Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis

Article abstract of DOI:10.1016/j.bmc.2016.02.022

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.

Full text of DOI:10.1016/j.bmc.2016.02.022

Bioorganic & Medicinal Chemistry 24 (2016) 1520–1527
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficacy of peptide nucleic acid and selected conjugates against
specific cellular pathologies of amyotrophic lateral sclerosis
Elisse C. Browne a, Sonam Parakh b,d, Luke F. Duncan a, Steven J. Langford c, Julie D. Atkin b,d
,
Belinda M. Abbott a,
⇑
a Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia
b Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia
c School of Chemistry, Monash University, Clayton 3800, Australia
d Department of Biomedical Sciences, Faculty of Medicine and Health Science, Macquarie University, Sydney 2109, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to
mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic
lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both
mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key
cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers
is reported for the first time.
Received 14 December 2015
Revised 9 February 2016
Accepted 17 February 2016
Available online 18 February 2016
Keywords:
Crown Copyright Ó 2016 Published by Elsevier Ltd. All rights reserved.
Peptide nucleic acid
Amyotrophic lateral sclerosis
Vitamin conjugation
1. Introduction
misfolded proteins accumulate within the ER. This triggers the
unfolded protein response (UPR), a signaling pathway that aims
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative
disorder which affects the upper and lower motor neurons of the
brain, brain stem and spinal cord.1 It causes progressive muscle
weakness, paralysis and death within 3–5 years of diagnosis and
there is currently no effective treatment. Around 10% of ALS cases
are familial (fALS), caused by genetic mutations. However, the
majority of ALS (90%) is sporadic, with no previous family history.2
Mutations in the superoxide dismutase 1 (SOD1) gene account for
approximately 20% of familial ALS cases and the most common
mutation in North America is A4V (alanine to valine).3,4 SOD1 is
a major cytosolic protein which catalyzes the reduction of harmful,
free superoxide radicals into molecular oxygen and hydrogen
peroxide.
The etiology of ALS remains unclear but the formation of intra-
cellular ubiquitin-positive inclusions containing misfolded pro-
teins is a characteristic pathological hallmark.5 Misfolded SOD1
inclusions are present in both sporadic human6 and familial ALS
patients, as well as transgenic SOD1G93A mice,7 the most widely
used animal disease model in preclinical studies. Stress in the
endoplasmic reticulum (ER) is also recognized to be an important
pathway to motor neuron death in ALS. ER stress is triggered when
to relieve the stress and thus restore homeostasis. However if ER
stress is prolonged, apoptosis is triggered. The transition of UPR
from cell survival to cell death is mediated by CCAAT-enhancer
binding protein homologous protein (CHOP),8 a transcription factor
which translocates to the nucleus when activated. We and others
previously demonstrated that ER stress is present in sporadic ALS
patient tissues9 as well as in cells expressing mutant SOD1 and
transgenic SOD1G93A mice.10–14 Novel pharmacological agents that
prevent the formation of misfolded mutant SOD1 inclusions and
inhibit the activation of CHOP in cells expressing mutant SOD1,
and hence the pro-apoptotic phase of UPR, would therefore have
therapeutic application in ALS.
Antisense agents such as peptide nucleic acids (PNA) are short
single stranded nucleic acid analogs designed to specifically bind
to complementary messenger ribonucleic acid (mRNA) targets
through Watson and Crick hydrogen bonding. As a result, these
antisense agents can silence a particular gene of interest. PNA is
a third generation oligonucleotide, which replaces the traditional
phosphate backbone of RNA/DNA with a peptide backbone made
up of repeating N-(2-aminoethyl)glycine units and the sugar moi-
ety replaced with a methylene carbonyl linker where the nucleic
bases are attached (Fig. 1).
The modified backbone of PNA gives rise to resistance to enzy-
matic degradation and leads to higher affinity binding, rates of
⇑
Corresponding author.
E-mail address: b.abbott@latrobe.edu.au (B.M. Abbott).
http://dx.doi.org/10.1016/j.bmc.2016.02.022
0968-0896/Crown Copyright Ó 2016 Published by Elsevier Ltd. All rights reserved.

E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
1521
Automated PNA synthesis was performed on PAL resin using
standard protocols24 and two PNA sequences were prepared as
previously described: H-GCACGACTT-NH2 (PNA1) and the comple-
mentary sequence H-AAGTCGTGC-NH2 (PNA2).21 Coupling of the
menadione analog 6 to the growing oligomer of PNA1 was then
undertaken using the same automated synthetic protocols. Purifi-
cation by reverse-phase high performance liquid chromatography
(RP-HPLC) gave the desired menadione-PNA conjugated oligomer
of 6-PNA1 (Fig. 2). Characterization was undertaken using
MALDI-TOF mass spectrometry where m/z calculated for
Figure 1. General structure of peptide nucleic acid (PNA) containing the four
nucleobases of adenine (A), thymine (T), guanine (G) and cytosine (C).
association and subsequently an increase in duplex stability as
there are no repulsive electrostatic interactions.15 Additionally,
the neutrality of the backbone significantly reduces the chance of
undesired nonspecific interactions such as binding to cellular pro-
teins.16 PNA exhibits low toxicity in cells and is stable of a wide pH
range, particularly toward the acidic end of the scale where DNA
can be denatured.
