Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides

Article abstract of DOI:10.1021/acs.joc.6b01829

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptide

Full text of DOI:10.1021/acs.joc.6b01829

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Article
A Switchable Protecting Strategy for Solid Phase
Synthesis of DNA and RNA Interacting Nucleopeptides
Maria Emilia Mercurio, Stefano Tomassi, Maria Gaglione, Rosita Russo, Angela Chambery, Stefania
Lama, Paola Stiuso, Sandro Cosconati, Ettore Novellino, Salvatore Di Maro, and Anna Messere
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01829 • Publication Date (Web): 28 Oct 2016
Downloaded from http://pubs.acs.org on November 1, 2016
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A Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting
Nucleopeptides
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Maria Emilia Mercurio§‡, Stefano Tomassi§‡, Maria Gaglione§, Rosita Russo§, Angela
Chambery§, Stefania Lama§§, Paola Stiuso§§, Sandro Cosconati§, Ettore Novellino†,
Salvatore Di Maro§* and Anna Messere§*
§Department of Environmental, Biological and Pharmaceutical Sciences and
Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy
†Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49,
80131 Napoli, Italy
§§Department of Biochemistry, Biophysics and General Pathology, Second University of
Naples, Via De Crecchio 7, 80127, Napoli, Italy
Abstract
Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears
nucleobases. They can recognize DNA and RNA targets modulating their biological
functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides
prevents their evaluation for biological and biomedical applications. Herein, we describe
an unprecedented approach that enables the synthesis of cationic both homo and hetero-
sequence nucleopeptides wholly on solid support with high yield and purity.
Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of
binding, thermodynamic stability and sequence specific recognition. Bio-stability assay
and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature
acceptable serum stability and ability to cross the cell membrane.
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Introduction
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MicroRNAs1 (miRNAs), recently uncovered effectors for RNA interference (RNAi) gene
regulation mechanism, result dysregulated in several human diseases, representing an
attractive target for therapeutic applications.2,3 Inhibition of overexpressed miRNAs may
be achieved by 20-23 in length anti-miRNA oligonucleotides (AMOs). Unfortunately,
their application is often hampered by limited bio-stability, low binding affinity, poor
cellular delivery and nonspecific stimulation of the immune system.4 Recently, a more
attractive strategy based on the targeting of the sole miRNA “seed region”5 by
octanucleotide congeners,6-8 enabled to partially address the aforementioned limitations.
In this respect, Peptide Nucleic Acids (PNAs) represent the most promising DNA mimics
(Figure 1).9 PNAs bind the single-stranded DNA and RNA with high affinity and
specificity following the Watson-Crick base pairing rules and form very stable duplexes
even in low ionic strength solutions. These properties, together with the high bio-stability
of PNAs allowed for broad applications, such as inhibition of target microRNA function
both in vitro and in vivo.10,11 On the other hand, problems still exist in the use of PNAs as
real therapeutics, mainly due to their poor cell membrane permeability, low aqueous
solubility and sequence dependent self-aggregation. In an effort to overcome these
limitations, several strategies were explored, including PNA conjugation,12,13
formulation14,15 and chemical modifications of the PNA backbone.16-18 As part of the
ongoing studies aiming to develop alternative structures to PNAs, a new class of
molecules known as nucleopeptides gained considerable biological relevance.19-21
Nucleopeptides combine both the polyarginine cell penetrating peptide (CPP)22 features
critical for cell uptake, and the specificity of recognition of nucleic acids (Figure 1). They
differ from conventional conjugates because the delivery and recognition elements are
integrated into a single component. Indeed, nucleopeptides hybridizing DNA and RNA
are polymers in which the repeating unit is made up by a nucleobase-substituted amino
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acid and an underivatized residue. A previous study reported the binding properties
towards Poly-deoxy-adenylic acid (polydA) and Poly-adenylic acid (polyA) of a
thymine-functionalized nucleopeptide, composed of alternating thymine-bearing L-Lys
and underivatized L-Arg residues.23
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Figure 1. Nucleic acid, PNA and Nucleopeptide backbone units comparison
Unfortunately, the tremendous potential of this new class of molecules has not been fully
disclosed yet, mainly because of the lack of a versatile synthetic strategy that can assist
for the synthesis of both homo- and hetero-nucleobase sequences. While so far the
described strategy allowed only the synthesis of thymine homo-sequences, we can
anticipate that obtaining hetero-sequences is not an easy task considering the need to
prepare in solution the four fully protected nucleo-amino acids.
