative events that occur during the α-syn aggregation
2

P. Ryan, et al.
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reaction. As the effects of O-GlcNAcylation are site-specific [18], we
were interested in developing a panel of small glycopeptides resulting
from shifting the glycoside to sites presumed to be less mechanistically
engaged in the recognition interaction, i.e. the N-terminal, to couple the
disruptive nature of the O-GlcNAc residue with the associative nature of
the sequence. The peptides examined here were C-terminally amidated
and N-terminally acetylated, consistent with previous reports [20].
Here we describe efforts to construct and evaluate 10-/12-mer glyco-
peptides, derived from the NAC segment of α-syn, to probe their effects
on wt α-syn aggregation. This study was conducted with a view to
deciphering the role of O-GlcNAc on aggregation inhibition or me-
chanisms underlying the aggregation event.
disposable polypropylene syringes (Torviq) equipped with Teflon sin-
ters.
Loading of Fmoc-amino acids onto Rink Amide (RAM) resin: Rink
Amide (RAM) resin (0.450 mmol/g loading, 467 mg, 0.210 mmol) was
swollen in dry DMF (5 mL) for 5 min at room temperature before being
treated with 10 vol% piperidine/DMF (5 mL) solution and shaken for
5 min at room temperature. The procedure was repeated with a fresh
portion, after which the resin was washed with DMF (5 × 4 mL), DCM
(5 × 4 mL) and then DMF (5 × 4 mL). After, a solution of the Fmoc-
protected amino acid (0.84 mmol, 4 equiv.) PyBOP (0.84 mmol, 4
equiv.) and N-methylmorpholine (1.68 mmol, 8 equiv.) in DMF (3 mL)
was mixed with the resin and shaken for 1 h. After, the resin was wa-
shed with DMF (5 × 4 mL), DCM (5 × 4 mL) and then DMF (5 × 4 mL).
The desired peptides were then assembled following iterative Fmoc-
SPPS procedures. To determine resin loading, a solution of piperidine in
DMF (4 mL, 10% v/v) was added to the resin before being shaken for
3 min. The drained Fmoc deprotection solution was retained in a 10 mL
volumetric flask and the resin washed with fresh piperidine in DMF
(10% v/v) such that the total volume did not exceed 10 mL. The effi-
ciency of the initial loading was quantitatively determined by mea-
surement of the dibenzofulvene-piperidine adduct using Varian Cary
4000- UV–Vis spectrophotometer (λ = 301 nm). Amino acid loading
onto the resin was most often quantitative. The resin was subsequently
washed with DMF (10 × 5 mL), DCM (10 × 5 mL), and DMF
(10 × 5 mL).
2. Materials and methods
2.1. General
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded using a Bruker Avance DPX 400 spectrometer at a frequency of
400.2 MHz. Carbon nuclear magnetic resonance (13C NMR) spectra
were recorded on a Bruker Avance DPX 400 spectrometer at a fre-
quency of 100 MHz. The spectra are reported as parts per million (ppm)
downfield shift using the solvent peak as internal reference. The data
are reported as chemical shift (δ), multiplicity, relative integral, cou-
pling constant (JHz) and assignment where possible. Low resolution
mass spectra were recorded on a Finnigan LCQ Deca ion trap spectro-
meter (ESI). High resolution mass spectra were recorded on a Bruker
7 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
(FTICR).
Fmoc deprotection: Pre-loaded resin was treated with 10 vol% pi-
peridine/DMF (5 mL) solution and shaken for 5 min at room tempera-
ture before being filtered and treated again with a fresh 10 vol% pi-
peridine/DMF solution. The efficiency of the initial loading was
quantitatively determined by measurement of the piperidine-fulvene
adduct using UV–Vis spectrophotometry (λ = 301 nm). The resin was
subsequently washed with DMF (5 × 4 mL), DCM (5 × 4 mL) and DMF
(5 × 4 mL).
