Synthesis and improved cross-linking properties of c5-modified furan bearing pnas

Article abstract of DOI:10.3390/molecules22112010

Over the past decades, peptide nucleic acid/DNA (PNA:DNA) duplex stability has been improved via backbone modification, often achieved via introducing an amino acid side chain at the α- or γ-position in the PNA sequence. It was previously shown that interstrand cross-linking can further enhance the binding event. In this work, we combined both strategies to fine-tune PNA crosslinking towards single stranded DNA sequences using a furan oxidation-based crosslinking method; for this purpose, γ-L-lysine and γ-L-arginine furan-PNA monomers were synthesized and incorporated in PNA sequences via solid phase synthesis. It was shown that the L-lysine γ-modification had a beneficial effect on crosslink efficiency due to pre-organization of the PNA helix and a favorable electrostatic interaction between the positively-charged lysine and the negatively-charged DNA backbone. Moreover, the crosslink yield could be optimized by carefully choosing the type of furan PNA monomer. This work is the first to describe a selective and biocompatible furan crosslinking strategy for crosslinking of γ-modified PNA sequences towards single-stranded DNA.

Full text of DOI:10.3390/molecules22112010

molecules
Article
Synthesis and Improved Cross-Linking Properties of
C5-Modified Furan Bearing PNAs
Joke Elskens 1,†, Alex Manicardi
Roberto Corradini *
1,2,†
ID
, Valentina Costi , Annemieke Madder *
2
1,
ID
and
2
,
1
Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular
Chemistry, Ghent University, Krijgslaan 281-S4, 9000 Gent, Belgium; Joke.Elskens@Ugent.be (J.E.);
Alex.Manicardi@Ugent.be (A.M.)
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma,
Parco Area delle Scienze 17/A, 43124 Parma, Italy; valentina.costi@studenti.unipr.it
Correspondence: Annemieke.Madder@Ugent.be (A.M.); roberto.corradini@unipr.it (R.C.);
Tel.: +32-9-264-4472 (A.M.); +39-0521-905-410 (R.C.)
2
*
†
These authors contributed equally to the work.
Received: 21 October 2017; Accepted: 9 November 2017; Published: 20 November 2017
Abstract: Over the past decades, peptide nucleic acid/DNA (PNA:DNA) duplex stability has been
improved via backbone modification, often achieved via introducing an amino acid side chain at the
α-
or -position in the PNA sequence. It was previously shown that interstrand cross-linking can further
γ
enhance the binding event. In this work, we combined both strategies to fine-tune PNA crosslinking
towards single stranded DNA sequences using a furan oxidation-based crosslinking method; for this
purpose,
γ
-L-lysine and
γ
-L-arginine furan-PNA monomers were synthesized and incorporated
-modification had a
in PNA sequences via solid phase synthesis. It was shown that the L-lysine
γ
beneficial effect on crosslink efficiency due to pre-organization of the PNA helix and a favorable
electrostatic interaction between the positively-charged lysine and the negatively-charged DNA
backbone. Moreover, the crosslink yield could be optimized by carefully choosing the type of furan
PNA monomer. This work is the first to describe a selective and biocompatible furan crosslinking
strategy for crosslinking of γ-modified PNA sequences towards single-stranded DNA.
Keywords: PNA; backbone modification; furan; crosslinking; chiral monomers
1
. Introduction
Peptide nucleic acids (PNAs), first described by Nielsen et al. [1], are a class of nuclease-resistant
synthetic oligonucleotide mimics based on repeating N-(2-aminoethyl)-glycine units. The uncharged
backbone of the PNA reduces electrostatic repulsion upon hybridization, resulting in very strong
binding affinity towards complementary oligonucleotides (ONs) [
in antisense [ ] and antigene strategies [ ]. Furthermore, PNAs have been successfully used as
artificial restriction DNA cutters [ ] or artificial RNA restriction enzymes [10 11], as probes for mRNA
2]. PNAs have various applications
3–
5
6,7
8,
9
,
detection [12,13], as anti-microRNA agents [14–17], and in microarray applications [18–21].
Further enhancement of oligonucleotide binding can be achieved by introducing modifications
in the backbone structure of the PNA strand [22]. Backbone modification with amino acid side
chains is one of the most reported and studied modifications of the original structure of the PNA
monomer [23
–
29], of which
γ-substitution is the most popular. Ly et al. reported on L-serine- and
L-alanine-modified
γ
-PNA that show increased binding affinity towards DNA [30]; this was shown
to be the result of a constrained conformation of the single strand, as recently demonstrated using
molecular dynamics simulations [31], which leads to a lesser entropy loss in the formation of the
PNA:DNA duplex. The use of γ-L-Lys-PNA building blocks has been reported as a synthetic handle
Molecules 2017, 22, 2010; doi:10.3390/molecules22112010
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2010
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for the attachment of various functional groups [32] including fluorophores and peptides. Additionally,
guanidine based - and -PNAs have been developed to improve cellular uptake [33 35]. Glazer et al.
developed tail-clamp PNAs for gene editing purposes, featuring a polyethylene glycol -modification
α
γ
–
γ
to improve water solubility without changing electrostatic interactions. These PNAs could be delivered
in vivo via polymer nanoparticle formulation [36].
The high stability in the formation of PNA:ON duplexes might be advantageously combined
with the use of interstrand crosslinking for therapeutic purposes. In 2006, Zhilina et al. reported on
the development of PNA–nitrogen mustard conjugates, used for DNA alkylation and interstrand
crosslinking towards the human epidermal growth factor receptor 2 (HER2/neu) promotor [37].
The use of 2-amino-6-vinylpurine as a crosslink moiety allowed selective crosslinking towards
single-stranded DNA and RNA [38]. Alternatively, ‘switch-on’ crosslinking methods have been
developed, where the pro-reactive moiety is activated upon an external trigger [39
crosslinking of PNA:DNA duplexes induced by -irradiation has been reported [42]. Unfortunately,
these methodologies often require harsh reaction conditions [40 42], long reaction times [38],
or impart problems like unspecific [37 42] and reversible [41] crosslinking, toxicity [39], and cellular
damage [37 40 42]. In our research group, a furan oxidation-based crosslinking strategy was developed
and successfully implemented for interstrand crosslinking of nucleic acids [43 44], crosslinking of DNA
–41]. Also,
γ
,
,
,
,
,
and RNA with proteins [45], and crosslinking of peptides with proteins [46] while ensuring a high
selectivity and crosslinking yield. Furan oxidation can either be achieved via N-bromosuccinimide
or via a biocompatible method using singlet oxygen. Recently, we also reported on the successful
crosslinking of furan-modified peptide nucleic acids towards single- and double-stranded DNA [47].
Here, we became interested in further increasing the efficiency of furan–PNA crosslinking towards
single-stranded DNA sequences, by exploiting the use of modified backbones.
2
. Results and Discussion
2
.1. Design and Synthesis of the Backbone-Modified PNA Monomers
In previous work we focused on the introduction of a furan moiety in the middle of a PNA strand
to form an interstrand crosslink (ICL) with target ssDNA and dsDNA by means of the use of three
different building blocks, where the furan unit was connected to the normal PNA backbone through
three different geometries [47]. In the current study, we explore the possibility of introducing the
furan modification in backbone-modified PNAs, in order to confer additional properties to the PNA
strand, such as improved solubility, increased binding stability, and increased target selectivity by
means of the introduction of positively-charged side chains. For this purpose, arginine and lysine side
chains were selected as candidates for backbone modification, especially to try and compensate for
the stability loss in the cases where the furan moiety was linked to the backbone and replaced the
nucleobase unit, thus leading to a loss of the Watson–Crick base pairing ability.
C5(
)-modified ones (from D-amino acids) in view of the higher stability of the resulting duplexes
with target DNA and RNA [21 35]. Moreover, unlike in the case of C2 modification, C5-modified PNA
γ)-modified-PNA backbones derived from L-amino acids were selected over the
C2(
α
,
derivatives can be coupled to the PNA chain without extensive risk of epimerization, thus avoiding
the need to apply submonomer-based strategies, earlier developed for C2 derivatives [48].
The synthesis of the required monomers for PNA synthesis, depicted in Scheme 1, starts from the
commercially available amino acids Fmoc-Arg(Pbf) and Fmoc-Lys(Boc) which are converted to the
corresponding Fmoc-protected aminoaldehydes 2a and 2b via reduction of the corresponding Weinreb
amide (1a and 1b, respectively). These aldehydes were then submitted to reductive amination in order
to obtain the corresponding protected modified backbone building blocks 3a and 3b. EDC/DhBtOH
coupling of the desired carboxylic acids followed by selective methyl ester deprotection allowed the
synthesis of the desired monomers 5a–f.

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Scheme 1. Synthesis of the 5L-modified monomers. (a) HBTU, DIPEA, N,O-dimethylhydroxylamine,
DMF; (b) LiAlH , THF; (c) Glycine methylester hydrochloride, NaBH CN, AcOH, DIPEA, MeOH;
4
3
(
d) R CH COOH, EDC·HCl, DhBtOH, DIPEA, DMF; (e) Ba(OH) , H O:THF 1:1.
2
2
2
2
2
.2. PNA Synthesis
A series of six new PNAs were synthesized (see Figure 1, PNA 2–3, PNA 5–6, PNA 8–9);
the sequence chosen for the study is identical to the one used in earlier work [47] and was selected for
its complementarity to a specific region of the MYCN gene that was reported to be efficiently targeted
for blocking gene expression [37]. The synthesis of the different PNA sequences was performed using
a standard Fmoc/Bhoc solid phase synthesis protocol on Rink Amide ChemMatrix resin.
In analogy with previous work, the synthesis of the
PNA 8 ) proceeded through incorporation of the different monomers bearing the azide function
designed starting from 2-azidoacetic acid for PNA 5 or from 2-(5-azidomethyluracil-1-yl)acetic
acid for PNA 8 ) in the growing chain. The full length PNAs were then cleaved from the resin and
f and T(f) series (see Figure 1, PNA 5–6 and
–
9
(
–6
–9
submitted to post-synthesis copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) in the
presence of 3-(furan-2-yl)-N-(prop-2-yn-1-yl)propanamide (Figure 2a). For comparison the sequences
of PNA 1, 4 and 7, synthesized in a previous work [47], were used.
Figure 1.
(
a) Peptide nucleic acid (PNA) and DNA sequences used in this study; (b) Structure of the
X monomers employed in this study with varying furan side- chain structure (F, f, T(f)) and varying
backbone modification none (O), Lys side chain (K) or Arg side chain (R).

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For the synthesis of the
F
series (PNA 2
–3
) we previously described that the furan unit must be
protected from C2-alkylation by the benzhydrylic carbocation generated during the cleavage of the
product from the resin [47]. A strategy exploiting a different cleavage cocktail mixture has already
been proposed. However, this approach only allowed for mitigation of the formation of the undesired
alkylated product but did not fully prevent it. We further found this procedure to be very sensitive
to cleavage solution storage conditions (data not shown). For these reasons we decided to develop a
protection protocol that had already been shown to allow efficient protection of maleimide residues
during incorporation into oligonucleotides [49]. We here reasoned that a similar protection protocol
might be applicable for the protection of the furan moiety during PNA synthesis. This approach is
based on the temporary protection of the furan ring by on-resin formation of a [4 + 2] cycloadduct
with maleimide, under thermal Diels–Alder (DA) conditions, and the subsequent retro-DA reaction in
solution, after cleavage (Figure 2b). The chosen strategy relies on the actual possibility of tuning the
DA/retro-DA conditions, by carefully choosing the maleimide N-substituents [50].
Figure 2. (a) General protocol for the synthesis of PNA 4–9; (b) general protocol for the synthesis of
PNA 1–3. SPPS: solid phase peptide synthesis, CuAAC: Cu-catalyzed azide alkyn cyclodaddition.
The formation of the DA adduct was first optimized on a short PNA sequence, where it could
be observed that the relative amount of maleimide, its concentration, and the time of reaction are
crucial factors in achieving protection of the furan unit while avoiding the formation of unidentified
by-products of reaction with the maleimide (see Supplementary Materials Figures S1–S3). Optimized
conditions for the protection of the aromatic system from electrophilic substitution were obtained by
prolongation of the DA reaction time (from 5 to 7 h) to ensure good DA-adduct formation with reduced
by-product formation, and the use of a modified cleavage cocktail (10% m-cresol, 10% thioanisole
in TFA) to avoid damage to any remaining unreacted furan. An example of the analysis of a time

