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Petersson and R. H. Mach, Chem. Commun., 2020, DOI: 10.1039/C9CC09849F.
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Synthesis and Characterization of High Affinity Fluorogenic -

Synuclein Probes
Zsofia Lengyel-Zhanda†, John J. Ferrieb†, Bieneke Janssena, Chia-Ju Hsieha, Thomas Grahama, Kui-
Published on 22 February 2020. Downloaded by LA TROBE UNIVERSITY on 2/22/2020 2:59:01 PM.

Received 00th January 20xx,

Accepted 00th January 20xx ying Xua, Conor M. Haneyb‡, Virginia M.-Y. Leec, John Q. Trojanowskic, E. James Peterssonb* and
Robert H. Macha*

ChemComm Accepted Manuscript

DOI: 10.1039/x0xx00000x

Fluorescent small molecules are powerful tools for imaging - fibrils.5 Although antibodies are the currently favored choice for
synuclein pathology in vitro and in vivo. In this work, we explore detection of various amyloids,6 they are not suitable for
benzofuranone as a potential scaffold for the design of fluorescent studying the location and availability of binding sites in -Syn
-synuclein probes. These compounds have high affinity for fibrils, information that is crucial for the development of
synuclein, show fluorescent turn-on upon binding to fibrils, and imaging probes and therapeutic agents targeting -Syn
display different binding to Lewy bodies, Lewy neurites and glial inclusions. Besides antibodies, fluorescent small molecules are
cytoplasmic inclusion pathologies in post-mortem brain tissue. often used in imaging due to their favorable properties: small
These studies not only reveal the potential of benzofuranone size, high sensitivity and physicochemical properties that can be
compounds as -synuclein specific fluorescent probes, but also readily fine-tuned.7 For the detection of amyloid fibrils, the
have implications for the ways in which synucleinopathies are most widely utilized fluorescent probes are Thioflavin S, the
conformationally different and display distinct small molecule benzothiazole dye Thioflavin T (ThT) and the sulfonated azo dye
binding sites. Congo Red.8 Despite their favorable absorbance and fluorescent
properties, these probes have significant drawbacks. They have
Insoluble protein aggregates are pathological hallmarks of low affinity for alpha-synuclein fibrils, often give false positive
many neurodegenerative disorders and have been used for results and they cannot distinguish between -Syn inclusions.9
identification at time of autopsy.1 Parkinson’s disease (PD) and These deficiencies have led to increased efforts to develop
multiple system atrophy (MSA) are characterized by the better probes for imaging -Syn fibrils, since a small molecule
abnormal deposition of -synuclein (-Syn) in multiple brain that labels LB pathology with high affinity and does not label
regions. In PD patients, -Syn forms amyloid fibrils and GCIs would greatly advance research for PD.10 As of today, a
accumulates in spherical shaped cytoplasmic inclusions, called number of fluorescent molecules have been reported for
Lewy bodies (LBs), and rod-shaped neuritic inclusions, called binding to -Syn fibrils.11 Despite the progress that has been
Lewy neurites (LNs).2 By contrast, -Syn is found within glial achieved in recent years, many of the reported fluorescent
cytoplasmic inclusions (GCIs) in multiple system atrophy probes fail to distinguish between different forms of -Syn (LB
(MSA)3, and it has been reported that MSA patients possess vs GCIs) and have low affinity.5b
pathological -Syn with biochemical and biological properties Recently, we reported a series of (benzylidene)indolin-2-
distinct from those found in Lewy bodies and neurites.4 one derivatives that could serve as lead compounds for PET
Currently, the detection of -Syn and other amyloid tracer development.12 Most of the described compounds
deposits in vitro is commonly based on measuring enhanced showed modest affinity for -Syn fibrils and only a few
fluorescence emission of probes upon binding to amyloid displayed dissociation constants lower than 20 nM. Encouraged
by the results, we set out to explore binding and fluorescent
a. Department
properties of the benzofuranone analogues of these
of Radiology, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, United States compounds. Benzofuranone derivatives are promising scaffolds
b. Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania
for the design of fluorescent probes, due to their favorable
19104, United States
c. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, absorption and fluorescence properties.13 Herein we describe
University of Pennsylvania, Philadelphia, PA 19104, United States the synthesis and characterization of various benzofuranone
† These authors contributed equally. analogues and we demonstrate for the first time that a small
‡ current address: Department of Chemistry and Biochemistry; University of North