HPLC: Analytical reverse-phase HPLC was performed on a system
consisting of a Shimadzu BM‐20A Prominence communications bus
control module, two Shimadzu LC‐20 AD UFLC liquid chromatograph
pumps fitted with a solvent mixer, a Shimadzu DGU‐20A3 Prominence
degasser, a Shimadzu SIL‐20A HT UFLC Prominence chilled auto-
sampler module, a Shimadzu CTO‐20 AC Prominence column oven, a
Shimadzu SPD‐M20A Prominence Diode array detector module with an
Alliance series column heater at 30 °C and 2996 photodiode array de-
tector. A Waters Sunfire 5 μm, 2.1 × 150 mm column was used at a flow
rate of 1 mL∙min−1 using a mobile phase of 0.1% TFA in water (Solvent
A) and 0.1% TFA in acetonitrile (Solvent B) and a linear gradient of
either 0–100 %B or 0–50 %B over 10 min. The results were analysed
using LabSolutions software. Preparative reverse-phase HPLC was per-
formed using a the same Shimadzu system only coupled with a
Shimadzu SPD-20AC dual wavelength detector operating at 214 and
254 nm and a Gilson FC 204 fraction collector calibrated to collect only
peaks with intensities exceeding 100 mAU (λ = 214 nm) which were
sub-fractionated into 0.2 min interval fractions. A Waters Sunfire 5 μm,
19 × 150 mm column was used at a flow rate of 7 mL min−1 using a
mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA acet-
onitrile (Solvent B) using a linear gradient of 0–100% B over 60 min
unless mentioned otherwise. HPLC-ESI/MS (LCMS) was performed on
an Agilent 1290 HPLC (with PDA) coupled in series to an Agilent 6530
Q-TOF operating in positive mode using Agilent Jet Stream ESI ion
Glycosylated and unglycosylated amino acid coupling: Amino acids
were coupled using a mixture of appropriate protected amino acid
(0.84 mmol,
4 equiv.), PyBOP (0.84 mmol, 4 equiv.) and NMM
(1.68 mmol, 8 equiv.) in DMF (3 mL) which was added to the resin and
shaken. After 1 h the resin was washed with DMF (5 × 4 mL), DCM
(5 × 4 mL) and then DMF (5 × 4 mL). Glycopeptides were synthesised
at a 0.07 mmol scale, incorporation of the glycosylated amino acid
building block 16 was conducted using a mixture of Fmoc-Ser(O-
GlcNAc)-OH (0.091 mmol, 1.3 equiv), HATU (0.112 mmol, 1.6 equiv.),
HOAt (0.112 , 1.6 equiv.), and NMM (0.224, 3.2 equiv.), in DMF (4 mL)
which was added to the resin and shaken. After 16 h the resin was
washed with DMF (5 × 4 mL), DCM (5 × 4 mL) and DMF (5 × 4 mL).
Capping: Following each coupling, unreacted sequences were
capped by treating the resin with 10 vol% acetic anhydride/pyridine
(5 mL) solution before being shaken for 5 min at room temperature,
filtered and subsequently washed with DMF (5 × 4 mL), DCM
(5 × 4 mL) and DMF (5 × 4 mL).
Resin cleavage and ether precipitation: The resin was washed
thoroughly with DCM (10 × 4 mL) and subsequently treated with a
solution of TFA/TIS/H2O (90:5:5 v/v/v, 4 mL) and was shaken for 1 h
at room temperature. The resin was filtered and washed with DCM
(2 × 2 mL) before the resultant filtrate was evaporated to dryness. Cold
diethyl ether (2 mL) was added to the precipitate, suspended, trans-
ferred to a 2 mL Eppendorf tube, and subsequently centrifuged at
1400 rpm for 1 min. The supernatant was decanted and discarded and
the precipitate was dried under high-vacuum.
source. Separations were achieved using
a Waters Sunfire 5 μm,
2.1 × 150 mm column and a flow rate of 1 mL∙min−1. A mobile phase
of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in
acetonitrile (Solvent B) using a linear gradient of 0–100% or 0–50% B
over 8–10 min was used. Commercial materials were used as received
unless otherwise noted. Amino acids, coupling reagents and resins were
obtained from Mimotopes. Dichloromethane and methanol were dis-
tilled from calcium hydride. DMF was obtained as peptide synthesis
grade from Auspep or Labscan.