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course experiment of the retro-DA reaction is shown in Figure S4. The optimized conditions were
subsequently used for the more precious PNA 1 , without isolating the intermediates. In Figure 3,
–
3
a typical result obtained using this strategy is shown, where the 5
heated at 90 C in the presence of a 0.25 M solution of N-(N-Boc-2-aminoethyl)maleimide in DMF (1:10
µ
mol scale resin bearing PNA 3 was
◦
furan:maleimide ratio). The resulting resin was then cleaved and the crude was dissolved in 5 mL
◦
milliQ and heated for 5 h at 90 C in order to induce the retro DA. As can be seen from the MS trace
(Figure 3b) and from the de-convoluted spectrum (Figure 3c), only the signals relative to the desired
product are present, and no significant formation of undesired products of alkylation (∆MW: 166.2 Da)
or overreaction with maleimide (∆MW: 140.1 Da) were present.
Figure 3.
(
a
) UPLC-ESI-MS of crude PNA 3 after the Diels–Alder (DA)/retro-DA; (
b) MS trace of
the 2.40–5.00 min region; and (c) de-convoluted spectrum from the MS trace (expected MW: 3187.8).
The peak present in the 8.00–9.00 min region of the UPLC-MS corresponds to a column wash step.
2
.3. Evaluation of PNA:DNA Duplex Stabilities
The effect of introduction of the backbone modification was studied through evaluation of the
melting temperature of the complex formed between the synthesized PNA strands and the target
DNA. All four base permutations facing the modification were studied (detailed sequence info for
m
DNA strands used for the melting temperature (T ) evaluation are reported in Figure 1a). The results
of these experiments are summarized in Table 1.
Analysis of the T
ability of the PNA is maintained, the introduction of the backbone modification (with the concurrent
positive charge) is beneficial for the complex stability (cf. T for PNA 8 ON 1 versus PNA 7 ON
). There is no significant difference between Lys (PNA 8) versus Arg (PNA 9) side chains, suggesting
m
data shows that in the T(f) series (PNA 7–9), where the base discrimination
m
–
9/
–
4
/
1–4
that the different steric requirements of these two side chains do not play a crucial role in the
complex formation. The stabilizing effect appears higher in the T:C (with ON 2) and T:G (with
ON 3) mismatched sequences, where partial recognition of the nucleobases is still possible.
A similar stabilizing effect was also observed for the
PNA 4 ON 1 ), where significant differences in stability are found between the different nucleobases,
which are however similar for PNA with either the Lys or the Arg side chain. In previous studies,
it was already hypothesized that the longer linker present in the building block allowed the aromatic
m
f series (cf. T for PNA 5–6/ON 1–4 versus
/
–4
f

Molecules 2017, 22, 2010
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ring to interact with the nucleobases through
π
-
π
interaction. This interaction can force the furan ring
to remain confined inside the helical duplex, while pointing the side chains toward the solvent.
Table 1. Melting temperatures (T
buffer at pH 7. Strand concentration: 5
stands for the modified monomer (O, K, R, F, f, T (f
m
) for PNA:DNA, measured by UV absorbance at 260 nm in PBS
M. PNA sequence: Ac-GGG CA GAT CT-Gly-NH , where
) see Figure 1 for structural details), DNA sequence:
-AGA TCN TGC CC-3 , where N=A (ON 1), C (ON 2), G (ON 3) or T (ON 4). Numbers inside
parentheses indicate the T difference with respect to T s obtained for the sequences with unmodified
backbone (PNA 1 PNA 4 and PNA 7). Data for PNA ref (Ac-GGGCATGATCT-Gly-NH ) are from [47].
µ
X
X
2
0
0
5
m
m
,
2
Light grey lines indicate PNAs bearing a lysine side chain modification, dark grey lines indicates PNAs
bearing an arginine side chain modification.
ON 1, N=A
ON 2, N=C
ON 3, N=G
ON 4, N=T
PNA 1, X=O, F
PNA 2, X=K (∆Tm), F
PNA 3, X=R (∆Tm), F
PNA 4, X=O, f
PNA 5, X=K (∆Tm), f
PNA 6, X=R (∆Tm), f
PNA 7, X=O, T(f)
47.4
47.2
48.0
47.2
47.3 (−0.1)
45.7 (−1.3)
50.0
50.3 (+0.3)
51.4 (+1.4)
69.0
50.4 (+3.2)
48.6 (+1.4)
49.8
50.8 (+1.0)
50.9 (+1.1)
51.0
50.7 (+2.7)
43.7 (−4.3)
59.5
62.3 (+2.8)
63.4 (+3.9)
55.8
50.4 (+3.2)
48.7 (+1.3)
51.2
54.8 (+3.6)
55.0 (+3.8)
57.8
PNA 8, X=K (∆Tm), T(f)
PNA 9, X=R (∆Tm), T(f)
PNA ref, X=O, T
71.0 (+2.0)
71.0 (+2.0)
68.9
54.0 (+3.0)
54.5 (+3.5)
50.0
59.6 (+3.8)
59.7 (+3.9)
55.4
59.4 (+1.6)
58.3 (+0.5)
56.8
Surprisingly, in the
m
F series (cf. T for PNA 2–3/ON 1–4 versus PNA 1/ON 1–4) the introduction
of the positively charged side chain is not always beneficial for the complex stability. In this context
the insertion of the bulkier and less flexible arginine side chain modification leads to a destabilization
of the duplex, especially when a hindering purine nucleobase is placed in the complementary strand.
In contrast, when a pyrimidine nucleobase is present, a small stabilization is observed. This behavior
is not present when the lysine side chain is introduced. Taken together, these results suggest that the
furan unit in the
F building blocks adopts a conformation that can be perturbed by the introduction of
the bulkier arginine side chain.
2
.4. Evaluation of the Crosslink Reactivity of Furan–PNAs towards Single Stranded DNA Sequences
Next, the crosslink properties of the synthesized PNA sequences towards complementary single
stranded DNAs ON 1
1 equivalent every 15 min, 4 equivalents in total) to trigger the oxidation of the furan ring, and the
results were analyzed through denaturing polyacrylamide gel experiments (PAGE).
It can be seen in Figure 4 that when furan building block PNA 1 ) was used, crosslinking was
–4 were evaluated at a 10 µM strand concentration, using N-bromosuccinimide
(
F
(
–3
most efficient when lysine-modified PNA 2 was hybridized to ON 1 (adenine) and ON 2 (cytosine),
resulting in the appearance of a slow mobility band on the gel (Figure 4a), with a light preference for
the formation of the crosslink in presence of ON 1. In addition, with furan building block f (PNA 4–6),
crosslinking seemed to be more efficient with the PNA strands containing the building block with
the lysine side chain (PNA 5) and arginine modification (PNA 6), again with the selective formation
of a slow migrating band only with ON 1 and ON 2, but in this case with a small preference toward
the cytosine counterpart (Figure 4b). Finally, in the case of furan building block T (f)
no crosslink is formed and the smearing of the bands which results in an apparent slower migration
band (PNA 8 ON 1 and PNA 9 ON 1) has to be attributed to the remaining PNA:DNA duplex which is
(PNA 7–9),
:
:
not denatured under the analysis conditions. (Figure 4c). For experiments using target ON 3, bearing
guanine opposite to the furan unit, only very faint bands (with PNA 2 and PNA 6) and smears were
observed; in none of the crosslink experiments with ON 4 (bearing a thymine base) clearly visible slow
mobility bands related to cross-linked species could be observed.

Molecules 2017, 22, 2010
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The results obtained in the
f
series (PNA 4
–6
) suggest that the introduction of a positive charge
(PNA 5–6) in the PNA backbone is beneficial for the formation of the crosslink as the slow migration
bands appears more intense compared to those obtained with PNA 4. Moreover, the migration
difference observed in the different lanes is in line with the insertion of a positive charge in the ICL
product. The identity of the ICL product was further confirmed by HPLC purification and MALDI
analysis (see supplementary materials, Figure S16).
In contrast, in the
is introduced (PNA 3), possibly due to the fact that the bulkier modification is perturbing the
orientation which the monomer needs to adopt in order to react with the nucleophile placed on the
opposed strand. This is also in line with the results obtained in the T evaluation, where the arginine
side chain showed a destabilization effect on the duplex. The identity of the formed ICL product in
the PNA 2 ON 1 experiment was also confirmed by MALDI analysis (see Supplementary Materials,
F series (PNA 1–3) no ICL formation is observed when the arginine modification
F
m
–
Figure S17). A schematic representation of the putative mechanism of ICL formation [51] for this series
of PNA is depicted in Figure S18 (supplementary material).
For the T(f) series (PNA 7–9) no ICL was expected as the furan moiety is pointing in the major
groove, and the introduction of the modification is only reflected in the degree of smearing of the band
and appearance of a new band due to the PNA:DNA complexes of increased stability. Under these
conditions, the most stable duplex appears to be that formed by the PNA 8, with the L-Lys side chain
(Figure 4c).
Next, strand displacement experiments were performed. Since crosslinking only occurred
with adenine or cytosine as complementary base (which is in line with previous results in dsDNA
crosslinking), it was chosen to only perform these experiments with ON 1 and ON 2. The DNA duplex
was first allowed to form by annealing the two complementary strands, before the addition of the PNA
◦
strand. The solution was then left to equilibrate for 14 h at 37 C before activation of the furan.
Figure 4. Denaturing polyacrylamide gel electrophoresis analysis of the crosslink experiments.
(
a
) Crosslink experiment between ON 1
between ON 1 and PNA 4 (building block
(building block )); ( ) Strand displacement experiment between ON 1
–9; (e) Strand displacement experiment between ON 2:cON duplex and PNA 1–9.
–
4
and PNA 1
); ( ) Crosslink experiment between ON 1
cON duplex and PNA
–
3
(building block
F
); (
b
) Crosslink experiment
–
4
–
6
f
c
–4
and PNA
7
1
–
9
T(
f
d
:
Results are shown in Figure 4d–e and confirm the results obtained in the initial screening
experiments to determine the ability of the different PNA strands for ICL formation toward ssDNA.
◦
As previously reported, due to its high stability (T
m
= 50.2 C), the targeting of the complex ON 1
:
cON
is very difficult and only a very faint band appears when PNA 2 is used. The slow mobility band

Molecules 2017, 22, 2010
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observed with PNA 7 and PNA 8 can again be attributed to the PNA:DNA duplex instead of the
crosslinked product due to the smearing of the DNA band. On the contrary, targeting of the ON 2
:
cON
◦
complex proved to be facilitated by its reduced stability (T
m
= 33.9 C) and both PNA 1 and PNA 2
were shown to form an ICL. The introduction of the arginine side chain seems to be deleterious for
both furan building blocks, while the introduction of the lysine modification gives conflicting effects,
improving the ability of the building block
using building block . It is clear that the mere introduction of an additional positive charge in the
backbone through building block modification does not guarantee a positive effect on duplex stability
and crosslink capacity. In the case of PNA 7 , the strand displacement experiments revealed a new
band which can be attributed to the PNA:DNA duplex (Figure 4d) with charge differing for PNA 7
and , whereas for PNA 9 only a smear was observed; in addition, a band of even lower mobility could
be observed, which can be due to intermolecular cross-linking or to duplex-duplex interactions.
F to form ICL, while inhibiting crosslink formation when
f
–8
8
3
. Materials and Methods
3
.1. General
First, 2-((5-azidomethyl)uracil-1-yl)acetic acid [52] and 3-(2-furyl)propionic acid [47] were
synthesized accordingly to previously reported procedures. The synthesis of PNA 1
and PNA 7 was reported earlier [47].
, PNA 4,
All solvents and chemical reagents were purchased from Sigma-Aldrich (Overijse, Belgium),
Acros (Geel, Belgium), Carlo Erba (Milano, Italy), TCI (Zwijndrecht, Belgium), Novabiochem (Overijse,
Belgium), Link Technology (Bellshill, UK) in the highest purity available. Non-modified DNA
sequences were purchased from Eurogentec (Seraing, Belgium).
1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance 400 (Bruker, Brussels, Belgium)
spectrometer operating at room temperature. Chemical shifts are reported in parts per million relative
to the residual solvent peak. Multiplicities are reported as singlet (s), broad singlet (br s), doublet (d),
doublet of doublets (dd), triplet (t), or multiplet (m).
LC-MS data were collected on an Agilent 1100 Agilent Technologies, Machelen, Belgium Series
◦
instrument with a Phenomenex Kinetex C18 100 Å column (150
×
4.6 mm, 5
µ
m at 35 C) connected
to an ESMSD type VL mass detector (quadrupole ion trap mass spectrometer, Agilent Technologies,
Machelen, Belgium) with a flow rate of 1.5 mL/min was used with the following solvent systems:
(
A): 0.1% HCOOH in H O and (B) MeCN. The column was flushed with 100% (A) for 2 min, then a
2
gradient from 0 to 100% (B) over 6 min was used, followed by 2 min of flushing with 100% (B).
UPLC-ESI-Q data were collected on a Waters Acquity UPLC system (Waters, Sesto San Giovanni,
◦
Italy) equipped with a Waters Acquity UPLC BEH C18 column (50
×
2.1 mm, 1.7
µ
m) at 35 C. A flow
rate of 0.25 mL/min was used with the following solvent systems: (A): 0.2% formic acid (FA) in H O
2
and (B): 0.2% FA in MeCN. The column was flushed for 0.9 min with solvent (A), then a gradient from
0
to 50% (B) in 5.7 min. PNA purification was performed on a Agilent 1100 Series instrument equipped
◦
with a Phenomenex Jupiler C18 (5
0
µm, 300 Å, 250
×
10 mm) at 40 C with a linear gradient from H O
2
.1% TFA to 50% MeCN 0.1% TFA in 30 min at a flow rate of 4.0 mL/min.
Concentrations of DNA and PNA solutions were measured with a Trinean DropSense96 UV-VIS
droplet reader (Trinean, Gent, Belgium). Purification of the crosslinked samples was performed on an
Agilent 1200b System (Agilent Technologies, Machelen, Belgium) equipped with a Waters X-bridge
◦
1
30 Å Oligonucleotide C18 column (2.5
µM, 4.6 mm
×
50 mm) at a column temperature of 50 C.
Acetonitrile and 0.1 M TEAA buffer with 5% acetonitrile were used as mobile phase and were applied
through a gradient of 0–18% MeCN in 20 min.
3
.2. Monomer Synthesis
Synthesis of N-methyl-N-methoxy-N
equipped with a CaCl valve, Fmoc-L-Arg(Pbf)-OH (4.00 g, 6.2 mmol, 1 eq) was dissolved in 20 mL of
α
-Fmoc-Nω-Pbf-L-arginine-amide (1a). In a round-bottom flask
2