The major limitation of PNA as an efficient antisense drug is its
low phospholipid solubility due to the hydrophilic nature of the
molecule, PNA does not readily cross cell membranes. Hydrophobic
vitamins such as vitamin K and vitamin E are hypothesized as good
candidates for conjugation to PNA. The main function of vitamin K
is post-translational modification during the biosynthesis of vita-
min K dependent proteins.17 The synthetic form of this vitamin,
vitamin K3 (menadione) has been found to readily cross the blood
brain barrier as it is a small lipophilic molecule.18 Despite its larger
size, highly lipophilic vitamin E (tocopherol) has also been found to
cross the blood brain barrier.19 While the main task of this vitamin
is as an antioxidant, vitamin E has also been shown to alleviate
oxidative stress by promoting normal cell function.20
We have previously described the synthesis and conjugation of
tocopherol (vitamin E) analogs to PNA for the investigation of
effects on hybridization.21 In this further study, we present the
synthesis of a menadione (vitamin K) analog, its conjugation to
the same nine nucleobase PNA oligomer and the subsequent
hybridization results. In addition, this novel vitamin K conjugated
oligomer, along with the previously synthesized three vitamin E
derived conjugated PNA oligomers and the unconjugated PNA oli-
gomer, were studied using cellular assays to determine their
effects on the formation of mutant SOD1 inclusions and induction
of on ER stress.
C
119H150N55O33 requires [M+H]+ 2734.069 and m/z of [M+H]+
2734.375 was found.
In order to determine the suitability of the conjugated mena-
dione-PNA oligomer 6-PNA1, the thermodynamics of hybridization
to both the complementary PNA (PNA2) and DNA oligomers was
undertaken to ensure conjugation did not impair hybridization
(Tables 1 and 2). Two complementary methods were chosen for
thermodynamic analysis of the duplexes, UV monitored melting
curves (UVM)25,26 and isothermal titration calorimetry (ITC).27
Data for the control duplexes of PNA1/PNA2 and PNA1/DNA was
previously recorded21 and is provided here to facilitate the com-
parison of the data obtained with the conjugated duplexes. The
change in the thermodynamic differences between the two meth-
ods agree well with each other.
In the case for both conjugated duplexes, conjugation of the
menadione analog does not appear to significantly affect the stabil-
ity of the duplexes as reflected in the recorded thermal melting
temperature (Tm). As expected, lower thermal stability and thus
affinity was observed for the mixed duplexes of PNA and DNA
oligomers.28,29 However, the duplex binding affinity, which is
related to the enthalpy change (
jugated duplexes to the same extent (approximately 73 kJ molꢀ1
on average). As a result of this, the free energy ( G°) is also
DH°) is reduced in both of the con-
D
reduced leading to less favorable duplex formation at 37 °C with
the conjugated duplex despite no change in Tm. The reduction in
D
H° indicates that there is a weaker interaction between the two
oligomers, most likely a result of steric bulk minimizing the inter-
action of the base pairs adjacent to the conjugate. However, the
conformations of the base pairs or stacking patterns may also have
an effect along with counter ion uptake and release and hydration
factors may also have an effect. Despite the change in the enthalpy
and free energy change for this duplex, the thermal melting tem-
perature remains relatively unaffected overall.
2. Results and discussion
2.1. Synthesis and thermodynamic studies of the menadione-
PNA conjugate
2.2. PNA1, 6-PNA1 and 7-PNA1 inhibit the formation of mutant
SOD1 inclusions in neuronal cell lines
The menadione analog was synthesized by the adaptation of the
methods of Abell et al.22 Commercially available menadione was
directly brominated using molecular bromine in the presence of
sodium acetate and glacial acetic acid to yield the brominated
adduct 1,23 followed by reduction of the dione using potassium
hydroxide and in situ methylation with dimethyl sulfate to give
2 in 45% yield over the two steps (Scheme 1). Alkylation of 2 using
ethyl bromoacetate was achieved via a copper transmetalation
reaction through a diaryl cuprate intermediate from treatment
with n-butyllithium and copper bromide dimethyl sulfide. Increas-
ing the number of equivalents of ethyl bromoacetate from 1.1 to
1.5 and extending the reaction time from 5 to 18 h resulted in an
optimized yield of 55%, a considerable improvement over the pre-
viously reported 34% yield for this step.22 Reduction of the ethyl
ester 3 using lithium aluminum hydride proceeded in high yield
to give the corresponding alcohol 4 which was succinylated using
the anhydride to afford 5. Ceric ammonium nitrate (CAN) was uti-
lized to oxidatively deprotect the methoxy groups and restore the
quinone functionality that is characteristic of the vitamin K family
affording 6 in 84% yield.