Carolina Wilmington; Wilmington, NC 28403 molecule can distinguish between LB, LN and GCI pathology in
Electronic Supplementary Information (ESI) available: [details of any supplementary human post-mortem brain tissue.
information available should be included here]. See DOI: 10.1039/x0xx00000x

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The synthesis of the target compounds began with the radioligands by increasing concentrations ofView competitor
Article Online
preparation of Tg-47 by reacting 5-hydroxybenzofuran-3-one compounds, using fixed concentrations of fibrils and
DOI: 10.1039/C9CC09849F
with 4-methoxycinnamaldehyde, which was then converted to radioligand. Tg-51-53 displayed remarkably high affinity for site
trifluoromethanesulfonate ester Tg-49 (Scheme 1). Next, we 2 of -Syn fibrils and a slightly lower affinity for site 9, with Tg-
incorporated various substituents on the aromatic ring to 52 being the most potent and having an inhibitory
determine if these groups would improve the affinity and concentration (IC50) of 1.18 nM for site 2 and an inhibitor
fluorescence properties of these compounds. A Suzuki cross constant (Ki) of 2.32 nM for site 9 (Table 1 and Figure S4b in ESI).
coupling between triflate Tg-49 and the appropriate The affinity of Tg-52 is several orders of magnitude higher than
(hetero)aryl boronic acid was performed to obtain the commonly used dye, thioflavin T (Table S2 in ESI). Replacing
benzofuranone analogs Tg-51-53. The methylcarbamate the pyridine ring with an N-methyl carbamate group (Tg-54)
analogue Tg-54 was synthesized by reacting Tg-47 with N- resulted in decreased affinity for site 2 and slightly lower affinity
methylcarbamoyl chloride. The final compounds were obtained to site 9 compared to Tg-51-52. This is likely due to the lack of
Published on 22 February 2020. Downloaded by LA TROBE UNIVERSITY on 2/22/2020 2:59:01 PM.

as yellow/orange solids with purity >95%.  stacking between the pyridine group and the tyrosine 39
residue of the fibrils, which could further stabilize the

ChemComm Accepted Manuscript

Scheme 1. Synthesis of benzofuranone analogues.
interaction. The results are consistent with the docking studies
and confirm that these benzofuranone derivatives are suitable
-Syn ligands with high affinity.
Table 1. Comparison of IC50 and Ki values for binding to site 2 or site 9 in Syn fibrils.

Site 2 Site 9
Compound IC50 (nM)* Ki (nM)*
Tg-51 25.8 (13.4-51.1) 18.3 (8.10-42.7)
Tg-52 1.18 (0.394-4.03) 2.32 (1.56-3.42)
Tg-53 38.6 (16.1-92.0) 33.8 (18.0-64.9)
Tg-54 222 (102-492) 35.1 (25.0-49.7)

*Values were determined by competition binding assay with [3H]-Tg-190b or [3H]-

BF2846. 95% confidence intervals for IC50 and Ki values are shown in parentheses
(n=3).