Molecules 2017, 22, 2010
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0
0
dry DMF together with N,N,N ,N -Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
(
◦
HBTU, 3.52 g, 9.3 mmol, 1.5 eq). Subsequently, the solution was cooled to 0 C with an ice bath,
N,N-Diisopropylethylamine (DIPEA, 3.8 mL, 21.7 mmol, 3.5 eq) was added, and the mixture was
allowed to react for 30 min. Next, N,O-dimethylhydroxylamine hydrochloride (1.21 g, 12.4 mmol, 2 eq)
◦
was added to the solution. The reagents were allowed to react for 10 min at 0 C and 3 h 20 min at room
temperature. Next, the reaction mixture was diluted with 200 mL EtOAc and washed with saturated
KHSO (2
×
200 mL), saturated NaHCO (2
×
200 mL), and brine (200 mL). The organic phase was
4
3
dried over Na SO and the solvent was evaporated under reduced pressure. 1a was obtained as a
pale yellow foam (6.189 mmol, 99.8% yield). TLC (AcOEt) Rf: 0.29, MP ( C): 106.2–108.3 C; H-NMR
2
4
◦
◦
1
(
(
(
(
CDCl , 400 MHz) δ (ppm): 7.77 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 6.7 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.31
3
t, J = 6.6 Hz, 2H), 6.09 (3H, br s), 5.84 (d, J = 8.4 Hz, 1H), 4.80–4.65 (1H, m), 4.52–4.31 (1H, m), 4.20
1H, t, J = 6.9 Hz), 3.74 (3H, s), 3.21 (3H, s), 2.94 (2H, s), 2.60 (3H, s), 2.53 (3H, s), 2.10 (3H, s), 1.84–1.54
6H, m), 1.46 (6H, s); 13C-NMR (CDCl , 100 MHz)
δ
(ppm): 172.1 (31), 158.8, 156.8, 156.0, 143.7, 141.3,
3
1
2
38.4, 132.9, 132.4, 127.8, 127.1, 125.1, 124.6, 120.0, 117.5, 86.4, 67.2, 61.7, 50.2, 47.1, 43.2, 41.0, 32.1, 30.8,
8.6, 24.7, 19.3, 17.9, 12.5; MS (ESI, MeOH): m/z calcd. for C H N O S [M]: 691.30397, found: 692.3
3
6
45
5
7
+
+
+
[M + H] , 1383.5 [2M + H] ; HRMS (LTQ Orbitrap, MeOH) m/z found: 692.31076 [C H N O S] ;
36 46 5 7
−
1
FT-IR (ATR)
ν (cm ): 3322.4 (w), 2952.0 (m), 2936.1 (m), 2356.7 (s), 2334.6 (s), 1700.0 (s), 1652.4 (s),
1
615.5 (s), 1543.3 (s), 1448.5 (s), 1247.7 (m), 1104.6 (s), 1088.6 (s), 993.7 (s), 783.4 (s), 757.3 (s).
Synthesis of N-methyl-N-methoxy-N -Fmoc-N -Boc-L-lysine-amide (1b). In a round-bottom flask equipped
α
ω
with a CaCl valve, Fmoc-L-Lys(Boc)-OH (3.0 g, 6.4 mmol, 1 eq) was dissolved in 20 mL of dry DMF
2
◦
together with HBTU (3.64 g, 9.6 mmol, 1.5 eq). Subsequently, the solution was cooled to 0 C with an
ice bath, DIPEA (3.9 mL, 22.4 mmol, 3.5 eq) was added, and the mixture was allowed to react for 30
min. Next, N,O-dimethylhydroxylamine hydrochloride (1.25 g, 12.8 mmol, 2 eq) was added to the
◦
solution. The reagents were allowed to react for 10 min at 0 C and 3 h 20 min at room temperature.
Next, the reaction mixture was diluted with 200 mL EtOAc and washed with saturated KHSO (2
×
4
2
00 mL), saturated NaHCO (2
×
200 mL), and brine (200 mL). The organic phase was dried over
3
Na SO and the solvent was evaporated under reduced pressure. 1b was obtained as a white foam in
2
4
◦
◦
1
quantitative yield. TLC (AcOEt) Rf: 0.29, MP ( C): 50.2–50.5 C; H-NMR (CDCl , 400 MHz)
δ
(ppm):
.78 (2H, d, J = 7.6 Hz), 7.63 (2H, t, J = 7.5 Hz), 7.42 (2H, t, J = 7.5 Hz), 7.34 (2H, tdd, J = 7.4 Hz, 2.9 Hz,
.1 Hz), 5.55 (1H, d, J = 9.1 Hz), 4.81–4.72 (1H, br s), 4.65–4.53 (1H, s br), 4.39 (2H, d, J = 7.2 Hz), 4.24
1H, t, J = 7.0 Hz), 3.80 (3H, s), 3.25 (3H, s), 3.14 (2H, t, J = 6.7), 1.86–1.70 (2H, m), 1.69–1.58 (1H, m),
3
7
1
(
1
.56–1.49 (2H, m), 1.49–1.36 (10H, m); 13C-NMR (CDCl , 100 MHz)
δ
(ppm): 172.6 (22), 156.2, 156.0,
3
1
44.0, 141.3, 127.7, 127.0, 125.1, 119.9, 79.1, 67.0, 61.6, 50.7, 47.2, 40.3, 32.5, 32.1, 29.6, 28.4, 22.5; MS (ESI,
+
+
MeOH): m/z calcd. for C H N O [M]: 511.26824, found: 512.2 [M + H] , 1023.4 [2M + H] 534.2
2
8
37
3
6
+
+
+
[M + Na] , 550.2 [M + K] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 534.25746 [C H N O Na] ;
28 37 3 6
−
1
FT-IR (ATR)
ν (cm ): 3316.1 (w), 2972.7 (m), 2938.8 (m), 2863.8 (m), 2359.3 (s), 1700.0 (m), 1655.0 (s),
1513.1 (s), 1450.0 (s), 1390.9 (s), 1365.0 (s), 1246.2 (s), 1165.1 (s), 1040.4 (s), 990.9 (s), 860.9 (m), 758.7 (s),
7
38.9 (s).
Synthesis of Nα-Fmoc-Nω-Pbf-L-argininal (2a). In a three-neck flask connected with a nitrogen line
through a bubble counter, 1a (1.0 g, 1.44 mmol, 1 eq) was dissolved in 20 mL dry THF and cooled
◦
down to 0 C with an ice bath. LiAlH (60.33 mg, 1.58 mmol, 1.1 eq) was dissolved in 1.5 mL dry
4
THF and slowly added to the solution. The reaction was allowed to react for 30 min under stirring.
Next, the reaction was quenched by addition of 20 mL saturated KHSO . The organic fraction was
4
evaporated under reduced pressure, after which the resulting water phase was diluted with 200 mL
EtOAc and 200 mL saturated KHSO . The organic phase was washed with saturated KHSO (2
×
4
4
1
00 mL) and brine (2
×
100 mL). The organic phase was dried with Na SO and the solvent removed
2 4
under reduced pressure. 2a was obtained as a white foam (1.28 mmol, 88.6% yield). TLC (AcOEt) Rf:
◦
◦
1
0
.56, MP ( C): 95.5–97.7 C; H-NMR (CDCl , 400 MHz)
δ
(ppm): 9.53 (1H, s), 7.77 (2H, d, J = 7.6 Hz),
3
7.60 (2H, d, J = 8.4 Hz), 7.41 (2H, t, J = 6.5 Hz), 7.32 (2H, m), 6.56 (3H, br s), 5.67 (1H, br s), 5.26 (1H, d,

Molecules 2017, 22, 2010
10 of 20
J = 9.9 Hz), 4.39 (2H, d, J = 7.2 Hz), 4.21 (1H, t, J = 7.2 Hz), 3.77–3.63 (1H, m), 3.19 (1H, m), 2.96 (2H, s),
.57 (3H, s), 2.51 (3H, s), 2.11 (3H, s), 1.89–1.50 (6H, m), 1.47 (6H, s); 13C-NMR (CDCl , 100 MHz)
2
δ
3
(
8
[
6
ppm): 158.0, 155.9, 155.3, 144.4, 144.3, 141.2, 138.0, 134.3, 132.0, 128.1, 127.5, 125.8, 124.9, 120.6, 116.9,
6.8, 66.1, 55.4, 51.7, 47.1, 42.9, 28.8, 24.3, 19.4, 18.1, 12.7; MS (ESI, MeOH): m/z calcd. for C H N O S
34
40
4
6
+
+
M]: 632.26686, found: 633.3[M + H] , 1265.6 [2M + H] ; HRMS (LTQ-Orbitrap, MeOH) m/z found:
+
−1
33. [C H N O S] ; FT-IR (ATR) ν (cm ): 3414.2 (m), 3341.4 (m), 2967.8 (m), 2936.1 (m), 1704.6 (s),
34 41 4 6
1
8
684.8 (s), 1628.8 (m), 1576.9 (s), 1510.0 (s), 1448.5 (s), 1241.0 (m), 1086.9 (m), 990.2 (s), 869.7 (s), 852.4 (s),
18.9 (s), 758.8 (s), 738.1 (s).
Synthesis of N -Fmoc-N
α
ω
-Boc-L-lysinal (2b). In a three-neck flask connected with a nitrogen line through
◦
a bubble counter, 1b (1.44 g, 2.8 mmol, 1 eq) was dissolved in 20 mL dry THF and cooled down to 0 C
with an ice bath. LiAlH (117.7 mg, 3.1 mmol, 1.1 eq) was dissolved in 1.5 mL dry THF and slowly
4
added to the solution. The reaction was allowed to react for 32 min under stirring. Next, the reaction
was quenched by addition of 20 mL saturated KHSO . The organic fraction was evaporated under
4
reduced pressure, after which the resulting water phase was diluted with 200 mL EtOAc and 200 mL
saturated KHSO . The organic phase was washed with saturated KHSO (2
×
100 mL), and brine
4
4
(
2
×
100 mL). The organic phase was dried with Na SO and the solvent removed under reduced
2
4
1
pressure. 2b was obtained as a white foam (1.92 mmol, 68.7% yield). TLC (AcOEt) Rf: 0.56; H-NMR
CDCl , 400 MHz) (ppm): 9.61 (1H, s), 7.80 (2H, d, J = 7.5Hz), 7.63 (2H, d, J = 7.4 Hz), 7.43 (2H, t, J =
.3 Hz), 7.38–7.31 (2H, m), 4.80–4.55 (2H, m), 4.44 (2H, d, J = 7.0 Hz), 4.26 (1H, t, J = 6.9 Hz), 3.43–3.36
2H, m), 3.15 (1H, br s), 1.91–1.37 (15H, m); 13C-NMR (CDCl , 100 MHz)
(ppm): 197.0, 156.5, 143.8,
41.3, 127.7, 127.1, 125.0, 120.0, 80.4, 66.9, 57.7, 49.7, 47.1, 42.8, 28.7, 28.4, 24.00; MS (ESI, MeOH): m/z
(
7
(
1
δ
3
δ
3
+
+
+
calcd. for C H N O [M]: 452.23112, found: 453.2 [M + H] ; 475.2 [M + Na] , 491.1 [M + K] ; HRMS
26
32
2
5
+
(
LTQ-Orbitrap, MeOH) m/z found: 507.24656 [C H N O Na] .
27 36 2 6
Synthesis of Nα-Fmoc-Ψ(L-arginine-(Nω-Pbf))-glycine methyl ester (3a). In a 50 mL round-bottom flask,
2a (200 mg, 0.316 mmol, 1 eq) was dissolved in 10 mL dry MeOH, together with glycine methyl
◦
ester hydrochloride (79.4 mg, 0.632 mmol, 2 eq) and reacted for 15 min at 0 C and 15 min at room
temperature. Next, NaBH CN (41.7 mg, 0.66 mmol, 2.1 eq) and acetic acid (36.9 L, 0.66 mmol, 2.1 eq)
µ
3
were added. The pH of the solution was checked to be around 4.5. The reaction mixture was stirred
overnight at room temperature. Next, the organic solvent was removed, and the crude was taken
up with 20 mL EtOAc and washed with saturated NaHCO (2
×
20 mL) and brine (2
×
20 mL).
3
The organic phase was dried over Na SO and evaporated under reduced pressure. The product
2
4
was purified using flash chromatography (DCM:MeOH 96:4 to DCM:MeOH 94:6), which yielded 3a
◦
◦
1
as a white foam (0.184 mmol, 58.3% yield). TLC (AcOEt) Rf: 0.28, MP ( C): 69.5–71.4 C; H-NMR
(
CDCl , 400 MHz)
δ
(ppm): 7.76 (2H, d, J = 7.0 Hz), 7.60 (2H, d, J = 8.0 Hz), 7.40 (2H, t, J = 7.2 Hz), 7.31
3
(
2H, m), 6.13 (3H, s), 5.51 (1H, d, J = 7.3 Hz), 4.41 (2H, d, J = 4.4 Hz), 4.20 (1H, t, J = 6.7 Hz), 3.73 (3H, s),
3
2
.70–3.62 (1H, m), 3.46 (1H, d, J = 17.6 Hz), 3.38 (1H, d, J = 16.7 Hz), 3.33–3.11 (2H, m), 2.94 (2H, s),
.73–2.62 (2H, m), 2.60 (3H, s), 2.53 (3H, s), 2.09 (3H, s), 1.86 (1H, s), 1.70–1.47 (4H, m), 1.45 (6H, s); MS
+
+
(
ESI, MeOH): m/z calcd. for C H N O S [M]: 705.31962, found: 706.3 [M + H] , 1411.7 [2M + H] ;
37 47 5 7
+
−1
HRMS (LTQ-Orbitrap, MeOH) m/z found: 706.3258 [C H N O S] ; FT-IR (ATR)
ν (cm ): 3325.6
37
48
5
7
(
(
m), 2936.1 (m), 2917.2 (m), 1718.4 (m), 1700.8 (m), 1622.6 (m), 1560.7 (s), 1546.8 (s), 1448.8 (s), 1242.3
m), 1153.9 (s), 1089.3 (s), 1033.6 (s), 993.1 (s), 851.7 (s), 807.7 (s), 781.3 (s), 760.1 (s), 739.4 (s).
Synthesis of N
α-Fmoc-Ψ(L-lysine-(Nω-Boc))-glycine methyl ester (3b). In a 50 mL round-bottom flask,
2
b
(861 mg, 1.90 mmol, 1 eq) was dissolved in 10 mL dry MeOH, together with glycine methyl
◦
ester hydrochloride (477.8 mg, 3.81 mmol, 2 eq) and reacted for 15 min at 0 C and 15 min at room
temperature. Next, NaBH CN (251.1 mg, 4.00 mmol, 2.1 eq) and acetic acid (228 L, 4.00 mmol,
.1 eq) were added. The pH of the solution was checked to be around 4.5. The reaction mixture was
stirred overnight at room temperature. Next, the organic solvent was removed, and the crude was
taken up with 20 mL EtOAc and washed with saturated NaHCO (2 20 mL) and brine (2 20
mL). The organic phase was dried over Na SO and evaporated under reduced pressure. The crude
µ
3
2
×
×
3
2
4