Glycopeptide deacetylation: Dry precipitate was suspended in dry
methanol (1 mL) and mixed at room temperature before treatment with
sodium methoxide in methanol (0.5 M, 50 μL, pH = 10). The solution
was allowed to stir for 2 h before being neutralised with glacial acetic
acid and evaporated to dryness. A solution of H2O/ACN (1:1 v/v, 5 mL)
was added to the precipitate, suspended, and centrifuged at 1400 rpm
for 1 min. The supernatant was decanted and lyophilised to afford
2.2. Solid phase synthesis
All solid-phase peptide syntheses were carried out manually in
3

P. Ryan, et al.
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precipitate ready for RP-HPLC purification. Details on the purity of the
(glyco)peptides are presented in Table S1 and Figs. S1–S22 (analytical
HPLC trace and HRMS data) of the supplementary material.
2.2.8. Synthesis of Ac-Ser(O-GlcNAc)-68Gly-D-Ala-D-Val-D-Val-D-Thr-Gly-
D-Val-D-Thr-D-Ala-D-Val77-NH2 (8)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
19.4 min). The title compound (2.6 mg, 3%) was a white lyophilisate.
Analytical HPLC: Rt 4.1 min (0–100 %B over 10 min, λ = 214 nm);
2.2.1. Synthesis of Ac-71Val-Thr-Gly-Val-Thr-Ala-Val-Ala-Gln-Lys-Thr-
Ala82-NH2 (1)
+
HRMS (ESI) 1226.6243 ([M+Na]+), calcd. for C51H89N13NaO20
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
19.7 min). The title compound (22.4 mg, 41%) was a white lyophilisate.
Analytical HPLC: Rt 5.9 min (0–100 %B over 10 min, λ = 214 nm);
1226.6239.
2.2.9. Synthesis of Ac-77Val-Ala-Thr-Val-Gly-Thr-Val-Val-Ala-Gly68-Ser
(O-GlcNAc)-NH2 (9)
+
HRMS (ESI) 1186.6817 ([M+H]+), calcd. for C51H92N15O17
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
20.1 min). The title compound (26.1 mg, 30%) was a white lyophilisate.
Analytical HPLC: Rt 7.1 min (0–100 %B over 10 min, λ = 214 nm);
1186.6790.
2.2.2. Synthesis of Ac-Ser(O-GlcNAc)-71Val-Thr-Gly-Val-Thr-Ala-Val-Ala-
Gln-Lys-Thr-Ala82-NH2 (2)
+
HRMS (ESI) 1226.6244 ([M+Na]+), calcd. for C51H89N13NaO20
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
18.2 min). The title compound (15.4 mg, 15%) was a white lyophilisate.
Analytical HPLC: Rt 5.5 min (0–100 %B over 10 min, λ = 214 nm);
HRMS (ESI) 1476.7922 ([M+H]+), calcd. for 1476.7904+ 1476.7904.
1226.6239.
2.2.10. Synthesis of Ac-68Gly-Ala-Val-Val-Thr-Gly-Val-Thr-Ala-Val77-Ser
(O-GlcNAc)-NH2 (10)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
19.2 min). The title compound (23.5 mg, 27%) was a white lyophilisate.
Analytical HPLC: Rt 6.8 min (0–100 %B over 10 min, λ = 214 nm);
2.2.3. Synthesis of Ac-68Gly-Ala-Val-Val-Thr-Gly-Val-Thr-Ala-Val77-NH2
(3)
HRMS (ESI) 1226.6243 ([M+Na]+), calcd. for C51H89N13NaO20
+
The peptide was prepared following Fmoc-SPPS procedures and
purified by preparative RP-HPLC (5–95 %B over 60 min; Rt 21.5 min).
The title compound (26.2 mg, 40%) was a white lyophilisate. Analytical
HPLC: Rt 7.4 min (0–100 %B over 10 min, λ = 214 nm); HRMS (ESI)
1226.6239.
2.2.11. Synthesis of Ac-D-77Val-D-Ala-D-Thr-D-Val-Gly-D-Thr-D-Val-D-Val-
936.5119 ([M+Na]+), calcd. for C40H71N11NaO13 936.5125.
+
D-Ala-Gly68-Ser(O-GlcNAc)-NH2 (11)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
20.3 min). The title compound (15.3 mg, 18%) was a white lyophilisate.