In this study, we aimed at bridging this gap by describing the development of a facile and
powerful synthetic method that enables, for the first time, the synthesis of both homo-
and hetero- cationic nucleopeptides wholly on solid support with a high yield and a high
degree of purity. Here, the DNA and RNA binding properties of synthesized
nucleopeptides were also investigated by CD, UV spectroscopic techniques and
compared with the corresponding DNA and RNA sequences. A further characterization
of the RNA hybrid complex was carried out by RP-HPLC and ESI Q-TOF MS
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spectrometry. Finally, cellular uptake and human serum bio-stability properties were
investigated using laser scanning confocal microscopy and HPLC and MALDI/TOF MS
analyses, respectively.
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Results and Discussions
Rationale design and building blocks synthesis
With aim to obtain new molecules able to bind specific nucleic acid targets and to cross the cellular
barriers, we designed nucleopeptides alternating cell-penetrating components (cationic residues)
and recognition elements (nucleobases). In DNA and RNA isomorphous nucleopeptides, two
aminoacids span the length of six bonds in the sugar-phosphate backbone (Figure 1).24 Thus, a
dipeptide residue bearing a nucleobase represents the monomeric unit mimicking a single
nucleotide. In our nucleopeptides, the monomer was designed selecting L-Lys and L-Arg residues.
In particular, L-Lys was employed to introduce the suitable derivatized nucleobase, while L-Arg
was chosen due to the large number of studies indicating this amino acid as main component of the
CPP. Moreover, to validate our synthetic approach, we preferred L-amino acids for the backbone
building because of cost and availability-related reasons.
The preparation of nucleopeptides relies on two orthogonal protecting groups at α and ε positions of
L-Lys residues that can be easily switched based on the need to synthesize homo- or hetero-
sequences (Figure 2). As first protecting group, we adopted Fluorenylmethyloxycarbonyl (Fmoc),
which is the most widely used for the solid phase peptide synthesis, because it is rapidly and
quantitatively removed by mild base treatments of the peptide resin. As orthogonal protecting
group, we employed 2-nitrobenzenesulfonyl (oNBS), that was first described by Fukuyama,25 and
then extensively adopted by Kessler and Miller both for the selective alkylation of peptide on solid
support and as a Fmoc alternative in the synthesis of unalkylated peptides.26,27 oNBS is easily and
quantitatively removed by treating the peptide resin with a K2CO3/thiophenol in DMF.25 The switch
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of these two protecting groups led to the key intermediates X-L-Lys(Y)-OH, where X = Fmoc and
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Y = oNBS for the synthesis of homo-nucleopeptides, and X =
oNBS and Y = Fmoc for the
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synthesis of hetero-nucleopeptides.
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Figure 2. Retrosynthetic analysis of homo- (PATH A) and hetero-nucleopeptide monomers (PATH
B)
Fmoc-L-Lys(oNBS)-COOH was commercially available, while oNBS-L-Lys(Fmoc)-COOH28 (2)
was prepared in excellent yields by reacting the commercially available H-L-Lys(Fmoc)-COOH
with oNBS-Cl in the presence of DIPEA as base (Scheme 1a). To functionalize the ε amino groups
of L-Lys, thymine acetic acid (4) was synthesized as previously described (Scheme 1b), while bis-
Boc protecting group was selected as strategy for the exocyclic amino functions of the cytosine (7),
adenine (10) and guanine (14) nucleobases (Scheme 1c-e). The synthesis of the challenging Boc-
protected nucleobase acetic acids (7, 10, 14) was carried out in solution modifying previously
reported procedures.29 In particular, we improved the yields of the alkylation step of the
nucleobases by using Cs2CO3 instead than more harmful NaH, which is the base usually described
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for introducing the methyl bromoacetate moiety.29 In addition, the displacement of the chlorine at
the C-6 of the purine ring (12) with oxo-group (13) was performed with 1,4-diazabicyclo-
[2.2.2]octane (DABCO) in presence of potassium acetate and triethylamine in dioxane followed by
treatment with a saturated aqueous solution of NaHCO3, in order to assure the full conversion of
12-acetate intermediate into 13. Compared to the previous protocols,30 we significantly improved
the methodology obtaining higher yields of nucleobase acetic acid derivatives and reducing the use
of toxic reagents and solvents such as volatile trimethylamine and DMF. In addition, caesium
acetate that is usually described for the DABCO based reaction31 was replaced with the less
expensive potassium acetate.