Molecules 2017, 22, 2010
11 of 20
was purified using flash chromatography (DCM to DCM:MeOH 96:4), which yielded 3b as a white
1
foam (1.66 mmol, 87.2% yield). TLC (AcOEt) Rf: 0.26; H-NMR (CDCl , 400 MHz)
δ
(ppm): 7.79 (2H,
3
d, J = 7.5 Hz), 7.63 (2H, d, J = 7.1 Hz), 7.42 (2H, t, J = 7.4 Hz), 7.34 (2H, t, J = 7.4 Hz), 5.13 (1H, d,
J = 7.6 Hz), 4.61 (1H, br s), 4.43 (2H, d, J = 8.8 Hz), 4.24 (1H, t, J = 6.7 Hz), 3.78–3.63 (4H, m), 3.49
(
1
1H, d, J = 15.3 Hz), 3.40 (1H, d, J = 16.8 Hz), 3.19–3.03 (2H, m), 2.80–2.64 (2H, m), 1.86 (1H, br s),
.59–1.30 (15H, m); 13C-NMR (CDCl , 100 MHz)
δ
(ppm): 172.8, 156.4, 156.1, 144.0, 141.3, 127.7, 127.0,
3
1
25.1, 119.9, 79.1, 66.5, 53.4, 52.8, 51.8, 50.7, 47.4, 40.2, 32.6, 29.8, 28.4, 23.0; MS (ESI, MeOH): m/z calcd.
+
+
+
for C H N O [M]: 525.28389, found: 526.2 [M + H] , 548.3 [M + Na] , 1051.5 [2M + H] ; HRMS
29
39
3
6
+
(LTQ-Orbitrap, MeOH) m/z found: 526.29116 [C H N O ] .
29 40 3 6
Synthesis of Fmoc-PNA(5L-Arg(Pbf))-N -OMe (4a). In a round-bottom flask, 2-azidoacetic acid
3
(
0
63.6
µ
L, 0.85 mmol, 2 eq) and 3-hydroxy-1,2,3-benzotriazin-4 (3 h)-one (DhBtOH) (138.7 mg,
◦
.85 mmol, 2 eq) were dissolved in 5 mL of dry DMF and cooled to 0 C. Then,
1
2
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC
·
HCl) (162.9 mg, 0.85 mmol,
◦
eq) and DIPEA (148.2
µL, 0.85 mmol, 2 eq) were added and stirred for 10 min at 0 C, followed
◦
by 20 min at room temperature. Next, the reaction was cooled again to 0 C and 3a was added
◦
(
300.5 mg, 0.425 mmol, 1 eq). The reaction was stirred for 5 min at 0 C and for 5 h at room temperature.
The reaction mixture was then diluted with EtOAc (100 mL) and washed with saturated KHSO4
100 mL), saturated NaHCO (2 100 mL), and brine (100 mL). The organic phase was dried over
Na SO and the solvent was evaporated under reduced pressure. The crude was purified using flash
(2
×
×
3
2
4
chromatography (AcOEt:Hexane 8:2 to AcOEt) and yielded 4a as a white foam (0.272 mmol, 63.9%
◦
1
yield). TLC (AcOEt) Rf: 0.28, MP ( C): 85.1–85.3; H-NMR (CDCl , 400 MHz, main rotamer) δ (ppm):
3
7
.77 (2H, d, J = 7.6 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.40 (2H, t, J = 7.5 Hz), 7.31 (2H, m), 6.28 (3H, s), 5.62
(
(
(
1H, d, J = 7.6 Hz), 4.43–4.30 (2H, m), 4.22–4.13 (1H, m), 4.06 (2H, s), 3.86 (2H, s), 3.82–3.77 (1H, m), 3.74
3H, s), 3.44–3.12 (4H, m), 2.94 (2H, s), 2.60 (3H, s), 2.53 (3H, s), 2.10 (3H, s), 1.75–1.50 (4H, m), 1.45
6H, s); 13C-NMR (CDCl , 100 MHz, main rotamer)
δ
(ppm): 169.2, 168.6, 159.2, 157.0, 155.9, 143.8, 141.3,
3
1
4
38.7, 132.7, 127.8, 127.1, 125.1, 124.9, 120.0, 117.8, 114.7, 86.6, 66.8, 52.9, 52.4, 51.5, 50.4, 49.8, 47.2, 43.2,
1.0, 28.6, 25.2, 25.0, 19.3, 17.5, 12.5 (21); MS (ESI, MeOH): m/z calcd. for C H N O S [M]: 788.33158,
39
48
8
8
+
+
found: 789.2 [M + H] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 789.33886 [C H N O S] ; FT-IR
3
9
49
8
8
−
1
(
ATR)
ν
(cm ): 3449.1 (m), 3426.9 (m), 3340.3 (w), 2939.3 (w), 2104.1 (s), 1745.9 (s), 1714.5 (s), 1653.8 (s),
1
9
616.2 (s), 1547.4 (s), 1450.1 (s), 1369.3 (s), 1241.1 (m), 1217.8 (m), 1154.4 (s), 1103.5 (s), 1088.7 (s),
92.2 (s), 808.4 (m), 758.5 (s), 740.0 (s).
Synthesis of Fmoc-PNA(5L-Lys(Boc))-N -OMe (4b). In a round-bottomed flask, 2-azidoacetic acid
3
(
72.6
µ
L, 0.97 mmol, 2 eq) and DhBtOH (158.2 mg, 0.97 mmol, 2 eq) were dissolved in 5 mL of dry DMF
◦
and cooled to 0 C. Then, EDC
were added and stirred for 10 min at 0 C, followed by 20 min at room temperature. Next, the reaction
was cooled again to 0 C and 3b was added (255.0 mg, 0.48 mmol, 1 eq). The reaction was stirred for
5
·
HCl (185.9 mg, 0.97 mmol, 2 eq) and DIPEA (169.2
µL, 0.97 mmol, 2 eq)
◦
◦
◦
min at 0 C and for 3 h at room temperature. The reaction mixture was then diluted with EtOAc
(
(
100 mL) and washed with saturated KHSO (2
100 mL). The organic phase was dried over Na SO and the solvent was evaporated under reduced
×
100 mL), saturated NaHCO (2
×
100 mL), and brine
4
3
2
4
pressure. The crude was purified using flash chromatography (AcOEt:Hexane 6:4) and yielded 4b as
◦
1
a pale yellow foam (0.360 mmol, 74.2% yield). TLC (AcOEt) Rf: 0.28, MP ( C): 51.3–51.6; H-NMR
CDCl , 400 MHz, main rotamer) (ppm): 7.79 (2H, d, J = 7.4 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.43 (2H,
t, J = 7.3 Hz), 7.35 (2H, t, J = 7.3 Hz), 5.05 (1H, d, J = 5.7 Hz), 4.60 (1H, br s), 4.52–4.39 (2H, m), 4.22
(
δ
3
(
(
1H, d, J = 6.8 Hz), 4.01 (2H, s), 3.82 (2H, s), 3.78 (3H, s), 3.72–3.62 (1H, m), 3.29–3.18 (2H, m), 3.17–3.07
2H, m), 1.58–1.37 (15H, m); 1 C-NMR (CDCl , 100 MHz, main rotamer)
3
δ
(ppm): 169.1, 168.4, 156.5,
3
1
56.1, 143.8, 141.3, 127.7, 127.1, 125.0, 120.0, 79.1, 66.6, 52.8, 52.4, 50.5, 49.9, 49.2, 47.3, 40.1, 32.4, 29.8,
+
2
8.4, 22.8; MS (ESI, MeOH): m/z calcd. for C H N O [M]: 608.29585, found: 648.39 [M + K] ; HRMS
31
40
6
7
+
−1
LTQ-Orbitrap, MeOH) m/z found: 631.28507 [C H N O Na] ; FT-IR (ATR) ν (cm ): 3350.5 (w),
31 40 6 7
(