Analytical HPLC: Rt 7.1 min (0–100 %B over 10 min, λ = 214 nm);
2.2.4. Synthesis of Ac-Ser-68Gly-Ala-Val-Val-Thr-Gly-Val-Thr-Ala-Val77
-
NH2 (4)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5–95 %B over 60 min; Rt
20.6 min). The title compound (16.6 mg, 23%) was a white lyophilisate.
Analytical HPLC: Rt 7.1 min (0–100 %B over 10 min, λ = 214 nm);
HRMS (ESI) 1226.6251 ([M+Na]+), calcd. for C51H89N13NaO20
+
1226.6239.
+
HRMS (ESI) 1023.5452 ([M+Na]+), calcd. for C43H76N12NaO15
2.3. Expression and purification of α-synuclein
1023.5445.
Escherichia coli BL21 (DE3) cells containing the human α-syn gene
inserted into a pET-22b (+) vector (Novagen, Merck, MA, USA) were
induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. α-syn was
first purified following a non-chromatographic protocol reported by
Volles and Lansbury [22]. Further purification using anion exchange
followed protocols reported by Ventura and co-workers [23].
2.2.5. Synthesis of Ac-Ser(O-GlcNAc)-68Gly-Ala-Val-Val-Thr-Gly-Val-Thr-
Ala-Val77-NH2 (5)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5 to 95 %B over 60 min; Rt
19.2 min). The title compound (5.3 mg, 6%) was a white lyophilisate.
Analytical HPLC: Rt 9.7 min (0–100 %B over 12 min, λ = 214 nm);
2.4. In vitro inhibition of α-synuclein aggregation
+
HRMS (ESI) 1226.6240 ([M+Na]+), calcd. for C51H89N13NaO20
1226.6239.
A stock solution of ThT (5 mM) in glycine-NaOH buffer (pH 8.0) was
prepared and used with homogenous monomeric α-syn, made using
commonly employed protocols described by Rahimi et al. [24]. Inhibi-
tion of α-syn aggregation was performed by incubation of a mixture of α-
syn monomers (80 μM) suspended in aggregation buffer (20 mM Tris-
HCl, pH 7.4, 200 mM MgCl2 and 0.05% NaN3) with or without candidate
inhibitor compounds (80 μM) at 37 °C with constant shaking (1000 rpm
in a thermomixer) for 48 h. For each treatment group, pre-incubated α-
syn samples (60 μL) were mixed with a ThT solution (240 μL, 50 μM), the
solution was dispensed in triplicate (n = 3) into 96-well plates which
were analysed using a fluorimeter (ex. 440 nm/em. 500 nm). The fluor-
escence intensity of samples was compared to an untreated negative
control and a positive control treated with epigallocatechin gallate
(EGCG, 400 μM) to obtain percentage inhibition values.
2.2.6. Synthesis of Ac-Ser(O-GlcNAc)-Gly-68Gly-Ala-Val-Val-Thr-Gly-Val-
Thr-Ala-Val77-NH2 (6)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5 to 95 %B over 60 min; Rt
19.3 min). The title compound (2.6 mg, 3%) was a white lyophilisate.
Analytical HPLC: Rt 9.7 min (0–100 %B over 12 min, λ = 214 nm);
HRMS (ESI) 1283.6461 ([M+Na]+), calcd. for C53H92N14NaO21
+
1283.6454.
2.2.7. Synthesis of Ac-68Gly-D-Ala-D-Val-D-Val-D-Thr-Gly-D-Val-D-Thr-D-
Ala-D-Val77-NH2 (7)
The glycopeptide was prepared following Fmoc-SPPS procedures
and purified by preparative RP-HPLC (5 to 95 %B over 60 min; Rt
21.4 min). The title compound (5.1 mg, 8%) was a white lyophilisate.
Analytical HPLC: Rt 7.4 min (0–100 %B over 10 min, λ = 214 nm);
2.5. Dot blot assay
HRMS (ESI) 936.5144 ([M+Na]+), calcd. for C40H71N11NaO13
Briefly, 2 μL samples were spotted on nitrocellulose membrane
(Amersham Protran, GE Healthcare) and dried at room temperature.
+
936.5125.