We next examined the effect of the PNA compounds in cells
expressing mutant SOD1 A4V. Mouse neuronal Neuro2a cells were
transfected with a previously generated construct encoding SOD1
A4V,30 tagged with Enhanced Green Fluorescent protein (EGFP)
to aid in visualizing the expressed protein. We previously demon-
strated that the presence of the EGFP tag does not affect the activ-
ity of the protein.30 Cells were also treated 4 h post transfection
either with dimethyl sulfoxide (DMSO) vehicle as a negative con-
trol, unconjugated PNA (PNA1 or its complementary sequence
PNA2), menadione-conjugate 6-PNA1 (Fig. 2), previously prepared
vitamin E derived-conjugates 7- PNA1, 8-PNA1 and 9-PNA1
(Fig. 3),21 or (+/ꢀ)-trans-1,2-bis(2-mercaptoacetamido)cyclohex-
ane (BMC), which we previously demonstrated significantly
reduces the formation of mutant SOD1 A4V inclusions and reduced
ER stress.13
Cells were transfected for 72 h and then examined by fluores-
cent microscopy with the percentage of cells bearing fluorescent
green mutant SOD1 inclusions quantified. Untransfected cells
(UTR) were included as a negative control to specifically determine

1522
E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
Scheme 1. Synthesis of a menadione analog. Reactions and conditions: (a) Br2, sodium acetate, glacial acetic acid, 3 days; (b) tetrabutylammonium iodide, tetrahydrofuran/
H2O (3:1), 2.2 M Na2S2O4(aq), 20 min then KOH, (CH3O)2SO2, 12 h; (c) n-butyllithium in hexanes, diethyl ether, 0 °C, 30 min then CuBrꢁMeS, 2.5 h then BrCH2COOCH2CH3, 0 °C,
2 h; (d) LiAlH4, diethyl ether, 35 min; (e) triethylamine, succinic anhydride, 4-(dimethylamino)pyridine, CH2Cl2, 12 h; (f) CAN, CH3CN/H2O (3:2), 0 °C, 30 min then room
temperature, 20 min.
2.3. PNA1 and 6-PNA1 reduces ER stress in neuronal cells
expressing mutant SOD1 A4V
Since PNA1, 6-PNA1 and 7-PNA1 reduced inclusion formation,
we proceeded to investigate whether the PNA oligomers could
inhibit ER stress induced by mutant SOD1 A4V. We examined the
Figure 2. Conjugated menadione-PNA oligomer of 6-PNA1.
pro-apoptotic phase of UPR using nuclear immunoreactivity to
CHOP, as previously described.13 Neuro2a cells were transfected
with the SOD1 A4V-EGFP construct and at 72 h post transfection,
cells were fixed and immunocytochemistry for CHOP was per-
the effects of transfection with mutant SOD1 and, as expected,
formed. Activation of CHOP, indicated by immunoreactivity in
rarely formed inclusions. However, in control DMSO only treated
the nucleus, was observed in 35% of transfected control cells that
populations, 19% of transfected cells formed inclusions
were treated with DMSO only (Fig. 5A and B, additional low mag-
(Fig. 4A and B, additional low magnification images provided as
nification images provided as Supplementary material). However,
Supplementary material) similar to our previous observations.30
in cells treated with 6-PNA1, there was a significant reduction in
As in an earlier study, treatment with BMC significantly reduced
the percentage of cells with nuclear immunoreactivity to CHOP
the percentage of cells bearing inclusions to 14% compared to
compared to DMSO-treated cells (25%, p <0.001), demonstrating
DMSO treated cells (p <0.05). Moreover, treatment of SOD1 A4V
that 6-PNA1 is protective against ER stress induced by mutant
expressing cells with 6-PNA1 significantly decreased the percent-
age of cells bearing inclusions to 15% (p <0.05). Furthermore, treat-
ment of cells with PNA1 and 7-PNA1 significantly reduced the
percentage of transfected cells bearing inclusions from 21% in
DMSO treated cells to 11% (p <0.01) and 14% (p <0.05) respectively
(Fig. 4A and C). There was a slight decrease in inclusion formation
in cells treated with compounds 8-PNA1 (17%) and 9-PNA1 (16%),
but this was not statistically significant. In contrast, treatment with
the negative control PNA, PNA2, did not alter the percentage of
cells bearing inclusions (20%) as expected. Hence this data indi-
cates that compounds PNA1, 6-PNA1 and 7-PNA1 were all equally
effective as BMC in preventing the formation of mutant SOD1
SOD1 A4V. Similarly, BMC treatment was also protective against
induction of UPR: the percentage of transfected cells with CHOP
activation was significantly decreased in this cell population
(30%, p <0.05).