We studied the fluorescence properties of these

benzofuranone analogs to determine their efficacy as probes
for biological applications. All compounds showed an
absorption maximum in the visible range in 50:50 tris
*Reagents and conditions: (a) HCl, EtOH, 70 °C, 3 h (52%) (b) pyridine, buffer/DMSO, and the absorbance maxima were all within ± 2
Tf2O, CH2Cl2, rt, 30 min (94%); (c) XPhos Pd G2, K3PO4, dioxane/H2O, nm (Figure 1a and Figure S5 in ESI). Curiously, the absorbance
50 °C, 20 min (Tg-51 76%; Tg-52 80%; Tg-53 49%); (d) TEA, DMAP, N- maximum of Tg-51 was blue-shifted by ~70 nm for absorbances
methylcarbamoyl chloride, rt, 2 h (49%).
measured at higher concentrations. Overall, the various
Previously we identified putative binding sites, sites 2 and 9 substitutions on the aromatic rings did not show a marked
(Figure S1b in Electronic Supplementary Information), for small effect on the absorption maxima (Table S3 in ESI). The spectra
molecules on -Syn fibrils.14 In order to access the binding site of all compounds display a bathochromic shift after binding to
of the benzofuranone derivatives and determine their binding fibrils, which has been observed for other fibril binding
energy, we performed computational docking studies. The NMR compounds. For ThT, spectral shifts in the molecule’s
structure of -Syn fibrils (PDB: 2N0A)15 was imported from the absorbance profile associated with fibril binding have been
Protein Data Bank and molecular blind docking studies were attributed to differences in the local dielectric forces
conducted via the AutoDock PyMOL plugin.16 All four experienced by the molecule when free in solution versus
compounds favored both sites 2 and 9 with Tg-54 being the bound to the fibril surface.17 Alternatively, binding of Congo Red
least and Tg-52 being the most potent binder (Table S1 in to fibrils results in an extension of the -system, producing the
Electronic Supporting Information). The interactions of the observed red-shift.18 Since, the absorbance maxima of these
compounds with binding sites 2 and 9 are shown in Figures S1- compounds revealed similar, ~10 nm shifts, due to both fibril
S3 in the Electronic Supplementary Information (ESI). binding and variations in solvent polarity for unbound
Next, we set out to determine the affinity of Tg-51-54 for - compounds, the bathochromic shift upon fibril binding is likely
Syn fibrils. Our recent work highlights two compounds, [3H]-Tg- the result of changes in local polarity (Figure S6 in ESI).
190b and [3H]-BF2846 (Figure S4a in ESI) that selectively bind to Moreover, the more pronounced shift observed from Tg-51
site 2 and site 9, respectively, and can be utilized in radioligand binding is also likely attributable to changes in local polarity, as
competition binding assays to measure the affinity of -Syn all compounds display significant hypsochromic shifts in the
ligands. The binding assay measured the displacement of these presence of high polarity solvents (Figures S6-S8 in ESI).

2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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DOI: 10.1039/C9CC09849F

Figure 1. (a) Absorbance and emission spectra of Tg-52 free in solution and bound to fibrils. Absorbance spectra free in solution (blue solid line) and bound to α-Syn fibrils (light-blue
solid line) and fluorescence excitation spectrum (dark red dashed line) and emission spectrum (red dashed line) were acquired with 1 μM of Tg-52 and 98 μM α-Syn fibrils. (b)
Published on 22 February 2020. Downloaded by LA TROBE UNIVERSITY on 2/22/2020 2:59:01 PM.

Determination of fluorescence turn-on of Tg-52 upon fibril binding. Fluorescence emission spectra were acquired for 1 μM Tg-52 in the presence of serial dilutions of α-Syn fibrils.
(c) Determination of absorbance and fluorescence sensitivity to environmental polarity. Absorbance (solid lines) and fluorescence spectra (dashed lines) of 20 μM and 5 μM Tg-52,

ChemComm Accepted Manuscript

respectively, in various solvent conditions.