4

P. Ryan, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 3. Purified α-syn (80 μM) was incubated at 37 °C alone or with compounds
(80 μM) before analysis by ThT fluorescence (λex = 440 nm, λem = 500 nm) at
48 h. Representative results from 3 independent experiments are presented,
error bars represent standard deviation analysed using one-way ANOVA with
Dunnett’s post hoc comparison (*P < 0.05 vs control; **p < 0.01 vs control;
***p < 0.001 vs control).
of N-terminal O-GlcNAcylation of the control α-syn71-82 peptide. We
then selected a panel of glycopeptides modified at various positions
along the S68-V77 stretch (3–11, Scheme 1). To generate the glyco-
peptides, we incorporated a selectively-protected glycosylated amino
acid building block, Fmoc-L-Ser((Ac)3-β-D-GlcNAc)-OH (16), into Fmoc-
SPPS (Scheme 2). Briefly, commercially available glucosamine hydro-
chloride salt (12) was selectively protected over two steps to form 13.
Removal of the anomeric acetyl group, followed by reaction with tri-
chloroacetonitrile under basic conditions, afforded the glycosyl tri-
chloroacetimidate 14. The glycosyl acceptor, Fmoc-Ser(OH)-OtBu,
synthesised in one step from commercially available Fmoc-Ser-OH, was
then glycosylated stereoselectively using 14 with TMSOTf as a pro-
moter to give 15. Selective deprotection of 15 afforded the glycosylated
amino acid 16, usable in SPPS. Construction of the peptide sequences
was undertaken using Fmoc-SPPS using Rink Amide (AM) resin to yield
the C-terminal carboxamide. Protected amino acids were coupled using
PyBOP/NMM, however coupling of 16 onto the N-terminally available
peptide sequences was accomplished following a specialised procedure,
optimised to minimise the D/L-epimerization of the glycosylated serine
[26]. Serine was found to have a higher racemization rate than most
other natural amino acids [27]. Specifically, a mixture of 1-hydroxy-7-
azabenzotriazole (HOAt), (1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), and
NMM in DMF undergoes a rapid pre-incubation period prior to treat-
ment of the peptides on solid-support. The reaction was accomplished
over 16 h of mixing before the resin was washed with DMF and DCM. A
molar ratio of 3.3:3.65:3.7:4:1 Fmoc-Ser(Ac3GlcNAcβ)-OH/HATU/
HOAt/NMM/peptide has been shown to afford a 96.8% yield with only
a minimal (3.4%) amount of D-enantiomer produced [28]. Liquid
Chromatography tandem Mass Spectrometry (LCMS) was employed to
grade reaction success and confirm the identity of the glycopeptide
products. Upon completion of the glycopeptides on-resin, they were
released under acidic conditions and dried before the O-acetyl groups
were removed using sodium methoxide in methanol, pH = 10. Each of
the (glyco)peptides were purified by RP-HPLC and characterised by
HRMS and analytical HPLC.
Scheme 1. Contracted structures of peptide and glycopeptide modulators of α-
syn aggregation. Compounds 1 and 2 are derived from the α-syn71-82 stretch,
the remaining compounds (3–11) are derived from the α-syn68-77 stretch.
Nitrocellulose membrane was blocked at room temperature for 1 h with
3% BSA and then washed with Tris-buffered saline (TBST, 0.05%
Tween 20, 50 mM Tris–HCl, 150 mM NaCl, pH 7.5). Each membrane
was then incubated at 4 °C overnight with anti-α-synuclein antibody
(1:2,000, BD Biosciences) and anti-α-synuclein filament antibody
(1:8000, Abcam). Membranes were further washed in TBST and then
incubated with secondary anti-mouse and anti-rabbit IgG (1:20,000,
Thermo Scientific) for 2 h at room temperature. The blots were further
incubated with ECL reagent (Millipore) for 5 min and developed.