PNA1 also significantly reduced CHOP activation, to only 26%
transfected cells (p <0.05) compared to DMSO-treated cells (37%)
(Fig. 5A and C). In contrast, treatment with the negative control
PNA, PNA2, did not alter the percentage of cells with CHOP activa-
tion (40%) and hence ER stress. Treatment of cells with the remain-
ing PNA oligomers (7-PNA1, 8-PNA1 and 9-PNA1) significantly
reduced the percentage of transfected cells with ER stress when
compared with cells treated with PNA2 (31%, all p <0.05). However
inclusions in neuronal cells, the characteristic pathological hall-
this was not statistically significant when compared with cells
mark of ALS.
treated with DMSO only. As expected, very few untransfected cells
(UTR) displayed CHOP up-regulation. Hence these results reveal
Table 1
Thermodynamic data of duplex formation by UVMa,b
Table 2
Thermodynamic data of duplex formation by ITCa,b
Duplex
Tm (°C)
D
H°VH
d
D
H
D
G°VH
dD
Gc
(kJ molꢀ1
)
(kJ molꢀ1
)
c
c
Duplex
D
H°b (kJ molꢀ1
)
d
D
H
D
G°b (kJ molꢀ1
)
dD
Gc
PNA1/PNA2
6-PNA1/PNA2
PNA1/DNAd
6-PNA1/DNAd
64 ( 1)
66 ( 1)
57 ( 1)
55 ( 1)
ꢀ202 ( 6)
ꢀ123 ( 5)
ꢀ183 ( 13)
ꢀ106 ( 1)
ꢀ14.1 ( 1.7)
ꢀ7.8 ( 1.9)
ꢀ9.6 ( 2.4)
ꢀ6.6 ( 0.5)
PNA1/PNA2
6-PNA1/PNA2
PNA1/DNAd
6-PNA1/DNAd
ꢀ216 ( 4)
ꢀ143 ( 3)
ꢀ169 ( 1)
ꢀ107 ( 1)
ꢀ48.9 ( 0.7)
ꢀ42.7 ( 2.8)
ꢀ48.4 ( 3.2)
ꢀ41.8 ( 0.5)
+79
+77
+6.3
+3.0
+73
+62
+6.2
+6.6
a
b
c
a
b
c
Values obtained are an average of a minimum of 3 experiments.
Error corresponds to 1 standard deviation.
T = 310 K,
DNA sequence used is 50-AAGTCGTGC-30.
Values obtained are an average of a minimum of 3 experiments.
Error corresponds to 1 standard deviation.
T = 310 K.
DNA sequence used is 50-AAGTCGTGC-30.
d
d

E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
1523
Figure 3. Conjugated vitamin E derived-PNA oligomers.21
Figure 4. PNA1, 6-PNA1 and 7-PNA1 reduce mutant SOD1 A4V inclusion formation. Fewer mutant SOD1 A4V inclusions are formed in Neuro2a cells treated with PNA
compared to cells only treated with DMSO. (A) Fluorescent microscopy images of mutant SOD1 A4V-EGFP expressing in Neuro2a cells at 72 h post transfection. White arrow
indicates fluorescent green SOD1 inclusions, shown in first column. Nuclei are shown by Hoechst staining (blue, second column). Merge (third column) represents the
combined images from first and second columns. Top panel indicates cells treated with DMSO alone, second panel, BMC treated cells and third panel, 6-PNA1 treated cells.
The fourth and the fifth panels represent cells administered with PNA1 and 7-PNA1. Scale bar represents 10 lm. (B) Quantification of the percentage of cells bearing SOD1
A4V inclusions in BMC (positive control) and 6-PNA1 treated cells compared to DMSO-only treated cells. (C) Quantification of percentage of cells bearing SOD1 A4V inclusions
in PNA treated cells compared to DMSO-only treated cells. In each cell population, 100 transfected cells were examined for the presence of fluorescent green inclusions. UTR
represent control, untransfected cells. Data represented as Mean SD, n = 3, *p <.05 **p <.01 versus respective controls by one way ANOVA with Tukey’s post hoc test.

1524
E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
Figure 5. PNA1 and 6-PNA1 reduce activation of CHOP, and hence pro-apoptotic phases of UPR, in cells expressing mutant SOD1. Immunocytochemistry for CHOP was
performed in Neuro2a cells expressing mutant SOD1 A4V at 72 h post transfection (A) Fluorescent microscopy images of mutant SOD1 A4V-EGFP transfected Neuro2a cells.
White arrow indicates nuclear immunoreactivity to CHOP, indicating its activation, and hence induction of pro-apoptotic phases of UPR in these cells. Fluorescent mutant
SOD1 A4V (green) is shown in the first column, immunofluorescence detection of CHOP (red) in the second column, nuclei are shown by Hoechst staining (blue) in the third
column, and the fourth column represents merge image of all three. Top panel indicates cells treated with DMSO alone, second panel, BMC treated cells, third panel, 6-PNA1
treated cells, and the fourth panel represents cells administered with PNA1. Scale bar represents 10 lm. (B) Quantification of the percentage of cells with nuclear
immunoreactivity to CHOP in BMC (positive control) and 6-PNA1 treated cells compared to DMSO-only treated cells. (C) Quantification of the percentage of cells with nuclear
immunoreactivity to CHOP in PNA1, 7-PNA1, and 9-PNA1 treated cells compared to either DMSO-only treated cells or negative control PNA (PNA2) treated cells. In each cell
population, 100 transfected cells were examined for the presence of nuclear CHOP immunoreactivity. UTR represents control, untransfected cells. Data represented as
Mean SD, n = 3, *p <.05, **p <.01, ***p <.001 versus respective controls by one way ANOVA with Tukey’s post hoc test.
that the PNA compounds PNA1 and 6-PNA1 were protective
against the activation of CHOP, and hence induction of the pro-
apoptotic phases of the UPR, in neuronal cells expressing mutant
SOD1.