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Scheme 1. Synthesis of the nucleopeptide building blocks
Synthesis of homo and hetero-nucleopeptides
As described in Scheme 2, the homo-oligomeric nucleopeptide consisting of a cationic backbone of
12 amino acid residues (Lys-Arg)6 was assembled through progressive elongation reactions
following standard solid-phase peptide synthesis protocols by using the aminomethyl polystyrene
(AM-PS) resin functionalized with a RinkTM-amide linker, Fmoc-L-Lys(oNBS)-OH and Fmoc-L-
Arg(Pbf)-OH. Although, the introduction of a single oNBS protected amino acid has been
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previously described for backbone and side chain alkylations,26 very few data are available on its
employment as standard side chain protecting group. In our study, the full oNBS-protected
sequence, was obtained with a high yield and purity, indicating that oNBS is an appropriate
protecting group featuring high stability to reiterated cycles of Fmoc-deprotection and amino acid
couplings.
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At this step, the N-terminal function was acetylated and all the ε amino groups of L-Lys residues
were simultaneously released using a saturated DMF solution of K2CO3 in presence of thiophenol.
To perform a complete oNBS deprotection, the treatment needs to be repeated three times using
K2CO3 with a degree of purity ≥ 99.995%. The purity of K2CO3 has a tremendous impact on the
efficiency of the deprotection process, indeed, when K2CO3≤ 99.995 was employed, we noticed an
incomplete or no deprotection even increasing the reaction time and the number of treatments.
Next, the thymine residues previously functionalized with a methylene carbonyl linker (4) were
introduced by one pot reaction via amide linkage in presence of HBTU/HOBt for 3 h, affording
hexathymine nucleopeptide 21 with impressive yield and purity as verified by HPLC analysis (see
S.I.) and confirmed by MALDI-TOF MS (see S.I.).
The same one pot-strategy was adopted for the synthesis of peptide 23 (Scheme 2), consisting of 12
amino acid residues (Lys-Arg)6, bearing acetyl groups instead of thymine at the Ɛ position of L-Lys.
Also, using this approach we prepared nucleopeptide 22 (Scheme 2), an analogue of 21 in which L-
Arg residues were replaced by L-Lys. Both 22 and 23 were used as control to investigate the
interactions driving the recognition process of 21 with its targets. In particular, for the synthesis of
22, L-Arg was replaced by L-Lys, because it was the only alternative natural amino acid allowing to
preserve a comparable solubility to 21. Indeed, when L-Arg was substituted with L-Ala, L-Lys(Ac),
L-Ser, the resulting nucleopeptides suffered of very low solubility in aqueous media, that hampered
the binding studies (data not showed).
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Scheme 2. Synthetic route to homo-nucleopeptides 21-23
In addition two Fluorescein isothiocyanate (FITC)-labeled nucleopeptides (26 and 27)
were prepared adopting the same synthetic strategy (Scheme 3). The compound 26, the
(FITC)-labeled derivative of 21, was used to investigate the cellular uptake properties of
our compounds. On the other hand, compound 27 was designed by replacing arginines
with glutamines and used as negative control in the cell internalization experiment.
Glutamine is not considered an amino acid known to facilitate cell internalization, but it
holds the capability to form hydrogen bonds that could confer an acceptable aqueous
solubility for cell penetration experiments.