Molecules 2017, 22, 2010
12 of 20
2
941.5 (w), 2103.9 (s), 1747.8 (s), 1699.8 (m), 1662.1 (m), 1521.4 (s), 1450.4 (s), 1365.3 (s), 1247.5 (s), 1210.8
(s), 1168.4 (s), 1079.8 (s), 1022.9 (m), 759.2 (s), 739.4 (s).
Synthesis of Fmoc-PNA(5L-Arg(Pbf))-F-OMe (4c). In a round-bottomed flask, 3-(2-furyl)propionic acid
(
119.1 mg, 0.85 mmol, 2 eq) and DhBtOH (138.6 mg, 0.85 mmol, 2 eq) were dissolved in 5 mL of dry
◦
DMF and cooled to 0 C. Then, EDC
mmol, 2 eq) were added and stirred for 10 min at 0 C, followed by 20 min at room temperature. Next,
the reaction was cooled again to 0 C and 3a was added (300.0 mg, 0.43 mmol, 1 eq). The reaction was
stirred for 5 min at 0 C and for 3 h at room temperature. The reaction mixture was then diluted with
·
HCl (162.9 mg, 0.85 mmol, 2 eq) and DIPEA (148.2
µL, 0.85
◦
◦
◦
EtOAc (100 mL) and washed with saturated KHSO (2
and brine (100 mL). The organic phase was dried over Na SO and the solvent was evaporated under
×
100 mL), saturated NaHCO (2
×
100 mL),
4
3
2
4
reduced pressure. The crude was purified using flash chromatography (AcOEt:Hexane 8:2 to AcOEt)
◦
and yielded 4c as a white solid (0.347 mmol, 81.6% yield). TLC (AcOEt) Rf: 0.28, MP ( C): 75.1–75.3;
1
H-NMR (CDCl , 400 MHz, major rotamer) δ (ppm): 7.76 (2H, d, J = 7.6 Hz), 7.59 (2H, d, J = 7.5 Hz),
3
7
4
3
.39 (2H, t, J = 7.5 Hz), 7.32–7.21 (3H, m), 6.33–6.23 (4H, m), 6.02–5.96 (1H, m), 5.66 (1H, d, J = 7.4 Hz),
.36 (2H, d, J = 7.3 Hz), 4.20–4.10 (1H, m), 4.06 (2H, s), 3.73 (3H, s), 3.68–3.58 (1H, m), 3.35–3.24 (2H, m),
.21–3.09 (2H, m), 3.00–2.89 (4H, m), 2.60 (3H, s), 2.57–2.50 (5H, m, 36), 2.10 (3H, s), 1.70–1.48 (4H, m),
1
1
5
.45 (6H, s); 13C-NMR (CDCl , 100 MHz, major rotamer)
δ
(ppm): 173.9, 169.6, 159.0, 157.0, 155.9,
54.3, 143.8, 141.3, 141.1, 138.6, 132.6, 127.7, 127.1, 125.1, 124.7, 120.0, 117.6, 110.3, 105.4, 86.5, 66.7, 52.7,
2.2, 51.1, 50.4, 47.2, 43.2, 41.0, 31.4, 28.6, 25.6, 25.1, 23.5, 19.3, 18.0, 12.5; MS (ESI, MeOH): m/z calcd.
3
+
+
for C H N O S [M]: 827.35640, found: 828.2 [M + H] , 850.21 [M + Na] ; HRMS (LTQ-Orbitrap,
44
53
5
9
+
−1
MeOH) m/z found: 828.36368 [C H N O S] ; FT-IR (ATR)
ν
(cm ): 3341.4 (m), 2933.0 (m), 1718.2
m), 1623.7 (m), 1543.8 (m), 1449.6 (m), 1241.3 (m), 1212.4 (m), 1151.5 (m), 1087.6 (s), 1006.0 (s), 810.7 (s),
36.6 (s).
44
54
5
9
(
7
Synthesis of Fmoc-PNA(5L-Lys(Boc))-F-OMe (4d). In a round-bottom flask, 3-(2-furyl)propionic acid
(
135.9 mg, 0.97 mmol, 2 eq) and DhBtOH (158.2 mg, 0.97 mmol, 2 eq) were dissolved in 5 mL of dry
◦
DMF and cooled to 0 C. Then, EDC
mmol, 2 eq) were added and stirred for 10 min at 0 C, followed by 20 min at room temperature. Next,
the reaction was cooled again to 0 C and 3b was added (255.0 mg, 0.49 mmol, 1 eq). The reaction was
stirred for 5 min at 0 C and for 3 h at room temperature. The reaction mixture was then diluted with
·
HCl (185.9 mg, 0.97 mmol, 2 eq) and DIPEA (169.2
µL, 0.97
◦
◦
◦
EtOAc (100 mL) and washed with saturated KHSO (2
and brine (100 mL). The organic phase was dried over Na SO and the solvent was evaporated under
×
100 mL), saturated NaHCO (2
×
100 mL),
4
3
2
4
reduced pressure. The crude was purified using flash chromatography (AcOEt:Hexane 8:2) and yielded
◦
1
4
d
as a white solid (0.364 mmol, 75.0% yield). TLC (AcOEt) Rf: 0.39, MP ( C): 47.7–48.0; H-NMR
(
CDCl , 400 MHz, major rotamer) (ppm): 7.78 (2H, d, J = 7.6 Hz), 7.61 (2H, d, J = 8.6 Hz), 7.42 (2H,
δ
3
td, J = 7.4 Hz, 2.8 Hz), 7.33 (2H, t, J = 7.4 Hz), 7.28 (1H), 6.26 (1H, dd, J = 4.4 Hz, 2.5 Hz), 6.00 (1H,
dd, J = 12.1 Hz, 3.0 Hz), 5.22 (1H, d, J = 7.8 Hz), 4.60 (1H, br s), 4.41–4.34 (2H, m), 4.23–4.17 (1H, m),
4
.07–3.95 (2H, m), 3.81–3.60 (4H, m), 3.53–3.30 (2H, m), 3.18–3.06 (2H, m), 3.02–2.92 (2H, m), 2.54 (2H, t,
13
J = 7.6 Hz), 1.62–1.40 (15H, m); C-NMR (CDCl , 100 MHz, major rotamer)
δ
(ppm): 173.4, 169.6, 156.6,
3
1
4
56.1, 154.6, 143.9, 141.3, 141.1, 127.7, 127.1, 125.3, 120.0, 110.4, 105.4, 79.1, 66.6, 53.0, 52.6, 52.2, 49.7,
7.3, 39.8, 32.5, 31.3, 29.8, 28.4, 23.6, 22.8; MS (ESI, MeOH): m/z calcd. for C H N O [M]: 647.32067,
36
45
3
8
+
+
found: 648.3 [M + H] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 670.30989 [C H N O Na] ; FT-IR
36
45
3
8
−
1
(
(
7
ATR)
m), 1450.0 (m), 1365.1 (s), 1246.4 (s), 1210.6 (s), 1169.8 (s), 1076.9 (s), 1012.8 (s), 884.4 (s), 862.9 (m),
38.5 (s).
ν (cm ): 3312.4 (w), 2977.3 (m), 2934.5 (m), 2857.0 (s), 1748.5 (s), 1701.2 (m), 1641.5 (m), 1705.5
Synthesis of Fmoc-PNA(5L-Arg(Pbf))-T(N )-OMe
(4e).
In
a
round-bottom flask,
3
2
0
0
-((5-azidomethyl)uracil-1-yl)acetic acid (134.0 mg, 0.59 mmol, 1.1 eq) and DhBtOH (380 mg,
◦
.59 mmol, 1.1 eq) were dissolved in 2 mL of dry DMF and cooled to 0 C. Then, EDC
.59 mmol, 1.1 eq) and DIPEA (103.1
·
HCl (72.1 mg,
◦
µ
L, 0.96 mmol, 1.1 eq) were added and stirred for 10 min at 0 C,
◦
followed by 20 min at room temperature. Next, the reaction was cooled again to 0 C and 3a was

Molecules 2017, 22, 2010
13 of 20
◦
added (380 mg, 0.48 mmol, 1 eq). The reaction was stirred for 5 min at 0 C and overnight at room
temperature. The reaction mixture was then diluted with EtOAc (100 mL) and washed with saturated
KHSO (2
×
100 mL), saturated NaHCO (2
×
100 mL), and brine (100 mL). The organic phase was
4
3
dried over Na SO and the solvent was evaporated under reduced pressure. The crude was purified
2
4
using flash chromatography (AcOEt) and 4e was obtained as a yellowish solid (0.28 mmol, 74.7%
1
yield). TLC (AcOEt) Rf: 0.18, H-NMR (400 MHz, DMSO-d , major rotamer)
δ 11.58 (1H, s), 7.89 (2H,
6
d, J = 7.5 Hz), 7.67 (2H, t, J = 7.5 Hz), 7.60 (1H, d, J = 10.2 Hz), 7.41 (2H, t, J = 7.3 Hz), 7.37–7.24 (3H, m),
6
4
.66 (1H, br s), 6.40 (1H, br s), 4.83 (1H, d, J = 16.6 Hz), 4.68 (1H, d, J = 16.7 Hz), 4.40–4.20 (3H, m),
.03 (2H, s), 3.70 (2H, s), 3.61 (2H, s), 3.39–3.35 (1H, m), 3.04 (2H, s), 2.94 (2H, s), 2.48 (3H, s), 2.43 (3H,
13
s), 2.00 (3H, s), 1.55–1.28 (10H, m); C-NMR (100 MHz, DMSO-d6)
δ
169.7, 167.9, 163.9, 157.9, 156.5,
1
1
1
56.4, 151.1, 146.0, 144.5, 144.3, 144.2, 141.2, 138.8, 137.7, 134.7, 132.4, 131.9, 128.1, 127.5, 125.6, 124.8,
20.6, 117.7, 116.7, 107.8, 86.8, 65.7, 52.7, 52.2, 51.8, 50.0, 48.6, 48.6, 48.4, 47.3, 42.9, 28.7, 26.2, 19.4, 18.1,
+
2.7; MS (ESI, MeOH): m/z calcd. for C H N O S [M]: 912.35886, found: 913.7 [M + H] , 935.7
44
52 10 10
+
+
[M + Na] , 951.7 [M + K] .
Synthesis of Fmoc-PNA(5L-Lys(Boc))-T(N )-OMe
(
4f).
In
a
round-bottom flask,
3
2
0
-((5-azidomethyl)uracil-1-yl)acetic acid (215.0 mg, 0.96 mmol, 2 eq) and DhBtOH (155.8 mg,
◦
.96 mmol, 2 eq) were dissolved in 5 mL of dry DMF and cooled to 0 C. Then, EDC
·
HCl (183.1
mg, 0.96 mmol, 2 eq) and DIPEA (166.6
0
was added (251.0 mg, 0.48 mmol, 1 eq). The reaction was stirred for 5 min at 0 C and for 3 h at
room temperature. The reaction mixture was then diluted with EtOAc (100 mL) and washed with
µL, 0.96 mmol, 2 eq) were added and stirred for 10 min at
◦
◦
C, followed by 20 min at room temperature. Next, the reaction was cooled again to 0 C and 3b
◦
saturated KHSO (2
×
100 mL), saturated NaHCO (2
×
100 mL), and brine (100 mL). The organic
4
3
phase was dried over Na SO and the solvent was evaporated under reduced pressure. The crude
2
4
was purified using flash chromatography (AcOEt:Hexane 8:2 to AcOEt) and yielded 4f as a pale
◦
1
yellow foam (0.339 mmol, 71.0% yield). TLC (AcOEt) Rf: 0.41, MP ( C): 74.1–74.4; H-NMR (CDCl ,
3
400 MHz, major rotamer)
δ (ppm): 9.04 (1H, s), 7.78 (2H, d, J = 7.4 Hz), 7.62 (2H, d, J = 7.5 Hz), 7.41
(2H, t, J = 7.4 Hz), 7.33 (2H, m), 7.19 (1H, s), 5.11 (1H, d, 8.2 Hz), 4.81–4.58 (2H, m), 4.58–4.40 (3H,
m), 4.36–4.29 (1H, m), 4.13–3.94 (4H, m), 3.81 (3H, s), 3.82–3.74 (1H, m), 3.64–3.27 (2H, m), 3.21–3.06
2H, m), 1.59–1.32 (15 H, m); 1 C-NMR (CDCl , 100 MHz, major rotamer) δ (ppm): 169.5, 167.9, 162.7,
3
(
3
156.4, 150.5, 144.1, 143.8, 141.3, 127.8, 127.1, 125.1, 120.0, 109.4, 79.3, 66.5, 52.9, 52.4, 51.8, 50.4, 49.5, 47.9,
4
7
7.1, 40.0, 31.8, 29.7, 28.4, 22.9; MS (ESI, MeOH): m/z calcd. for C H N O [M]: 732.32313, found:
36 44 8 9
+
+
+
33.4 [M + H] ; 755.4 [M + Na] ; 771.4 [M + K] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 755.31235
+
−1
[
C H N O Na] ; FT-IR (ATR)
ν
(cm ): 3331.9 (m), 2974.1 (s), 2945.6 (s), 2929.8 (s), 2102.1 (s), 1672.4
m), 1517.8 (m), 1449.5 (m), 1364.9 (s), 1245.0 (s), 1212.6 (s), 1167.3 (s), 1103.5 (s), 1078.4 (s), 891.7 (m),
63.9 (m), 833.4 (s), 758.6 (s), 740.4 (s).
36
44
8
9
(
8
Synthesis of Fmoc-PNA(5L-Arg(Pbf))-N -OH (5a). Here, 4a (214.3 mg, 0.27 mmol, 1 eq) was solubilized
3
◦
in 11 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
.41 mmol, 1.5 eq) in 11 mL H O was added and left to react for 30 min at 0 C and a further 35 min at
·
8H O (128.5 mg,
2
2
◦
0
2
room temperature. The reaction was quenched with 1M HCl (0.95 mL, 0.95 mmol, 2.5 eq), the organic
fraction was evaporated under reduced pressure, and the pH of the solution was adjusted to 3.5.
◦
The product was allowed to precipitate for 2 h at 4 C. Then, 5a was collected as white solid through
◦
Buchner filtration and washed with H O (0.186 mmol, 68.6% yield). TLC (AcOEt) Rf: 0.17, MP ( C):
01.4–101.7; H-NMR (DMSO-d , 400 MHz, major rotamer)
2
1
1
δ
(ppm): 8.69–8.50 (1H, br s), 7.89 (2H, d,
6
J = 7.0 Hz), 7.66 (2H, d, J = 7.9 Hz), 7.41 (2H, t, J = 6.4 Hz), 7.33 (2H, m), 7.21 (1H, d, J = 8.3 Hz), 6.58–6.33
3H, br s), 4.29 (2H, m), 4.21 (1H, m), 3.97 (1H, d, J = 17.4 Hz), 3.87 (1H, d, J = 17.6 Hz), 3.67 (1H, m),
.61 (2H, s), 3.21 (2H, m), 3.02 (2H, s), 2.95 (2H, s), 2.49 (3H, s), 2.44 (3H, s), 2.00 (3H, s), 1.77 (2H, s), 1.40
8H, s); 13C-NMR (DMSO-d , 100 MHz, major rotamer)
(ppm): 170.7, 168.5, 158.1, 156.6, 156.4,144.3,
41.2, 137.7, 134.7, 131.9, 128.1, 127.5, 125.7, 124.8, 120.6, 116.7, 86.8, 67.5, 51.8, 50.0, 49.6, 48.1, 47.2, 42.9,
0.4, 28.7, 25.6, 19.4, 18.1, 12.7; MS (ESI, MeOH): m/z calcd. for C H N O S [M]: 774.31593, found:
(
3
(
δ
6
1
4
38
46
8
8