While the conjugation of vitamin K did not strengthen the cellular
effects over those observed by the unconjugated PNA, this work
has shown that conjugation which is not detrimental to activity
is possible and provides an important proof of concept in develop-
ing other conjugates. Further studies will be necessary to investi-
gate any benefits vitamin
delivery of PNA in an animal model.
K conjugation may have on the
3. Conclusions
The synthesis of a menadione-PNA conjugate was carried out to
facilitate thermodynamic studies by both UVM and ITC, which
resulted in the formation of the expected duplex indicating ther-
modynamic stability. Cellular studies were then carried out on
both unconjugated and conjugated PNA. We have shown for the
first time that compounds containing the peptide nucleic acid
sequence of H-GCACGACTT-NH2 are effective against specific cellu-
lar signaling pathways, which complements earlier studies investi-
gating the disease parameters in SOD1 mice but which did not look
any further at the specific mechanisms involved. The synthesized
PNA oligomers have demonstrated efficacy in reducing mutant
SOD1 inclusion formation and ER stress, which are two key cellular
pathological hallmarks in ALS, with the unconjugated PNA1 and
the menadione-conjugate 6-PNA1 found to be equally effective.
4. Experimental
4.1. Synthesis
NMR spectra were recorded on a Bruker Avance-300 Spec-
trophotometer (1H at 300.13 MHz and 13C at 75.47 MHz) or a Bru-
ker Avance-400 spectrometer (1H at 400.13 MHz and 13C at
100.62 MHz). All PNA and PNA conjugates were analyzed by Matrix
Assisted Desorption Ionization Time of Flight mass spectrometry
(MALDI-TOF-MS) using an Ultraflex III instrument (Bruker Dalton-
ics, Germany) and a-cyano-4-hydroxy cinnamic acid as the matrix.
Electrospray ionization (ESI) mass spectrometry was carried out
using an Esquire6000 ion trap mass spectrometer (Bruker Daltonics,

E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
1525
Germany). The samples were introduced at a flow rate of 4
lL/min
4.1.4. 2-(3-Methyl-1,4-dimethoxynapthelen-2-yl) ethanol 4
and a mass range of 50 – 3000 m/z was recorded. A scan rate of
5500 m/z/second was used with the temperature set at 300 °C.
The molecular ion peaks (m/z) were denoted [M+H]+. TLC was per-
formed using Merck Kieselgel 60 F254 plates. Drying and purifica-
tion methods for solvents and reagents were followed by
directions from Armarego and Chai.31 Melting points were col-
lected on hot stage Reichert ‘Thermopan’ apparatus. The DNA
sequence used in the thermodynamic experiments was purchased
from Sigma Aldrich.
The synthesis of this compound was adapted from the method
of Abell et al.22 The ester 3 (873 mg, 3.03 mmol) was vigorously
stirred in a solution of anhydrous ether (30 mL) under a bed of
argon. To this, LiAlH4 (288 mg, 7.57 mmol) was added and the
reaction stirred for 35 min. The reaction was quenched using a sat-
urated solution of aqueous NH4Cl (10 mL), the two layers were sep-
arated and the aqueous layer was extracted with EtOAc (30 mL).
The combined organic layers were washed with H2O (15 mL) fol-
lowed by brine (15 mL), dried with MgSO4 and the solvent
removed in vacuo. The resulting solid was purified on silica by
flash column chromatography eluting with EtOAc/hexane (1:4) to
afford 4 as an off white solid (692 mg, 93%), mp 68–70 °C (lit.22
66–69 °C). dH (300 MHz, CDCl3): 8.01–7.99 (2H, m, ArH), 7.46–
7.43 (2H, m, ArH), 3.89 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.83
(2H, t, J 7.2, CH2CH2OH), 3.11 (2H, t, J 6.9, ArCH2CH2), 2.78 (1H, s
(b), OH), 2.41 (3H, s, ArCH3). dC (75 MHz, CDCl3): 150.6, 150.3,
127.9, 127.4, 127.0, 126.5, 125.8, 125.5, 122.2, 62.6, 62.0, 61.4,
18.5, 12.6.
4.1.1. 2-Methyl-3-bromo-1,4-napthoquinone 1
The synthesis of this compound was based on the method of
Teeter et al.23 Menadione (5.5 g, 31.94 mmol), glacial acetic acid
(50 mL) and sodium acetate (11 g, 134.09 mmol) were heated in
a conical flask until the suspension dissolved. To this, bromine
(2 mL) was added and the flask was stoppered and placed in dark-
ness for 3 days. Water (300 mL) was added and the resulting solid
filtered, followed by recrystallisation from methanol to afford the
brominated product 1 as bright yellow crystals (5.86 g, 73%), mp
150–152 °C (lit.23 151–152 °C). dH (300 MHz, CDCl3): 8.15–8.09
(2H, m, ArH), 7.76–7.69 (2H, m, ArH), 2.38 (3H, s, ArCH3). dC
(75 MHz, CDCl3): 181.9, 171.5, 148.5, 139.0, 134.1, 133.9, 131.6,
131.2, 127.5, 127.1, 17.9.