In both cases, the FITC moiety was linked to the supported nucleopeptides using an
aminohexanoic spacer (Ahx) following a previously described procedure.31 We selected
allyloxycarbonyl (Alloc) as protecting group for the ε amine of the Ahx residue, based on
the stability of Alloc to the oNBS deprotection conditions.32
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Scheme 3. Synthetic route to FITC-homo-nucleopeptide 26 and 27
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The synthesis of hetero-sequence nucleopeptide 28 was achieved through a solid phase stepwise
strategy exploiting oNBS-L-Lys(Fmoc)-OH (2) intermediate (Scheme 4). After Fmoc removal of
the supported L-Arg, 2 was introduced following standard protocols for peptide synthesis. Next, the
Fmoc-protecting group at the ε position of the supported oNBS-L-Lys was removed and the
resulting free amine coupled with the suitable Boc-protected nucleobases (4, 7, 10, 14) by
HBTU/HOBt activation of the linker carboxy function. At this stage, the oNBS was removed from
the nucleobase functionalized dimeric Lys-Arg building block applying the developed oNBS
deprotection procedure described for the synthesis of homo-nucleopeptides. This enabled the
growth of cationic nucleopeptide bearing mixed sequence by repetitive coupling cycles with A)
Fmoc-L-Arg(Pbf)-OH, B) oNBS-L-Lys(Fmoc)-OH and C) protected nucleobase acetic acid
derivatives. Each coupling cycle was qualitatively monitored by Kaiser and 2,4,6-
Trinitrobenzenesulfonic acid (TNBS) tests,33,34 and quantitatively verified by measurement of UV
spectroscopic adsorption of the dibenzofulvene-piperidine adduct (≥ 98% yield). By using this
strategy, we have synthesized 28, containing the sequence ATAAGCTA, complementary to the
seed region of the biologically relevant miRNA 21,2 which was obtained in high yield (60% crude
overall yield) and high degree of purity (≥ 90%) as characterized by RP-HPLC (see S.I.) and
MALDI-TOF MS (see S.I.). The HPLC profile of crude 28 clearly indicates that reiterated oNBS
deprotection treatments with K2CO3/thiophenol did not produce any significant side reactions
during the synthesis of nucleopeptide 28. These results promoted oNBS as valid alternative
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protecting group in the synthesis of peptide nucleic acid analogues along with Boc-protected
nucleobases and unprotected thymine. All the final products, except 27, displayed high degree of
solubility in water without requiring the addition of organic solvents, such as ACN, MeOH and/or
DMSO. In the case of 27, the addition of 5% DMSO was required to have a complete dissolution in
water.
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Scheme 4. Synthetic route to hetero-nucleopeptide 28
Conformational analysis and hybridization properties
CD measurements were carried out for single stranded 21-23 and 28 at 6 µM concentration in 10
mM phosphate buffer (pH=7.4) at rt. As showed in Figure 3, no significant CD signals in the region
205-300 nm were observed suggesting that nucleopeptides 21-23 and 28 did not adopt any
structural pre-organization.
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Figure 3. CD spectra of 21 (green), 22 (blue), 23 (red), 28 (black)
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Next, we started evaluating the DNA and RNA binding properties of 21 by hybridization
experiments carried out with polydA and polyA single strands (ss), at 1:1 stoichiometry
ratio. CD profiles of 21/polydA and 21/polyA hybrids revealed a remarkable change both
in the intensity of the CD bands (hypochromic effect) and in their positions with respect
to DNA and RNA ss, suggesting that recognition events occurred (Figure 4).
Figure 4. a) CD spectra of polydA (red), 21/polydA sum (blue), 21(green), 21/polydA
hybrid (violet) ; (b) CD spectra of polyA (red), 21/polyA sum (blue), 21 (green),
21/polyA hybrid (violet)
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Consistent UV and CD melting temperature (Tm) measurements indicated that 21 binds
the DNA and RNA targets, giving well-defined curves with higher Tm than those
observed for natural duplexes (Table 1).