3. Results and discussion
It has previously been shown that α-syn, when incubated in the
presence of the NAC-derived sequence corresponding to residues 71–82
of α-syn (1, Scheme 1), undergoes aggregation at an accelerated rate
[20]. We synthesised a peptide corresponding to this segment using
iterative Fmoc-based solid-phase peptide synthesis (SPPS) with PyBOP/
NMM. The ability of the peptide to promote aggregation was then
verified by incubating α-syn (80 μM), expressed by E. coli and purified
using anion exchange, with or without 1 (80 μM) under constant agi-
tation for 120 h before being analysed by thioflavin T (ThT) fluores-
cence. We were particularly interested in the effect of the peptide at the
end-point of the experiment rather than any effects on the rate of ag-
gregation as ThT has been found previously to alter aggregation ki-
netics [25]. A selection of different conditions was used to compare the
effect of different buffers on catalysing the reactions. Under all condi-
tions, pH was maintained at 7.4 and NaN3 was used as an antiseptic
agent. It was surmised that 1 worked effectively at increasing fibril
formation when using Tris(hydroxymethyl)aminomethane chloride
(Tris-Cl) as a buffer (pH = 7.4), and MgCl2 as an inducer of aggregation
(see Fig. 3).
Following construction of the (glyco)peptides, their effects on α-syn
aggregation was studied using ThT fluorescence assay. Mixtures of the
candidate peptides (80 μM) were co-incubated with α-syn (80 μM) at
37 °C in a 1:1 ratio for 48 h with constant shaking. The fluorescence of
the peptide analogues alone was also examined as some sequences were
speculated to promote fluorescence even in the absence of α-syn, po-
tentially skewing results. It was found that the fluorescence measured
Given that 1 was able to significantly promote α-syn aggregation,
we next set out to design and synthesise a selection of glycopeptides
derived from segments spanning the NAC domain of α-syn. We first
selected 2 (Ac-S(O-GlcNAc)VTGVTAVAQKTA-NH2) to probe the effects
5

P. Ryan, et al.
BioorganicChemistryxxx(xxxx)xxxx
Scheme 2. Reagents and conditions: (a) i: TrocCl, K2CO3, H2O, rt, 1 h; ii: Ac2O, pyridine, rt, 16 h, 61% over 2 steps; (b) i: ethylenediamine, AcOH, THF, rt, 15 h, 79%;
ii: CCl3CN, DBU, DCM, rt, 20 min, 60%; (c) Fmoc-Ser-OtBu, TMSOTf, DCM, −78 °C, 1 h, 97%; (d) i: Zn, Ac2O, rt, 2 h, 78%; ii: TFA, DCM (1:1), rt, 2 h, quant.
in the case of co-incubation with 4. When functionalised with Ser(O-
GlcNAc), the effects of the α-syn68-77 sequence on ThT fluorescence
were significantly altered. N-terminally O-GlcNAcylated 5 and its ana-
logue 6, bearing a glycine spacer residue, reduced α-syn-mediated ThT
fluorescence by 52% and 33% respectively. Though less effective, the
retro-inverso analogue 10 was able to significantly decrease ThT fluor-
escence also. Finally, compounds 7, 8, 9 and 11 did not reduce ThT
fluorescence under these conditions.
The effects displayed by 1 and 2 here are in agreement with ob-
servations made previously for a centrally O-GlcNAcylated peptide
[20]. Findings suggest that, when O-GlcNAcylated, the α-syn protein is
incorporated into the aggregation reaction with a reduced efficiency
[17]. The results here suggest that O-GlcNAcylated 2 may be excluded
from the aggregation reaction in a similar manner. It is possible
therefore, that the decrease in fluorescence observed in samples con-
taining either 5 or 6 results from their incorporation into the ag-
gregation reaction. The decrease in fluorescence may result from the
formation of heterogenous α-syn assemblies with decreased propensity
to aggregate. We expect that the peptides may be arresting aggregation
via interaction with the precursors to aggregation, species such as the
oligomers. Mass spectra of the α-syn samples with and without com-
pound were recorded and compared to determine whether the peptides
were establishing strong binding interactions with the monomeric
peptide, however the findings suggested that none of these peptides
formed complexes with the monomer in any detectable manner (for
spectra and methods, see SI).