4.1.5. 2-(3-Methyl-1,4-dimethoxynaphthalen-2-yl)ethyl
hydrogen succinate 5
The synthesis of this compound was adapted from the method
of Abell et al.22 To a stirred solution of alcohol 4 (490 mg,
1.99 mmol) in CH2Cl2 (25 mL),
a solution of TEA (400 lL,
4.1.2. 2-Bromo-3-methyl-1,4-dimethoxynapthelene 2
2.88 mmol), succinic anhydride (420 mg, 4.18 mmol) and DMAP
(22 mg, 0.18 mmol) in CH2Cl2 (15 mL) was added drop-wise and
the reaction stirred overnight. The solvent was removed in vacuo
and the resulting residue was dissolved in CH2Cl2 (30 mL) and
washed with 10% HCl (20 mL) followed by H2O (2 ꢂ 20 mL), dried
with MgSO4 and the solvent removed in vacuo to give 5 (680 mg,
98%) as a yellow oil. dH (300 MHz, CDCl3): 8.06–7.99 (2H, m,
ArH), 7.46–7.44 (2H, m, ArH), 4.28 (2H, t, J 7.5, OCH2CH2), 3.90
(3H, s, OCH3), 3.85 (3H, s, OCH3), 3.16 (2H, t, J 7.2, ArCH2), 2.93
(1H, s, OH), 2.67–2.64 (4H, m, CH2CH2), 2.44 (3H, s, ArCH3). dC
(75 MHz, CDCl3): 178.0, 172.2, 151.0, 150.2, 128.0, 127.1, 126.6,
126.2, 125.9, 125.5, 122.3, 122.2, 64.0, 62.2, 61.3, 28.9, 28.3, 26.7,
12.5.
The synthesis of this compound was adapted from the method
of Abell et al.22 To a stirred solution of 1 (1 g, 3.97 mmol), TBAI
(176 mg, 0.48 mmol) in water (3.6 mL) and tetrahydrofuran
(10.9 mL), a solution of sodium dithionite (4.16 g, 23.9 mmol) in
water (10.9 mL) was added and the reaction stirred for 20 min.
To this, a solution of potassium hydroxide (15.8 M, 5.8 mL) was
added drop-wise and stirred for a further 5 min, at which time
dimethylsulfate (4.5 mL, 47.8 mmol) was then added drop-wise
and the reaction was stirred overnight. The product was extracted
with CH2Cl2 (4 ꢂ 50 mL), dried with MgSO4 and solvent removed in
vacuo. The orange mass was recrystallized from methanol to afford
2 in 62% yield (2.78 g) as light orange crystals, mp 83–84 °C (lit.22
82–84 °C). dH (300 MHz, CDCl3): 8.09–8.03 (2H, m, ArH), 7.55–7.45
(2H, m, ArH), 3.96 (3H, s, OCH3), 3.87 (3H, s, OCH3), 2.52 (3H, s,
ArCH3). dC (75 MHz, CDCl3): 150.5, 149.8, 127.8, 127.6, 127.3,
126.5, 126.2, 122.4, 122.4, 117.2, 61.6, 61.3, 16.7.
4.1.6. 2-(3-Methyl-1,4-naphthoquinon-2-yl)ethyl hydrogen
succinate 6
The synthesis of this compound was adapted from the method
of Abell et al.22 To a cooled solution of 5 (990 mg, 2.85 mmol), in a
2:1 solution of CH3CN:H2O (11 mL), a solution of ceric ammonium
nitrate (3.92 g, 7.14 mmol) in 1:1 CH3CN:H2O (13 mL) was added
drop-wise and the reaction stirred at 0 °C for 30 min, followed by
20 min at room temperature. The solution was diluted with H2O
(50 mL) and the bright yellow solution was extracted with CH2Cl2
(6 ꢂ 40 mL). The combined organic solution was washed with H2O
(50 mL), dried with MgSO4 and the solvent removed in vacuo to
afford 6 as a yellow-orange oil (762 mg, 84%). dH (400 MHz, CDCl3):
8.08–8.04 (2H, m, ArH), 7.71–7.67 (2H, m, ArH), 4.28 (2H, t, J 6.6,
OCH2), 3.00 (2H, t, J 6.6, ArCH2), 2.65–2.55 (4H, m, CH2CH2), 2.22
(3H, s, ArCH3). dC (100 MHz, CDCl3): 184.9, 184.3, 177.7, 172.0,
145.5, 142.6, 133.5, 133.5, 132.0, 131.9, 126.3, 63.0, 28.7, 26.7,
13.0.
4.1.3. Ethyl 2-(3-methyl-1,4-dimethoxynapthalen-2-yl)acetate 3
To a cooled solution of 2 (1.96 g, 6.97 mmol) in anhydrous ether
(12 mL) under an atmosphere of argon, n-butyllithium (1.6 M in
hexane, 5.0 mL, 8.02 mmol) was added drop-wise and stirred at
0 °C for 30 min. Copper bromide dimethyl sulfide complex
(1.00 g, 4.88 mmol) was added and the solution stirred for 2.5 h.