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Since homopyrimidine/homopurine sequences are known to form triplex structures, CD
spectra were also performed on the complex 21/polydA in 2:1 stoichiometric ratio. CD
profiles exhibited the presence of a negative dichroic band at 228 nm and the
disappearance of the band at 248 nm observed in the duplex spectrum (Figure 5a),
revealing the formation of triplex structure, as confirmed by CD tritation and Job’s plot
(Figure 5b and 5d). Two melting transitions at 25 and 40 °C were observed by UV
experiment (Figure 5c).
Figure 5. a) CD spectra of 21/polydA hybrid 1:1 stoichiometric ratio (green) and
21/polydA hybrid 2:1 stoichiometric ratio (blue); b) CD titration of the 21/polydA hybrid
from 0:1 to 2:1 stoichiometric ratio; c) UV melting profile of 21/polydA hybrid (2:1
stoichiometric ratio); d) Job’s plot of the 21/polydA hybrid from 0:1 to 2:1 stoichiometric
ratio
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In principle, the binding and the high thermal stability of cationic nucleopeptides toward
DNA and RNA targets could arise from non-specific electrostatic interactions between
the nucleopeptide guanidinium residues and the target phosphate backbone.18 To assess
the role of the base-pairing into the hybridization process, we performed CD binding
experiments with 23, which bears acetyl groups instead of the thymine nucleobases. CD
profiles of 23/polydA and 23/polyA turned out to be different from single stranded
polydA and polyA and sum spectra, showing changes in signal intensities, probably due
to the interactions between the cationic backbone of 23 and anionic nucleic acids. Mostly,
the CD spectra of the hybrids 23/polydA and 23/polyA were different from CD profile of
the hybrids 21/polydA and 21/polyA and from those of polydA and polyA, suggesting
that ionic-directed binding occurred but the hybridization process was mainly controlled
by base pairing rules (Figure 6). In addition, the dramatic changes observed in the CD
profiles of hybrids involving 21, those were not detected in the case of 23, seem to
indicate the involvement of both thymine and L-Arg residues in the target recognition
mode.
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Figure 6. a) CD spectra of PolydA (red); 23/polydA (blue) and 21/polydA (green); b) CD
spectra of PolyA (red); 23/polyA (blue) and 21/polyA (green)
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Further investigations performed with the control nucleopeptide 22, confirmed that the L-
Arg residues are involved but did not drive the binding process. Indeed, comparison
between 21-22/polydA and 21-22/polyA hybrid spectra (Figure 7) pointed out that L-Arg
residues may provide extra interaction sites in RNA recognition process. Also, the
resulting hybrids showed different thermal stability (Table 1). The reasons behind this
different behaviour of nucleopeptides bearing L-Arg (21) and L-Lys (22) require further
studies.
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Figure 7. (a) CD Spectra of polydA (blue), 22/polydA (green) and 23/polydA (red)
hybrids; (b) CD Spectra of polyA (blue), 22/polyA (green) and 23/polyA (red) hybrids
Finally, the base-specific interaction of 21 with the complementary target (polydA) was
confirmed by CD experiments carried out with 21/non-complementary (T)6 and
21/polydA.35 As showed in Figure 8, CD spectra revealed that the DNA conformation
was conserved when 21 was annealed with non-complementary target, providing a
further evidence of the key role of thymines into the binding process.
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Figure 8. CD spectra of 21/ polydA (red); T6 DNA/polydA (black); 21/T6 DNA (green).
Based on the recent data reporting as anti-miRNA approach the targeting of the seed
region by 8mer oligonucleotide analogues,5-8 we investigated the binding properties of
the hetero-nucleopeptide 28, consisting of the eight nucleobases sequence complementary
to miR21 seed region. First, CD spectra and Tm of 28 were measured after hybridization
with DNA and RNA (8 mer) complementary strands. Next, CD and Tm data were
collected to evaluate the binding of 28 to the target miR21. CD spectrum of 28/DNA
duplexes showed hypochromic effect, a positive band at 270 nm accompanied by a
negative band near 240 nm, typical of DNA/DNA duplexes, suggesting a conformational
similarity of hybrid and natural DNA duplexes (Figure 9). In contrast, differences in the
spectroscopic behaviour can be observed for RNA duplexes, reflecting significant
structural changes. When compared to the CD spectrum of RNA/RNA duplex, 28/RNA
hybrid exhibited a slight bathochromic shift of the positive band (from 264 to 270 nm)
and the reduction of the weak negative band at 245 nm (Figure 9). These results clearly
pointed out that 28 adopts different conformations in DNA and RNA hybrids, which
could contribute to their different thermal stability.