Molecules 2017, 22, 2010
14 of 20
+
−
7
ν
75.2 [M + H] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 773.30756 [C H N O S] ; FT-IR (ATR)
38
45
8
8
−
1
(cm ): 3339.2 (w), 2971.0 (m), 2929.8 (m), 2106.0 (s), 1704.0 (m), 1617.3 (m), 1547.0 (s), 1449.8 (s),
1
7
419.3 (m), 1241.8 (m), 1153.5 (s), 1089.7 (s), 1033.7 (s), 993.3 (s), 969.0 (s), 814.1 (m), 782.7 (s), 760.0 (s),
39.6 (s).
Synthesis of Fmoc-PNA(5L-Lys(Boc))-N -OH (5b). Here, 4b (203.2 mg, 0.33 mmol, 1 eq) was solubilized
3
◦
in 15 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
.50 mmol, 1.5 eq) in 15 mL H O was added and left to react for 30 min at 0 C and further 11 min
·
8H O (157.8 mg,
2
2
◦
0
2
at room temperature. The reaction was quenched with 1M HCl (1.2 mL, 1.2 mmol, 3.5 eq) and the
organic fraction was evaporated under reduced pressure and the pH of the solution was adjusted to
◦
3
.5. The product was allowed to precipitate overnight at 4 C. Then, 5b was collected as beige solid
through Buchner filtration and washed with H O (0.209 mmol, 63.3% yield). TLC (AcOEt) Rf: 0.19,
2
◦
1
MP ( C): 62.7–63.0; H-NMR (DMSO-d , 400 MHz, major rotamer)
δ
(ppm): 13.01–12.45 (1H, br s), 7.89
6
(2H, d, J = 7.5 Hz), 7.65 (2H, d, J = 7.7 Hz), 7.42 (2H, t, J = 7.4 Hz), 7.34 (2H, td, J = 7.4 Hz, 1.9 Hz),
7
.19 (1H, d, J = 9.1 Hz), 6.76 (1H, s), 4.37–4.25 (2H, m), 4.23–4.15 (2H, m), 4.04 (1H, d, J = 17.2 Hz), 3.99
(
1
1
1H, d, J = 38.3 Hz), 3.87 (1H, d, J = 17.3 Hz), 3.70–3.56 (1H, m), 3.28–2.97 (2H, m), 2.94–2.82 (2H, m),
.46–1.12 (15H, m) 13C-NMR (DMSO-d , 100 MHz, major rotamer)
δ (ppm): 170.7, 168.5, 156.6, 156.0,
6
44.3, 141.2, 128.1, 127.5, 125.6, 120.6, 77.8, 65.8, 52.0, 49.9, 49.8, 48.1, 47.3, 39.8, 31.7, 28.8, 25.6, 23.3; MS
+
(
ESI, MeOH): m/z calcd. for C H N O [M]: 594.28020, found: 595.4 [M + H] ; HRMS (LTQ-Orbitrap,
30
38
6
7
−
−1
MeOH) m/z found: 593.27182 [C H N O ] ; FT-IR (ATR)
ν
(cm ): 2926.7 (m), 2111.0 (s), 1714.3 (s),
699.9 (s), 1683.7 (s), 1655.1 (s), 1648.3 (s), 1527.5 (s), 1506.7 (s), 1449.1 (s), 1419.7 (s), 1366.0 (s), 1244.9
m), 1163.7 (m), 1102.7 (s), 1079.8 (s), 1020.7 (m), 949.7 (s), 799.2 (s), 759.0 (s), 737.4 (s).
30
37
6
7
1
(
Synthesis of Fmoc-PNA(5L-Arg Pbf))-F-OH (5c). 4c (286.8 mg, 0.35 mmol, 1 eq) was solubilized in
(
◦
1
5 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
·
8H O (164.0 mg, 0.52
2
2
◦
mmol, 1.5 eq) in 15 mL H O was added and left to react for 30 min at 0 C and further 46 min at
2
room temperature. The reaction was quenched with 1M HCl (1.2 mL, 1.2 mmol, 3.5 eq), the organic
fraction was evaporated under reduced pressure, and the pH of the solution was adjusted to 3.5. The
◦
product was allowed to precipitate for 2 h at 4 C. Then, 5c was collected as a white solid through
◦
Buchner filtration and washed with H O (0.223 mmol, 64.4% yield). TLC (AcOEt) Rf: 0.20, MP ( C):
08.2–108.4; H-NMR (DMSO-d , 400 MHz, major rotamer)
2
1
1
δ
(ppm): 8.81–8.59 (1H, br s), 7.88 (2H,
6
d, J = 7.5 Hz), 7.68 (2H, d, J = 6.8 Hz), 7.47 (1H, d, J = 10.6 Hz), 7.43–7.36 (2H, m), 7.33–7.24 (2H, m),
7
4
3
.14 (1H, d, J = 9.5 Hz), 6.60–6.40 (3H, br s), 6.34–6.27 (1H, m), 6.13–6.03 (1H, m), 4.35–4.24 (2H, m),
.23–4.13 (1H, m), 4.01 (1H, d, J = 16.8 Hz), 3.83 (1H, d, J = 17.8 Hz), 3.70–3.54 (3H, m), 3.33–3.21 (2H, m),
.08–2.96 (2H, m), 2.94 (2H, s), 2.83–2.71 (2H, m), 2.47–2.51 (3H, s) 2.43 (3H, s), 2.00 (3H, s), 1.80–1.73
(
2H, m), 1.39 (6H, s), 1.37–1.20 (2H, m); 13C-NMR (DMSO-d , 100 MHz, major rotamer)
δ (ppm): 172.3,
6
1
1
71.8, 157.9, 156.6, 156.4, 155.3, 144.3, 141.6, 141.2, 137.7, 134.8, 131.9, 128.1, 127.5, 125.6, 124.8, 120.6,
16.7, 110.8, 105.5, 86.7, 67.5, 52.4, 49.9, 48.2, 47.3, 42.9, 40.4, 31.1, 28.7, 25.6, 23.5, 19.4, 18.1, 12.7; MS
+
+
(ESI, MeOH): m/z calcd. for C H N O S [M]: 813.34075, found: 814.2 [M + H] , 836.1 [M + Na] ;
43 51 5 9
+
−1
ν (cm ): 3329.0
HRMS (LTQ-Orbitrap, MeOH) m/z found: 814.34803 [C H N O S] ; FT-IR (ATR)
43
52
5
9
(
(
w), 2967.8 (m), 2936.0 (m), 1715.1 (s), 1617.1 (m), 1545.7 (m), 1449.0 (m), 1406.0 (s), 1242.9 (m), 1151.9
m), 1088.5 (m), 1012.4 (s), 904.6 (s), 851.4 (s), 810.7 (s), 782.6 (s), 760.6 (s), 733.9 (s).
Synthesis of Fmoc-PNA(5L-Lys(Boc)-F-OH (5d). Here, 4d (219.6 mg, 0.34 mmol, 1 eq) was solubilized
◦
in 15 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
·
8H O (160.4 mg,
2
2
◦
0
.51 mmol, 1.5 eq) in 15 mL H O was added and left to react for 30 min at 0 C and further 46 min
2
at room temperature. The reaction was quenched with 1M HCl (1.2 mL, 1.2 mmol, 3.5 eq) and the
organic fraction was evaporated under reduced pressure and the pH of the solution was adjusted
◦
to 3.5. The product was allowed to precipitate overnight at 4 C. Then, 5d was collected as beige
solid through Buchner filtration and washed with H O (0.142 mmol, 42% yield). TLC (AcOEt) Rf:
2
◦
1
0
.19; MP ( C): 51.4–51.8; H-NMR (DMSO-d , 400 MHz, major rotamer)
δ
(ppm): 8.17–8.12 (1H, br
6
s), 7.88 (2H, d, J = 7.4 Hz), 7.65 (2H, d, J = 7.1 Hz), 7.52–7.36 (3H, m), 7.36–7.25 (2H, m), 7.21 (1H, d,

Molecules 2017, 22, 2010
15 of 20
J = 9.2 Hz), 6.75 (1H, s), 6.36–6.25 (1H, m), 6.10 (1H, d, J = 2.6 Hz), 4.28 (2H, m), 4.18 (1H, m), 4.12–3.78
2H, m), 3.74–3.62 (1H, s), 3.42–3.21 (2H, m), 2.87 (2H, s), 2.75 (2H, m), 2.58–2.46 (2H, m), 1.36 (15H,
(
13
m); C-NMR (DMSO-d , 100 MHz, major rotamer)
δ (ppm): 171.9, 171.2, 156.5, 156.0, 155.2, 144.3,
6
1
2
6
ν
41.6, 141.2, 128.0, 127.5, 125.5, 120.5, 110.8, 105.6, 77.8, 66.7, 52.6, 50.2, 48.1, 47.3, 40.4, 31.6, 30.7, 29.7,
+
8.7, 23.6, 23.3; MS (ESI, MeOH): m/z calcd. for C H N O [M]: 633.30502, found: 634.4 [M + H] ;
3
5
43
3
8
+
+
72.3 [M + K] ; HRMS (LTQ-Orbitrap, MeOH) m/z found: 656.29424 [C H N O Na] ; FT-IR (ATR)
(cm ): 3314.8 (w), 2937.2 (m), 2357.5 (s), 2344.2 (s), 1701.0 (m), 1636.7 (m), 1507.6 (m), 1449.8 (s),
365.9 (s), 1246.6 (m), 1166.2 (m), 1077.1 (s), 1014.1 (s), 859.5 (m), 759.6 (s), 736.3 (s).
35
43
3
8
−
1
1
Synthesis of Fmoc-PNA(5L-Arg(Pbf))-T(N )-OH (5e). Here, 4c (256.6 mg, 0.28 mmol, 1 eq) was solubilized
3
◦
in 10 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
.42 mmol, 1.5 eq) in 15 mL H O was added and left to react for 30 min at 0 C and then a further 46
·
8H O (133.0 mg,
2
2
◦
0
2
min at room temperature. The reaction was quenched with 1M HCl (0.84 mL, 0.84 mmol, 3.5 eq) and
the organic fraction was evaporated under reduced pressure and the pH of the solution was adjusted
◦
to 3.5. The product was allowed to precipitate for 2 h at 4 C. Then, 5c was collected as white solid
through Buchner filtration and washed with H O (0.271 mmol, 96.3% yield). TLC (EtOAc) Rf: 0.1;
2
1
H-NMR (400 MHz, DMSO-d , major rotamer)
δ 11.53 (1H, s), 7.89 (2H, d, J = 7.4 Hz), 7.67 (2H, t,
6
J = 8.3 Hz), 7.60 (1H, d, J = 6.9 Hz), 7.41 (2H, t, J = 7.4 Hz), 7.40–7.25 (3H, m), 7.06 (1H, 1 br s), 6.82
(
(
(
1H, br s), 6.54 (1H, br s), 4.52 (1H, s), 4.44–4.28 (2H, m), 4.22 (1H, d, J = 5.9 Hz), 4.03 (2H, s), 3.70–3.53
13
3H, m), 3.04 (2H, s), 2.94 (2H, s), 2.49 (3H, s), 2.42 (3H, s), 2.00 (3H, s), 1.55–1.20 (10H, m); C-NMR
100 MHz, DMSO-d6) 171.4, 168.0, 163.9, 157.9, 156.5, 156.3, 151.1, 146.4, 144.3, 141.2, 137.7, 134.7,
31.9, 130.1, 129.4, 129.0, 128.0, 127.5, 125.7, 124.8, 120.5, 116.7, 107.6, 86.7, 65.6, 51.7, 49.9, 49.4, 48.6,
δ
1
4
7.3, 42.9, 28.7, 25.6, 19.4, 18.1, 12.7; MS (ESI, MeOH): m/z calcd. for C H N O S [M]: 898.34321,
43 50 10 10
−
+
+
found: 897.7 [M
−
H] , 935.7 [M + Na] , 951.7 [M + K] ; HRMS (LTQ-Orbitrap, MeOH) m/z found:
−
8
97.3360 [C H N O S] .
43
49 10 10
Synthesis of Fmoc-PNA(5L-Lys(Boc))-T(N )-OH (5f). Here, 4f (232.8 mg, 0.32 mmol, 1 eq) was solubilized
3
◦
in 14 mL THF and cooled to 0 C with an ice bath. Next, a solution of Ba(OH)
.46 mmol, 1.5 eq) in 14 mL H O was added and left to react for 30 min at 0 C and a further 10 min
·
8H O (151.1 mg,
2
2
◦
0
2
at room temperature. The reaction was quenched with 1M HCl (1.1 mL, 1.1 mmol, 3.5 eq) and the
organic fraction was evaporated under reduced pressure and the pH of the solution was adjusted to 3.5.
◦
The product was allowed to precipitate overnight at 4 C. 5f was then collected as white solid through
◦
Buchner filtration and washed with H O (0.244 mmol, 77% yield). TLC (AcOEt) Rf: 0.21; MP ( C):
7.4–97.7; H-NMR (DMSO-d , 400M Hz, major rotamer)
2
1
9
δ
(ppm): 11.57 (1H, s), 8.10 (1H, m), 7.90 (2H,
6
d, J = 7.5 Hz), 7.69 (2H, d, J = 7.0 Hz), 7.60 (1H, s), 7.42 (2H, t, J = 7.4 Hz), 7.33 (2H, t, J = 7.4 Hz), 7.27
(
(
(
1H, d, J = 8.7 Hz), 6.81–6.72 (1H, m), 4.74 (2H, dd, J = 57.4 Hz, 16.7 Hz), 4.40–4.28 (2H, m), 4.26–4.19
1H, m), 4.13 (1H, d, J = 7.7 Hz), 4.17–3.87 (4H, m), 3.76–3.64 (1H, m), 3.63–3.50 (1H, m), 3.01–2.82
2H, m), 1.56–1.14 (15H, m); 13C-NMR (DMSO-d , 100 MHz, major rotamer)
δ
(ppm): 170.6, 167.7,
6
1
4
63.9, 156.6, 156.0, 151.1, 146.1, 144.4, 141.2, 128.1, 127.5, 125.7, 120.6, 107.7, 77.8, 65.8, 51.9, 51.5, 50.3,
8.6, 47.3, 47.2, 40.4, 32.0, 29.8, 28.7, 23.3; MS (ESI, MeOH): m/z calcd. for C H N O [M]: 718.30747,
35
42
8
9
+
+
+
found: 719.4 [M + H] ; 741.4 [M + Na] ; 757.4 [M + K] ; HRMS (LTQ-Orbitrap, MeOH) m/z found:
+
−1
7
1
(
41.29822 [C H N O Na] ; FT-IR (ATR)
ν
(cm ): 3317.9 (m), 2932.8 (m), 2100.8 (m), 1669.5 (m),
517.9 (m), 1450.4 (m), 1389.0 (s), 1365.3 (s), 1338.8 (s), 1246.0 (m), 1165.9 (m), 1104.4 (s), 1080.1 (s), 948.7
m), 864.4 (m), 760.2 (s), 740.4 (s).
35
42
8
9
3
.3. PNA Synthesis
The synthesis of the PNAs was performed using a standard Fmoc-based manual synthesis protocol
using 5a, 5b, 5c, 5d, 5e and 5f in addition to standard monomers, on a Rink amide ChemMatrix resin
loaded with Fmoc-Gly-OH as first monomer (0.2 mmol/g), using HBTU/DIPEA as an activating
mixture. Cleavage of the resin was performed using a TFA/m-cresol 9:1 solution for all the PNA