A chilled solution of ethyl bromoacetate (1.16 mL, 10.5 mmol) in
ether (8 mL) was then added slowly and the reaction stirred at
0 °C for 2 h, at which time the reaction was warmed to room tem-
perature and stirred for an additional 16 h. The reaction was
quenched with 3 M HCl (15 mL) and the two layers were separated.
The aqueous layer was extracted with ether (2 ꢂ 15 mL) and the
combined organic extracts were washed with H2O (30 mL), satu-
rated aqueous NaHCO3 (30 mL) and once more with H2O (30 mL).
The organic layer was dried with MgSO4 and evaporated in vacuo.
The crude orange oil was purified on silica by flash column chro-
matography, eluting with EtOAc/hexane (1:19) to yield 3 (1.10 g,
55%) as an orange oil. dH (300 MHz, CDCl3): 8.06–8.04 (2H, m,
ArH), 7.49–7.46 (2H, m, ArH), 4.19 (2H, q, J 7.2, OCH2CH3), 3.93
(2H, s, ArCH2), 3.91 (3H, s, OCH3), 3.86 (3H, s, OCH3), 2.37 (3H, s,
ArCH3), 1.27 (3H, t, J 7.2, OCH2CH3). dC (75 MHz, CDCl3): 171.7,
150.8, 150.1, 128.3, 127.1, 126.7, 126.0, 125.5, 124.1, 122.5,
122.3, 62.2, 61.4, 60.9, 33.1, 14.3, 12.7.
4.2. PNA synthesis and characterization by UVM and ITC
4.2.1. Synthesis
Bhoc- and Fmoc-protected PNA monomers (A, C, G and T) and 2-
aminoethoxy-2-ethoxyacetic acid (AEEA) were purchased from
ASM Research Chemicals and were used without further purifica-
tion. Automated synthesis was performed on an Expedite 8909
nucleic acid synthesizer, on a 2
lmol scale using Fmoc-PAL-PEG-
PS resin (0.19 mmol/g) from Applied Biosystems, following the

1526
E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
standard protocols.24 The synthetic procedure follows a deprotec-
tion and coupling strategy using solutions of 0.2 M PNA monomers
in N-methylpyrrolidone for all monomers except for the C mono-
mer (0.1 M) which was double coupled, deblocking solution (20%
piperidine in DMF) then activation using a base solution (0.18 M
HATU, 0.2 M DIPEA and 0.3 M 2,6-lutidine in DMF). Capping with
5% v/v acetic anhydride and 6% v/v 2,6-lutidine in DMF terminates
incomplete sequences. Conjugates (0.2 M in DMF) were coupled to
the PNA oligomer on resin following the same protocol. The PNA
was cleaved from the resin using TFA/m-cresol (4:1) then precipi-
tated and washed with ice cold ether and dried. The crude PNA was
4.2.5. Isothermal titration calorimetry
Calorimetric experiments were performed on a CSC 5300 Nano-
ITC 111 instrument at 25 °C, where one of the oligomer strands
(ꢄ0.1 mM, 100
l
L) was titrated into 1.4 mL of the complementary
strand (ꢄ5 M). Each injection was 4 or 5
l
l
L at 5 min intervals for
a total of 25 injections. Stirrer speed was set to 250 rpm. Solutions
were thoroughly degassed by sonification and absolute concentra-
tions determined as outlined above. The reference cell was filled
with degassed and deionized water. Isotherms were examined
using the software NanoAnalyze v2.0, whereby the binding con-
stant (Kb), intrinsic molar enthalpy change (DHb°) and stoichiome-
purified using a Phenomenex Jupiter C18 10
l
m, 250 mm ꢂ 10 mm
try of binding (n) were determined by means of best fit
(independent model) of the calorimetric data. The data was cor-
rected by subtracting the heat of dilution from the experiment.
Each duplex was titrated in triplicate, at a minimum.
column, with gradient elution using water (Eluent A) and acetoni-
trile (Eluent B) with 0.1% TFA. The pure PNA fractions were col-
lected, lyophilized and characterized by MALDI-TOF and ESI mass
spectroscopy as appropriate.