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Figure 9. CD Spectra of DNA/DNA (green) duplex vs 28/DNA (blue) hybrid and RNA/RNA
(black) duplex vs 28/RNA (red) hybrid.
As reported in Table 1, CD Tm measurements (for CD and UV melting profiles see S.I.) suggested
that 28 formed thermostable duplexes with its DNA and RNA partners, exhibiting higher Tm than
natural duplexes. Furthermore, the sequence specificity of 28 toward DNA and RNA targets was
assessed by evaluating its ability to form stable duplexes with DNA and RNA having a single base
mismatch (Table 1). The introduction of one mispairing, Adenine instead than Cytosine,
destabilized by -5 °C and -11 °C for DNA and RNA duplexes, respectively, confirming the essential
role of the base pairing in the duplex formation. The destabilization effect for mismatched RNA
was significantly higher than mismatched DNA duplex indicating an inherently high sequence
specificity of 28 toward RNA targets. Noteworthy, 28 showed excellent capabilities in
discriminating mismatched targets, those in turn could reduce the off-target effects due to undesired
sequence recognitions.
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Table 1. Tm values of 21, 22 and 28 with their respective nucleic acid targets.
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5
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9
HYBRID
NATURAL
DUPLEXES
Tm
Tm
(°C)
(°C)
DUPLEXES
10
11
12
13
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15
16
17
18
19
20
21
22
23
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26
27
28
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tttttt (21)/
TTTTTT/
polydA
36
40
18
38
35
30
36
25
25
35
-
polydA
ttttttt (21)/
TTTTTT/
polyA
polyA
tttttt (22)/
-
-
polydA
tttttt (22)/
-
polyA
ataagcta (28)/
3’TATTCGAT-5’
ataagcta (28)/
3’-TATTAGAT-5’
ataagcta (28)/
3’-UAUUCGAU-5’
ataagcta (28)/
3’-UAUUAGAU-5’
ataagcta (28)/
5’-ATAAGCTA-3’/
3’-TATTCGAT-5’
5’-ATAAGCTA-3’/
3’-TATTAGAT-5’
5’-AUAAGCUA-3’/
3’-UAUUCGAU-5’
5’-AUAAGCUA-3’/
3’-UAUUAGAU-5’
5’-AUAAGCUA-3’/
26
23
20
17
39
30
3’AGUUGUAGUCAGA
3’AGUUGUAGUCAGA
CUAUUCGAU-5’
CUAUUCGAU-5’
lowercase = nucleopeptide sequence; uppercase=natural nucleic acid sequence; bold = mir21 seed region sequence;
red = mismatch
The ability of 28 to form stable duplex complex with RNA was confirmed by HPLC and ESI Q-
TOF MS experiments. We were able to isolate 28/RNA hybrid by RP-HPLC as a well-defined peak
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with a different retention time with respect to 28 and ss RNA, which was successfully characterized
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by ESI Q-TOF MS (Figure 10 and S.I.).
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Figure 10. ESI-MS spectrum of 28/RNA duplex; insert: analytical RP-HPLC profiles of 28 (red), ss
RNA 8mer target (black) and 28/RNA duplex (blue); 0 to 15 % of ACN in 0.1 M TEAA aqueous
buffer in 30 min.
Furthermore, binding assay, Tm and strand invasion experiments were carried out with 28 and
miRNA target, to investigate its potential use in anti-miRNA approach using small oligomers
complementary to the miRNA seed region. Interestingly, 28 could form stable complex with
miRNA 21 and mostly, to selectively displace RNA strand from the RNA/RNA duplex. In fact, 8-
mer RNA/miR21 duplex CD spectrum changed in 28/miR21 hybrid spectrum in 60 min when
single strand 28 was added to pre-formed RNA/miR21 duplex solution (Figure 11a). Conversely, no
changes were detected when the single strand 28 was added to the DNA/DNA duplex, even after 24
h (Figure 11b). This behaviour opens new opportunities for the application of cationic
nucleopeptides in the modulation of biogenesis and maturation of RNA molecules.