Molecules 2017, 22, 2010
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strands except for PNA 2 and PNA 3, which were cleaved with a TFA/m-cresol/thioanisole 8:1:1
solution. The cleavage step was carried out for 1 h, twice.
General protocol for click reaction: different solutions were prepared: 200 mM solution of
3
-(furan-2-yl)-N-(prop-2-yn-1-yl)propanamide in MeOH, 200 mM solution of copper sulfate in H O,
2
and 200 mM solution of sodium ascorbate in H O. Reaction was carried out with a final PNA
2
concentration (from crude PNA) of 2 mM using a molar ratio alkyne/ascorbate/Cu(II) of 2:4:2.
The mixture was then left to react for 3 h before the purification.
General protocol for DA and retro-DA: for a 5 µmol scale of resin-loaded PNA in a 500 µL
Eppendorf tube, 200
µ
L of a 0.25 M solution of N-(N-Boc-2-amminoethyl)maleimide in DMF were
◦
added and the mixture was heated at 90 C for 7 h. The resin beads were then transferred to an
empty SPE tube equipped with PE frit, and washed carefully with DMF and DCM. The resin was then
submitted to a cleavage step and the resulting crude was solubilized in 5 mL milliQ (1 mM solution,
◦
calculated on the crude PNA) and heated at 90 C for 5 h.
3+
PNA 2 (HPLC-HRMS): Rt: 15.58; MW: 3158.2976; m/z found: 1054.1077 [MH ] , 790.8324
3
4
+
5+
[
[
MH ] , 632.8675 [MH ] ; PNA 3 (HPLC-HRMS): Rt: 15.93; MW: 3186.3051; m/z found: 1063.4428
4 5
3
+
4+
5+
6+
MH ] , 797.8342 [MH ] , 638.4686 [MH ] , 532.2249 [MH ] ; PNA 5 (HPLC-HRMS): Rt: 35.70;
3
4
5
3
6
4+
+
5+
6+
MW: 3296.3502; m/z found: 1100.1247 [MH3] , 825.3451 [MH4] , 660.4780 [MH5] , 550.5658 [MH6]
;
3+
4+
PNA 6 (HPLC-HRMS): Rt: 16.03; MW: 3324.3570; m/z found: 1109.4605 [MH ] , 832.3470 [MH ] ,
3
4
5
+
6+
6
1
66.0793 [MH ] , 555.2337 [MH ] ; PNA 8 (HPLC-HRMS): Rt: 15.60; MW: 3420.3795; m/z found:
5 6
3
+
4+
5+
6+
141.4685 [MH3] , 856.3526 [MH4] , 685.2841 [MH ] , 571.2374 [MH6]
;
PNA 9 (HPLC-HRMS): Rt:
5
3
+
4+
5+
1
5.77; MW: 3448.3870; m/z found: 1150.8037 [MH ] , 863.3548 [MH ] , 690.8850 [MH ] , 575.9053
3
4
5
6
+
[MH ] .
6
3
.4. Melting Temperature Measurements
Thermal denaturation profiles were measured by monitoring the absorbance at 260 nm from 18 C
◦
◦
◦
◦
◦
◦
to 90 C and from 90 C to 18 C with a heating rate of 1 C/min and recording every 0.1 C (3 cycles).
Measurement conditions: strand concentration = 5 M in pH 7.0 PBS buffer (100 mM NaCl, 10 mM
NaH PO ). Melting curves were recorded on a Varian Cary 300 Bio instrument equipped with a six-cell
µ
2
4
thermostated cell holder, and the melting temperatures were calculated from the first derivative of the
heating/cooling curves using the Cary 300 Bio software (v 3.00 (182), Varian, Machelen, Belgium).
3
.5. Crosslinking Experiments
All experiments were conducted in a total volume of 50 µL, at 10 µM strand concentration in
pH 7.0 PBS buffer (100 mM NaCl, 10 mM phosphates) and the complexes were allowed to form by
◦
slow annealing from 90 C to room temperature. Crosslink experiments were performed by adding a
freshly prepared stock solution of N-Bromosuccinimide (NBS, 0.5 nmol/2
µL) and to start the reaction,
1
equivalent (=0.5 nmol) of NBS was added. This was repeated every 15 min until four equivalents of
◦
NBS were added. Temperature was kept constant in an Eppendorf thermomixer comfort at 25 C.
For strand displacement experiments, the DNA duplex was first allowed to anneal before the
addition of the PNA strand, and the solution was left to equilibrate for 14 h at 37 C before running
◦
the crosslink experiment at the same temperature.
3
.6. Polyacrylamide Gel-Electrophoresis
Crosslink samples were analyzed on a 20% polyacrylamide gel (acrylamide:bisacrylamide 19:1)
prepared in 1
×
Tris-Borat-EDTA (TBE) buffer containing 7 M urea. The temperature of the gel was
◦
stabilized with a Julabo F12 at 25 C. The power supply used for gel electrophoresis was a consort
EV202 and a constant voltage of 230 V was used to run the gels. Gels were stained with SYBR gold
(
Thermo Fisher Scientific, Life Technologies, Merelbeke, Belgium) and pictures were taken with an
Autochemi imaging system (UVP). 4 L of the crosslink solution (10 M) were mixed with 16
formamide and from this mixture 8 µL were loaded on the gel.
µ
µ
µL

Molecules 2017, 22, 2010
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3
.7. RP-HPLC Purification and MALDI-TOF Analysis of Crosslinked Products
Prior to MALDI analysis, each crosslinked product was purified using reverse phase HPLC
(
1
RP-HPLC). Purification was performed on an Agilent 1200b System equipped with a Waters X-bridge
◦
30 Å Oligonucleotide C18 column (2.5
µM, 4.6 mm
×
50 mm) at a column temperature of 50 C.
Acetonitrile and 0.1 M TEAA buffer with 5% acetonitrile were used as mobile phase and were applied
through a gradient of 0–18% MeCN in 20 min. The masses of the PNA-DNA crosslinked samples
were analyzed by MALDI-TOF on an ABI Voyager DE-STR system equipped with a high performance
nitrogen laser (337 nm). PNA–DNA crosslinked samples were analyzed in linear, positive mode and
samples were prepared using 2,5-dihydroxybenzoic acid (15 mg in 100
with 1% TFA) as matrix.
µL milliQ and 50 µL MeCN
4
. Conclusions
The present results show that the synthesis of multifunctional PNA molecules with a cationic
substituent on the backbone and furan moieties instead of or on the nucleobase can be achieved by
careful choice of reaction conditions, with a Diels–Alder reaction being a part of the chemical toolbox for
temporary protection of the furan moieties. The crosslink properties of furan–PNA strands containing
modified PNA building blocks featuring a lysine or arginine side chain in addition to the furan moiety
were evaluated towards single stranded DNA sequences. The results show that the crosslink event
could be enhanced and optimized by varying the furan building block and side chain modification.
Overall, it was seen that the introduction of a lysine side chain in the furan-containing building
block resulted in a higher crosslink yield compared to non-modified or arginine-modified PNA. This
observation can be attributed to pre-organization of the PNA probe and a favorable electrostatic
interaction with the negatively charged DNA-backbone. Moreover, it cannot be excluded that the
lysine side chain helps in the crosslinking process. In addition, a correlation was seen between the
used furan building block and the preferred nucleobases for crosslinking. While crosslinking towards
adenine was favored with furan building block F, building block f is preferred cytosine crosslinking.
These results indicate that the crosslinking event can be fine-tuned for each individual target sequence.
Through the current screening, it has been further possible to define a suitable doubly modified PNA
building block containing a furan moiety as well as a positively charged lysine side chain that allows
enhancing cross-link formation of a PNA strand towards a complementary ssDNA strand in a strand
invasion scenario.
On the other hand, the positioning of the furan on the uracil base (T(f) series) generates a PNA
monomer still able to give Watson–Crick type of recognition with high sequence selectivity, which
can compete with DNA:DNA formation, but is not able to cross-link with the DNA, thus leaving the
activated furan moiety in the major groove. Though not useful for ICL, this reactive species could be
profitably used for the reaction of the PNA:DNA duplex with other groups, e.g., with proteins or small
molecules for labeling.
The results obtained in this study demonstrate the possibility to improve probe reactivity by
introduction of functional modifications such as positively charged side chains. It is however clear that
considerable effort is still needed in order to optimize the reaction yield and identifying the optimal
analytical tool that will help in a fast and reliable quantification of the ICL yield. Studies focusing on
the improvement of the efficiency of the crosslink formation by variation of the geometry of the furan
monomers and their placement along the strand are currently underway. The ICL extent obtained
in the current context was superior to that previously obtained with uncharged PNAs, but still far
from the quantitative reaction which would be needed for the blockage of aberrant genes in therapy.
For analytical assays however, even a slight increase in ICL yield can be highly beneficial.
Supplementary Materials: The following are available online: solid phase Diels–Alder optimization;
HPLC-DAD-HRMS chromatogram of synthesized PNAs; full PAGE images; MALDI analysis of the XL products;
and the mechanism of ICL formation.

Molecules 2017, 22, 2010
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Acknowledgments: An.M. acknowledges the FWO and the European Union’s Horizon 2020 research and
innovation programme under the Marie Skłodowska-Curie grant agreement No. 665501. Furthermore, financial
support from FWO-Vlaanderen (FWO.OPR.2016.0047) is gratefully acknowledged.
Author Contributions: Al.M., R.C. and An.M. conceived and designed the experiments. J.E., V.C. and Al.M.
performed the experiments. Al.M., A.M. and J.E. analyzed the data. Al.M. and J.E. wrote the paper with corrections
by An.M. and R.C.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
2
3
.
.
.
Egholm, M.; Buchardt, O.; Nielsen, P.E. Peptide nucleic acids (PNA). Oligonucleotide analogs with an achiral
peptide backbone. J. Am. Chem. Soc. 1992, 114, 1895–1897. [CrossRef]
Nielsen, P.E. Peptide nucleic acids (PNA) in chemical biology and drug discovery. Chem. Biodivers. 2010, 7,
7
86–804. [CrossRef] [PubMed]
Koppelhus, U.; Shiraishi, T.; Zachar, V.; Pankratova, S.; Nielsen, P.E. Improved cellular activity of antisense
peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain. Bioconjug. Chem. 2008, 19,
1
526–1534. [CrossRef] [PubMed]
4
.
.
Good, L.; Awasthi, S.K.; Dryselius, R.; Larsson, O.; Nielsen, P.E. Bactericidal antisense effects of peptide-PNA
conjugates. Nat. Biotechnol. 2001, 19, 360–364. [CrossRef] [PubMed]
5
Boutimah-Hamoudi, F.; Leforestier, E.; Sénamaud-Beaufort, C.; Nielsen, P.E.; Giovannangeli, C.;
Saison-Behmoaras, T.E. Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1
polypurine tract (PPT) containing RNA. Nucleic Acids Res. 2007, 35, 3907–3917. [CrossRef] [PubMed]
Tonelli, R.; Purgato, S.; Camerin, C.; Fronza, R.; Bologna, F.; Alboresi, S.; Franzoni, M.; Corradini, R.;
Sforza, S.; Faccini, A.; et al. Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human
neuroblastoma cells leading to cell growth inhibition and apoptosis. Mol. Cancer Ther. 2005, 4, 779–786.
6
.
[CrossRef] [PubMed]
7.
8.
9.
Janowski, B.A.; Kaihatsu, K.; Huffman, K.E.; Schwartz, J.C.; Ram, R.; Hardy, D.; Mendelson, C.R.; Corey, D.R.
Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids. Nat. Chem. Biol. 2005, 1,
2
10–215. [CrossRef] [PubMed]
Aiba, Y.; Hamano, Y.; Kameshima, W.; Araki, Y.; Wada, T.; Accetta, A.; Sforza, S.; Corradini, R.; Marchelli, R.;
Komiyama, M. PNA-NLS conjugates as single-molecular activators of target sites in double-stranded DNA
for site-selective scission. Org. Biomol. Chem. 2013, 11, 5233–5238. [CrossRef] [PubMed]
Yamamoto, Y.; Mori, M.; Aiba, Y.; Tomita, T.; Chen, W.; Zhou, J.M.; Uehara, A.; Ren, Y.; Kitamura, Y.;
Komiyama, M. Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile
manipulation of double-stranded DNA. Nucleic Acids Res. 2007, 35, 6–13. [CrossRef] [PubMed]
1
1
1
0. Ghidini, A.; Murtola, M.; Strömberg, R. Influence of conjugation and other structural changes on the activity
of Cu2+ based PNAzymes. Org. Biomol. Chem. 2016, 14, 2768–2773. [CrossRef] [PubMed]
1. Murtola, M.; Wenska, M.; Strömberg, R. PNAzymes That Are Artificial RNA Restriction Enzymes. J. Am.
Chem. Soc. 2010, 132, 8984–8990. [CrossRef] [PubMed]
2. Sonar, M.V.; Wampole, M.E.; Jin, Y.-Y.; Chen, C.-P.; Thakur, M.L.; Wickstrom, E. Fluorescence Detection of
KRAS2 mRNA Hybridization in Lung Cancer Cells with PNA-Peptides Containing an Internal Thiazole
Orange. Bioconjug. Chem. 2014, 25, 1697–1708. [CrossRef]
1
3. Holtzer, L.; Oleinich, I.; Anzola, M.; Lindberg, E.; Sadhu, K.K.; Gonzalez-Gaitan, M.; Winssinger, N. Nucleic
acid templated chemical reaction in a live vertebrate. ACS Cent. Sci. 2016, 2, 394–400. [CrossRef] [PubMed]
4. Fabani, M.M.; Abreu-Goodger, C.; Williams, D.; Lyons, P.A.; Torres, A.G.; Smith, K.G.C.; Enright, A.J.;
Gait, M.J.; Vigorito, E. Efficient inhibition of miR-155 function in vivo by peptide nucleic acids.
Nucleic Acids Res. 2010, 38, 4466–4475. [CrossRef] [PubMed]
1
1
5. Torres, A.G.; Fabani, M.M.; Vigorito, E.; Williams, D.; Al-Obaidi, N.; Wojciechowski, F.; Hudson, R.H.E.;
Seitz, O.; Gait, M.J. Chemical structure requirements and cellular targeting of microRNA-122 by peptide
nucleic acids anti-miRs. Nucleic Acids Res. 2012, 40, 2152–2167. [CrossRef] [PubMed]
1
6. Fabbri, E.; Manicardi, A.; Tedeschi, T.; Sforza, S.; Bianchi, N.; Brognara, E.; Finotti, A.; Breveglieri, G.;
Borgatti, M.; Corradini, R.; et al. Modulation of the Biological Activity of microRNA-210 with Peptide
Nucleic Acids (PNAs). ChemMedChem 2011, 6, 2192–2202. [CrossRef] [PubMed]