4.3. Cellular biology
4.2.2. Determination of solution concentration
All experiments were carried out in 10 mM sodium phosphate
buffer (pH 7.0). The concentrations of both PNA and DNA strands
were determined by UV absorption at a wavelength of 260 nm at
80 °C, using quartz cells with a 1 cm path length. The following
4.3.1. Constructs and cell lines
The mouse neuroblastoma Neuro-2a cell line (ATCC cell line
CCL-131) was used for all transient transfections. The SOD1A4V-
EGFP construct was as described previously.30
extinction coefficients were used;
DNA:G = 12220 Mꢀ1 cmꢀ1 DNA:C = 7600 Mꢀ1 cmꢀ1
T = 8700 Mꢀ1 cmꢀ1 PNA:A = 13700 Mꢀ1 cmꢀ1
PNA:G = 11700
Mꢀ1 cmꢀ1 PNA:C = 6600 Mꢀ1 cmꢀ1 PNAT = 8600 Mꢀ1 cmꢀ1 24
e
DNA:A = 15300 Mꢀ1 cmꢀ1
DNA:
,
e
,
e
,
e
4.3.2. Cell culture and transfection
,
e
,
e
Neuro2a cells were maintained in Dulbecco modified Eagle’s
medium with 10% fetal calf serum, 100 mg/ml penicillin and
100 mg/ml streptomycin. Transfections were performed using
Lipofectamine reagent 2000 (Invitrogen) according to the manu-
facturer’s protocol. Cells transfected with SOD1A4V-EGFP were
examined 72 h post-transfection by fluorescence microscopy and
cells with prominent EGFP-positive inclusions were counted as a
percentage of the total EGFP-positive, and hence transfected, cells.
Treatment with PNA derivatives were added from a stock dissolved
,
e
,
e
.
4.2.3. UV melting experiments
Melting curves were performed on a Varian Cary 100 Bio UV–
Vis spectrophotometer with a Cary temperature controller. The
duplexes formed during the ITC experiments were used directly
to obtain the melting curves, as previously undertaken in the liter-
ature.28 Samples were prepared by heating to 80 °C for 5 min, cool-
ing to 20 °C over 20 min and holding at 20 °C for a further 20 min.
The melting curves were measured at 260 nm, with the tempera-
ture increasing from 20 °C to 80 °C at a rate of 0.5 °C/min, with data
collection occurring every 0.2 °C. Each duplex melting curve was
performed in triplicate, at a minimum.
at 25
clohexane (BMC) (25
fected cell expressing SOD1 as
l
M in DMSO. (+/ꢀ)-trans-1,2-bis(2-mercaptoacetamido)cy-
M in DMSO) was also added to the trans-
positive control, 4 h post
l
a
transfection cells were analyzed for inclusion positive cells and
ER stress as previously described.13
4.2.4. Determination of thermodynamic parameters via UVM
4.3.3. Immunofluorescence and microscopy
The melting temperature (Tm) is dependent upon
a
, which is the
Cells were fixed with freshly prepared 4% (w/v) paraformalde-
hyde and incubated in the dark for 15 min. The cells were perme-
abilized with 0.2% (v/v) Triton-X in PBS for 5 min and blocked with
3% BSA in PBS for 30 min. After washing with 0.1% (v/v) Tween-20
in PBS, incubation with mouse anti-CHOP (1:50) (Abcam) antibody
was performed in PBS at 4 °C overnight. The secondary antibody,
AlexaFluor 568 conjugated rabbit anti-mouse IgG (1:250), was
added for 1 h and incubated in the dark at room temperature. After
washing, staining of nuclei was performed with Hoechst stain
33342 (Invitrogen) and mounted using fluorescent mounting
media. The slides were observed under 100ꢂ magnification on an
Olympus epifluorescent microscope. DAPI (nuclei), FITC (GFP fluo-
rescence) filters were used for viewing and images taken using
NIS-Elements BR 3.2 software.
fraction of the single strand in a duplex state, as described by
Marky and Breslaur25 is shown by Eq. A below:
As ꢀ A
a
¼
ðAÞ
ðAs ꢀ AÞ þ ðA ꢀ AdÞ
where A is absorbance at a given T, and As and Ad are the absorbance
from the single strand and the duplex respectively. The Tm of the
duplex is determined where
In order to calculate the van’t Hoff enthalpy, the equilibrium
constant (K) must be determined and expressed in terms of for
a = 0.5.
a
a non-complementary association, as shown in Eq. B, where CT is
the total strand concentration and n is the number of strands asso-
ciated with the complex.
a
K ¼
ðBÞ
4.3.4. Statistics
nꢀ1
n
ðCT=nÞ ð1 ꢀ Þ
a
Cells transfected with SOD1-EGFP were counted (100 total) and
the data was represented as Mean SD. Analyses were made using
ANOVA followed by Tukey’s post hoc test. (GraphPad Prism, San
Diego, CA, USA). p-Values of 0.05 or less were considered signifi-
Thus, the Gibbs free energy change can be determined (Eq. C)
where a plot of ln (K) versus 1/T will determine both the enthalpy
and entropy of the system (Eqs. D and E, respectively).
GvHꢃ ¼ ꢀRT lnðKÞ ¼
cant. p <0.05⁄, p <0.01⁄⁄, p <0.001⁄⁄⁄
.
D
D
D
D
Hꢃ ꢀ T
D
Sꢃ
ðCÞ
ðDÞ
ðEÞ
Acknowledgments
HvHꢃ ¼ slope ðlnðKÞ vs 1=TÞR
This work was supported by an Australian Research Council
Discovery Project (DP0771578). E.C.B. is a recipient of an Aus-
SvHꢃ ¼ intercept ðlnðKÞ vs 1=TÞR

E. C. Browne et al. / Bioorg. Med. Chem. 24 (2016) 1520–1527
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