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Figure 11. Strand invasion experiment of 28 in RNA/miR21 duplex; a) RNA duplex (green),
addition of 28 in RNA duplex (60 min, blue). b) Strand invasion experiment of 28 in DNA duplex;
DNA duplex (green), addition of 28 in DNA duplex (red 60 min, black 120 min, and blue 24 h).
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Bio-stability studies
Considering the potential of nucleopeptides to become real therapeutics, we evaluated the stability
of 21 when exposed to 90% human serum (Figure 12a). The inspections demonstrated that 21 starts
to be degraded after 30 min, but it can be detected up to 120 min, which indicates an higher stability
of 21 when compared the previously reported structural congeners.29 To better investigate the
proteolytic process that led to the degradation of 21, we characterized by MALDI-TOF MS
analyses the main breakdown products of nucleopeptide 21 (Figure 12b) at 30 min human serum
incubation. The results pointed out that the proteolytic process is mediated by endopeptidase
enzymes hydrolyzing the arginine peptide bonds. This observation can represent a useful
information that could drive the optimization process of these new compounds toward the synthesis
of more stable derivatives by introducing arginine surrogates or modification of the arginine amide
bonds (i.g. N-methylation).
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Figure 12. a) RP-HPLC analysis of 21 in 90% human serum at 37° C at different times; 0 to 90 %
ACN (0.1% TFA) in water (0.1 % TFA) over 30 min. The HPLC profile shows the results of one
representative experiment out of at least three independent ones. b) MALDI-TOF MS analyses of
the main breakdown products of 21 after 30 min human serum incubation
In vitro cell uptake
Although, several studies correlated the enhancement of peptide cell permeability with the
presence of arginine residues, to the best of our knowledge no investigation have been
performed with arginine-rich nucleopeptides. To assess the capability of our nucleopeptides
to penetrate into cells, LN229 glioblastoma cell line was incubated for 1 and 3h with 50 µM
concentration of 26 and processed for visualization by laser scanning confocal microscope.
Moreover, 27 bearing Gln residues instead than arginines was tested to assess the role of
arginines in the cell internalization process of nucleopeptides. As shown in Figure 13, for
compound 26, the green fluorescence was observed for both tested time intervals. In
particular, at 1h the 26-treated LN299 cells showed a strong nuclear localization indicating
that our nucleopeptide could cross nucleus membrane without the assistance of a nuclear
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localization signal peptides (NLS). Interestingly, after three hours, a diffusion of the
fluorescence signal was observed, probably triggered by interaction of 26 with polyA
targets, those are abundantly distributed also in the cell cytoplasm. As expected, no cellular
uptake was observed for 27, confirming that arginine is the essential component for cell
penetration.
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Figure 13. Laser scanning confocal microscope images of the distribuition of FITC-nucleopeptides
26 and 27 (50 µM) inside LN229 Glioblastoma cell line at 1 and 3h. The nuclei were stained with
DAPI (blue). Images were obtained by an LSM-410 Zeiss confocal microscope. Nucleopeptide 27
was employed as negative control.
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Conclusions
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9
In conclusions, the synergistic use of peptide and nucleic acid chemistry led to the development of a
powerful strategy for the facile solid-phase synthesis of homo- and hetero-cationic nucleopeptides
with high yield and purity. These new chemotypes exhibited intriguing advantageous properties in
terms of solubility in aqueous medium, thermodynamic stability and sequence specificity for RNA
and DNA, with effective mismatch discrimination especially for RNA. In order to translate these
promising results into an effective biological application, human serum stability and cellular uptake
studies provided preliminary but still solid evidences that our nucleopeptides feature acceptable
serum stability and ability to efficiently reach cellular compartments. We anticipate that our
synthetic method can be suitable for a variety of structural modifications of nucleopeptides, such as
introduction of D- and L-, α and β-amino acids, N-alkylation, cyclization and replacements of
nucleobases. This will finally allow for a fine optimization of these chemotypes to deliver their full
potential as new gene modulators.