Molecules 2017, 22, 2010
19 of 20
1
7. Brognara, E.; Fabbri, E.; Aimi, F.; Manicardi, A.; Bianchi, N.; Finotti, A.; Breveglieri, G.; Borgatti, M.;
Corradini, R.; Marchelli, R.; et al. Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in
breast cancer MDA-MB-231 cells. Int. J. Oncol. 2012, 41, 2119–2127. [CrossRef] [PubMed]
1
1
2
8. Kim, H.; Choi, J.; Cho, M.; Park, H. A PNA microarray platform for miRNA expression profiling using
on-chip labeling technology. Biochip J. 2012, 6, 25–33. [CrossRef]
9. Shi, H.; Yang, F.; Li, W.; Zhao, W.; Nie, K.; Dong, B.; Liu, Z. A review: Fabrications, detections and applications
of peptide nucleic acids (PNAs) microarray. Biosens. Bioelectron. 2015, 66, 481–489. [CrossRef] [PubMed]
0. Tedeschi, T.; Calabretta, A.; Bencivenni, M.; Manicardi, A.; Corrado, G.; Caramante, M.; Corradini, R.;
Rao, R.; Sforza, S.; Marchelli, R. A PNA microarray for tomato genotyping. Mol. Biosyst. 2011, 7, 1902–1907.
[
CrossRef] [PubMed]
1. Manicardi, A.; Calabretta, A.; Bencivenni, M.; Tedeschi, T.; Sforza, S.; Corradini, R.; Marchelli, R. Affinity
and selectivity of C2- and C5-substituted “chiral-box” PNA in solution and on microarrays. Chirality 2010
2 (Suppl. 1), E161–E172. [CrossRef] [PubMed]
2
2
2
2
,
2
2. Corradini, R.; Sforza, S.; Tedeschi, T.; Totsingan, F.; Manicardi, A.; Marchelli, R. Peptide nucleic acids with a
structurally biased backbone. Updated review and emerging challenges. Curr. Top. Med. Chem. 2011, 11,
1
535–1554. [CrossRef] [PubMed]
3. Sforza, S.; Tedeschi, T.; Corradini, R.; Marchelli, R. Induction of helical handedness and DNA binding
properties of peptide nucleic acids (PNAs) with two stereogenic centres. Eur. J. Org. Chem. 2007, 5879–5885.
[CrossRef]
4. Sforza, S.; Tedeschi, T.; Calabretta, A.; Corradini, R.; Camerin, C.; Tonelli, R.; Pession, A.; Marchelli, R.
A Peptide Nucleic Acid Embedding a Pseudopeptide Nuclear Localization Sequence in the Backbone
Behaves as a Peptide Mimic. Eur. J. Org. Chem. 2010, 2010, 2441–2444. [CrossRef]
2
2
2
5. De Cola, C.; Manicardi, A.; Corradini, R.; Izzo, I.; De Riccardis, F. Carboxyalkyl peptoid PNAs: Synthesis
and hybridization properties. Tetrahedron 2012, 68, 499–506. [CrossRef]
6. Bahal, R.; Sahu, B.; Rapireddy, S.; Lee, C.M.; Ly, D.H. Sequence-Unrestricted, Watson-Crick Recognition of
Double Helical B-DNA by (R)-MiniPEG-γPNAs. ChemBioChem 2012, 13, 56–60. [CrossRef] [PubMed]
7. Sacui, I.; Hsieh, W.-C.; Manna, A.; Sahu, B.; Ly, D.H. Gamma Peptide Nucleic Acids: As Orthogonal Nucleic
Acid Recognition Codes for Organizing Molecular Self-Assembly. J. Am. Chem. Soc. 2015, 150629121649000.
[CrossRef] [PubMed]
2
8. Zambaldo, C.; Daguer, J.P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for covalent inhibitors using
DNA-display of small molecule libraries functionalized with cysteine reactive moieties. Med. Chem. Commun.
2
016, 7, 1340–1351. [CrossRef]
2
3
9. Sforza, S.; Corradini, R.; Ghirardi, S.; Dossena, A.; Marchelli, R. Binding of A D-Lysine-Based Chiral PNA:
Direction Control and Mismatch Recognition. Eur. J. Org. Chem. 2000, 2905–2913. [CrossRef]
0. Dragulescu-Andrasi, A.; Rapireddy, S.; Frezza, B.M.; Gayathri, C.; Gil, R.R.; Ly, D.H. A simple
gamma-backbone modification preorganizes peptide nucleic acid into a helical structure. J. Am. Chem. Soc.
2
006, 128, 10258–10267. [CrossRef] [PubMed]
3
3
1. Verona, M.D.; Verdolino, V.; Palazzesi, F.; Corradini, R. Focus on PNA Flexibility and RNA Binding using
Molecular Dynamics and Metadynamics. Sci. Rep. 2017, 7, 42799. [CrossRef] [PubMed]
2. Englund, E.A.; Appella, D.H. Gamma-substituted peptide nucleic acids constructed from L-lysine are a
versatile scaffold for multifunctional display. Angew. Chem. Int. Ed. 2007, 46, 1414–1418. [CrossRef]
[PubMed]
3
3. Zhou, P.; Wang, M.; Du, L.; Fisher, G.W.; Waggoner, A.; Ly, D.H. Novel Binding and Efficient Cellular
Uptake of Guanidine-Based Peptide Nucleic Acids (GPNA) Novel Binding and Efficient Cellular Uptake of
Guanidine-Based Peptide Nucleic Acids (GPNA). Communication 2003, 12–14. [CrossRef]
3
4. Dragulescu-Andrasi, A.; Rapireddy, S.; He, G.; Bhattacharya, B.; Hyldig-Nielsen, J.J.; Zon, G.; Ly, D.H.
Cell-permeable peptide nucleic acid designed to bind to the 5’-untranslated region of E-cadherin transcript
induces potent and sequence-specific antisense effects. J. Am. Chem. Soc. 2006, 128, 16104–16112. [CrossRef]
[PubMed]
3
5. Manicardi, A.; Fabbri, E.; Tedeschi, T.; Sforza, S.; Bianchi, N.; Brognara, E.; Gambari, R.; Marchelli, R.;
Corradini, R. Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in
Leukaemic K562 Cells. ChemBioChem 2012, 13, 1327–1337. [CrossRef] [PubMed]

Molecules 2017, 22, 2010
20 of 20
3
3
3
3
4
6. Bahal, R.; Ali McNeer, N.; Quijano, E.; Liu, Y.; Sulkowski, P.; Turchick, A.; Lu, Y.-C.; Bhunia, D.C.; Manna, A.;
Greiner, D.L.; et al. In vivo correction of anaemia in -thalassemic mice by PNA-mediated gene editing
with nanoparticle delivery. Nat. Commun. 2016, 7, 13304. [CrossRef] [PubMed]
β
γ
7. Zhilina, Z.V.; Ziemba, A.J.; Nielsen, P.E.; Ebbinghaus, S.W. PNA-nitrogen mustard conjugates are effective
suppressors of HER-2/neu and biological tools for recognition of PNA/DNA interactions. Bioconjug. Chem.
2
006, 17, 214–222. [CrossRef] [PubMed]
8. Imoto, S.; Hori, T.; Hagihara, S.; Taniguchi, Y.; Sasaki, S.; Nagatsugi, F. Alteration of cross-linking selectivity
0
with the 2 -OMe analogue of 2-amino-6-vinylpurine and evaluation of antisense effects. Bioorg. Med.
Chem. Lett. 2010, 20, 6121–6124. [CrossRef] [PubMed]
9. Kornyushyna, O.; Stemmler, A.J.; Graybosch, D.M.; Bergenthal, I.; Burrows, C.J. Synthesis of a
metallopeptide-PNA conjugate and its oxidative cross-linking to a DNA target. Bioconjug. Chem. 2005, 16,
1
78–183. [CrossRef] [PubMed]
0. Ross, G.F.; Smith, P.M.; McGregor, A.; Turnbull, D.M.; Lightowlers, R.N. Synthesis of trifunctional
PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking.
Bioconjug. Chem. 2003, 14, 962–966. [CrossRef] [PubMed]
4
1. Liu, Y.; Rokita, S.E. Inducible alkylation of DNA by a quinone methide-peptide nucleic acid conjugate.
Biochemistry 2012, 51, 1020–1027. [CrossRef] [PubMed]
4
2. Gantchev, T.G.; Girouard, S.; Dodd, D.W.; Wojciechowski, F.; Hudson, R.H.E.; Hunting, D.J.
induced interstrand cross-links in PNA:DNA heteroduplexes. Biochemistry 2009, 48, 7032–7044. [CrossRef]
PubMed]
γ-radiation
[
4
3. Halila, S.; Velasco, T.; Clercq, P.D.; Madder, A. Fine-tuning furan toxicity: Fast and quantitative
DNA interchain cross-link formation upon selective oxidation of a furan containing oligonucleotide.
Chem. Commun. 2005, 936–938. [CrossRef] [PubMed]
4
4. De Beeck, M.O.; Madder, A. Unprecedented C-selective interstrand cross-linking through in situ oxidation
of furan-modified oligodeoxynucleotides. J. Am. Chem. Soc. 2011, 133, 796–807. [CrossRef] [PubMed]
5. Carrette, L.L.G.; Gyssels, E.; De Laet, N.; Madder, A. Furan oxidation based cross-linking: A new approach
for the study and targeting of nucleic acid and protein interactions. Chem. Commun. 2016, 52, 1539–1554.
4
[CrossRef] [PubMed]
4
6. Vannecke, W.; Ampe, C.; Van Troys, M.; Beltramo, M.; Madder, A. Cross-Linking Furan-Modified
Kisspeptin-10 to the KISS Receptor. ACS Chem. Biol. 2017, 12, 2191–2200. [CrossRef] [PubMed]
4
7. Manicardi, A.; Gyssels, E.; Corradini, R.; Madder, A. Furan-PNA: A mildly inducible irreversible interstrand
crosslinking system targeting single and double stranded DNA. Chem. Commun. 2016, 52, 6930–6933.
[
CrossRef] [PubMed]
8. Sforza, S.; Tedeschi, T.; Corradini, R.; Ciavardelli, D.; Dossena, A.; Marchelli, R. Fast, Solid-Phase Synthesis
of Chiral Peptide Nucleic Acids with a High Optical Purity by a Submonomeric Strategy. PNAS 2003
056–1063. [CrossRef]
4
,
1
4
5
5
9. Sánchez, A.; Pedroso, E.; Grandas, A. Maleimide-dimethylfuran exo adducts: Effective maleimide protection
in the synthesis of oligonucleotide conjugates. Org. Lett. 2011, 13, 4364–4367. [CrossRef] [PubMed]
0. Boutelle, R.C.; Northrop, B.H. Substituent Effects on the Reversibility of Furan–Maleimide Cycloadditions.
J. Org. Chem. 2011, 76, 7994–8002. [CrossRef] [PubMed]
1. Stevens, K.; Madder, A. Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA
inter-strand cross-linking with non-modified complements. Nucleic Acids Res. 2009, 37, 1555–1565. [CrossRef]
[PubMed]
5
2. Manicardi, A.; Accetta, A.; Tedeschi, T.; Sforza, S.; Marchelli, R.; Corradini, R. PNA bearing
5-azidomethyluracil: A novel approach for solid and solution phase modification. Artif. DNA PNA XNA
2
012, 3, 53–62. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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