The ability of 5 to inhibit α -syn fibril formation was validated using
dot blot analysis, a non-denaturing method for examining protein epi-
topes. Dot blot analysis has been employed previously to study α-syn
fibril formation and inhibitor-bound α-syn complexes structural con-
formations [29,30]. Equal volumes of each sample (2 μL) were spotted
on nitrocellulose membrane before being treated with one of either the
non-conformation dependent anti-α-syn antibody or the conformation
dependent anti-α-syn filament antibody prior to further treatment with
a detection antibody. Development of the blots confirmed that all
samples contained equivalent concentrations of α-syn protein (see
Fig. 4). Compound 5 was found to decrease the concentration of fi-
brillar α-syn by 50% relative to the untreated control.
Fig. 4. Purified α-syn (80 μM) was incubated at 37 °C alone or with compounds
(80 μM) before analysis by dot blot using a fibril-specific antibody at 48 h. Data
were presented in mean
control, n = 3.
standard error of the mean (SEM), ***p < 0.001 vs
following incubation of the (glyco)peptide compounds alone was neg-
ligible compared to the fluorescence of the sample containing α-syn
alone (See SI). Following incubation, each sample was mixed with ThT
(50 μM final concentration), aliquoted into three wells each of a 96-well
plate and read using a microplate reader.
Compounds 5 and 6 are the only glycopeptides identified to de-
crease α-syn-mediated ThT fluorescence to date. Compound 6 was no-
tably less effective at reducing fluorescence than 5. The structure of 6
differs from 5 by a single glycine residue, used as a conformationally
unrestrained spacer residue. The glycine residue also switches the face
of the peptide upon which the sugar is presented. In the structure of 6,
the glycoside is shifted further from the central binding residues,
hampering its participation in the interactions between the peptide and
As expected, 1 was found to significantly increase ThT fluorescence
while its N-terminally O-GlcNAcylated analogue, 2 did not. Compound
2 was not able to significantly decrease ThT fluorescence relative to the
untreated control either however. Indeed, no significant effect on ThT
fluorescence was observed upon co-incubation of α-syn with 2. Among
the compounds derived from α-syn68-77, unmodified 3 and serine-
modified 4 both increased ThT fluorescence by 12% and 19% respec-
tively relative to the control. This effect was only statistically significant
6

P. Ryan, et al.
BioorganicChemistryxxx(xxxx)xxxx
the native α-syn sequence. Similarly, shifting O-GlcNAc to the C-ter-
minus, as was achieved in 11, completely abolished inhibitory activity.
These observations suggest that the effects of O-GlcNAcylation are site-
dependent.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103389.
It is also interesting to note that 8 did not display similar inhibitory
effects as its structure resembles the D-isomer of α-syn68-77. Compound
8 was envisioned to effect α-syn aggregation in a similar manner to 5. A
number of D-isomeric Aβ-derived peptide sequences are reported to
inhibit wt Aβ fibrillogenesis more potently than their L-isomer analo-
gues [31]. Compound 9 was also unable to decrease ThT fluorescence,
though its proximity pattern of side chains remains the same as 5. Si-
milarly, 10 bearing a retro-inverso sequence was designed to assume a
side chain topology similar to that of the parent sequence yet it also
failed to decrease fluorescence as effectively as 5 or 6. Previous reports
have found that retro-inverting Aβ-binding sequences can massively
increase their association rate and binding affinity to Aβ while de-
creasing their dissociation rate [32]. Retro-inverso analogues are also
known sometimes to fail at adopting structures that mimic the functions
of the natural peptide, however. The lack of activity displayed by 9 and
10 could be due to a number of reasons. One explanation is that the
inverted hydrogen bonding patterns may yield compensatory adjust-
ments in the peptides local conformation which may render the se-
quence incompetent in participating in the aggregation reaction. The
fact that peptides 8, 9 or 10 were not effective at decreasing ThT
fluorescence suggests that the effects of O-GlcNAcylation are highly
dependent on the structural properties of the recognition sequence.
Similar studies have been conducted using O-glycosylated tau
fragments, explored for their capacities to inhibit truncated tau PHF6
aggregation. It was found that inhibition displayed by the glycopeptides
was dependent upon the nature of the glycoside unit [33]. Further
studies found that the extent to which glycosides on glycopeptides af-
fect the aggregation of the native counterparts also largely depends on
the scaffold to which they are linked [34]. Due to the ability of sugars to
hydrogen bond with hydrophilic amino acid side chains, they may
prove useful as agents for disrupting the self-interactions crucial for
amyloid formation.
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