Next, we measured the molar extinction coefficients () of samples from anecdotal cases of PD, AD and MSA patients. We
the various analogs free in solution and bound to fibrils (Figure chose Tg-52 to test as a biological probe due to its high affinity
S7 in ESI). The values of  at each compounds’ absorption for -Syn fibrils. Sections from frontal cortex tissue of PD and
maximum are summarized in Table S3 in ESI. The extinction AD cases and cerebellum tissue of MSA cases were incubated
coefficients of Tg-51 and Tg-52 were significantly smaller than with 10 µM Tg-52 and imaged using a fluorescence microscope
those of Tg-53 and Tg-54, however we observed that the (Figure 2.). Immunostaining with Syn303, a pan--Syn antibody
absorbance of all four compounds increased upon binding to - known to detect a variety of types of aggregates6b, was
Syn. Moreover, the extinction coefficients were highly sensitive performed on the same section to determine the protein
to changes in polarity, where low polarity solvents afforded species labeled by the fluorescent probe.
increases in absorbance (Figure S6 in ESI). The fluorescence
emission spectra of the compounds showed a maximum
between 522 and 534 nm. All four ligands are practically non-
fluorescent in water. Upon mixing with -Syn fibrils, a
pronounced increase in fluorescence intensity was observed
(Figure 1b and Figure S8 in ESI). The high turn-on associated
with binding of these compounds enabled us to facilely measure
their relative binding affinities by fluorometric titration. The
emission of compounds was measured in the presence of
varying concentrations of -Syn fibrils and the total intensity
from either excitation or emission spectra was plotted as a
function of fibril concentration (Figure S8 in ESI), with Tg-52
displaying the highest relative affinity (Table S4 in ESI). To
further characterize the spectroscopic properties of the
benzofuranone analogs, fluorescence quantum yields were
determined via comparison to ThT (Figure S9 in ESI). Tg-51
showed the highest quantum yield when bound to fibrils,
followed by Tg-53 (Table S3 in ESI). Thus, binding in the less
polar fibril pocket will lead to an increase in brightness due to
changes in extinction coefficient and quantum yield (Figure 1c
and S6 in ESI), but this does not fully account for the increase in
fluorescence observed during fibril binding. Therefore, we Figure 2. Fluorescence microscopy studies of compound Tg-52 in post-mortem samples
suspect that restriction of the numerous degrees of rotational of PD and MSA brain. Images show LBs (a-c), LNs (d-f) and GCIs (g-i) immunostained with
Syn303 antibody and Tg-52. The fluorescent compound shows high labeling of LBs, weak
and vibrational freedom associated with fibril binding reduces
labeling of GCIs and does not label LNs. [Scale bars, 50 µm (c), 65 µm (f) and 100 µm (i)]
non-emissive relaxation from the excited state, resulting in the
noted fluorescence turn-on associated with binding. Lastly, we Tg-52 displays high labeling of LBs in PD brain sections and a
measured the fluorescence lifetime of the designed compounds plaques in AD sections (Figure 2a-c and Figures S11-12 in ESI),
when bound to -Syn fibrils. (Figure S10 in ESI) The measured however it did not label LNs and displayed weak labeling of GCIs
lifetimes ranged between 1.00 and 1.16 ns, with Tg-52 in MSA brain sections (Figure 2d-I and Figure S11 in ESI),
exhibiting the longest lifetime of 1.16 ns (Table S3 in ESI). suggesting that these -Syn inclusions have distinct binding
The favorable fluorescence properties of these compounds sites available for small molecules. Understanding the location
and their high affinity for -Syn fibrils encouraged us to of these binding sites is crucial in designing therapeutic agents
investigate in vitro fluorescent staining of post-mortem brain

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

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ChemComm Accepted Manuscript

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This research was supported by the National Institutes of Shakerdge, S. S. Hou, W. E. Klunk, C. A. Mathis, E. E.
Health (NIH NS103873 to EJP and AG10124 and AG062418 to Nesterov, T. M. Swager, P. J. McLean and B. J. Bacskai, Mol.
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Instruments supported by the NIH and National Science Losytskyy, O. Tolmachev, Y. L. Slominskii, G. M. Segers-
Foundation include NMR (NSF CHE-1827457) and mass Nolten, V. Subramaniam and S. Yarmoluk, J. Fluoresc., 2012,
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spectrometers (NIH RR-023444 and NSF MRI-0820996). JJF T. K. Kwok, N. L. C. Leung, S. Chen, N. N. Vaikath, O. M. El-
thanks the NSF (DGE-1321851) and the Parkinson's Disease Agnaf and Y. Tang, Chem. Commun., 2015, 51, 1866-1869; (e)
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