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Experimental section
oNBS-L-Lys(Fmoc)OH (2). A solution of H-L-Lys(Fmoc)-OH (1.0 g, 2.72 mmol, 1 eq.) and
DIPEA (4.40 mL, 25.0 mmol, 9.2 eq.) in THF/H2O (100 mL, 1:1) was cooled down at 0 °C in an
ice bath then a solution of 2-nitrobenzensulfonyl chloride (0.66 g, 2.99 mmol, 1.1 eq.) in THF
(3 mL) was added dropwise and the reaction allowed to stir at rt for 1 h. The organic solvent was
evaporated under reduced pressure and the remaining solution acidified to pH 1 with HCl 0.1 N.
The aqueous phase was then extracted with EtOAc (3 x 60 mL), the combined organic layers were
dried with anhydrous Na2SO4, filtered and concentrated in vacuo. The crude mixture was purified by
silica gel chromatography (DCM/MeOH 95:5; 1 % AcOH) and fractions of interest were collected
1
to give 2 (1.46 g, 97 %) as white solid. H NMR (400 MHz, DMSO): δ 8.48 (bs, 1 H), 8.06 - 8.04
(m, 1H), 7.94 - 7.92 (m, 1H), 7.88 – 7.86 (m, 2H), 7.83 – 7.81 (m, 2H), 7.70 – 7.63 (m, 2H), 7.40 (t,
J = 7.2 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.24 (m, 1H), 4.39 - 4.21 (m, 3 H), 3.88 – 3.84 (m, 1H),
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2.94 – 2.89 (m, 2H), 1.75 – 1.58 (m, 2H), 1.34 – 1.10 (m, 4H) ppm. 13C NMR (400 MHz, DMSO):
δ 172.7, 156.1, 147.3, 144.0, 140.8, 134.0, 133.4, 132.5, 129.9, 127.6, 127.1, 125.2, 124.2, 120.1,
62.3, 55.9, 46.9, 31.5, 28.7, 22.4, 21.1 ppm. HRMS: Calculated: 554.19571 for C27H28N3O8S
[M+H+]. Found: 554.15875.
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2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic
acid
(4).
Thymine
(2 g,
15.86 mmol, 1 eq.) was dissolved in a solution of KOH (3.42 g, 61.07 mmol, 3.8 eq.) in water
(10 mL) and the reaction was allowed to stir at 50 °C for 5 minutes. After complete dissolution of
the reactants, a solution of bromoacetic acid (3.30 g, 23.80 mmol, 1.5 eq.) in water (5 mL) was
added dropwise and the mixture stirred at 50 °C overnight. The reaction was cooled down in ice
bath and pH slowly acidified with HCl 4N. The desired product 7 (2.19 g, 75 %) was obtained by
filtration as white solid and used without any further purification. Rf = 0.12 (DCM/MeOH, 75:25;
1
1 % AcOH). Rf = 0.15 (DCM/MeOH, 75:25; 1.5 % AcOH). H NMR (400 MHz, DMSO): δ 13.06
(bs), 11.33 (s, 1 H), 7.48 (s, 1H), 4.36 (s, 2H), 1.75 (s, 3H). 13C NMR (400 MHz, DMSO): δ 169.71,
164.42, 151.04, 141.85, 108.41, 48.47, 11.96 ppm. HRMS: Calculated: 185.05623 for C7H8N2O4
[M+H+]. Found: 185.05559.
Methyl 2-{4-[Bis(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)- yl}acetate (6). Compound
5 (1.70 g, 5.46 mmol, 1 eq.)33 and dry Cs2CO3 (1.96 g, 6.01 mmol, 1.1 eq.) were suspended in dry
CH3CN (15 mL) and left to stir at 0 °C for 10 minutes under Ar atmosphere. A solution of
BrCH2CO2Me (0.569 mL, 6.01 mmol, 1.1 eq.) in dry CH3CN (3 mL) was added dropwise and the
reaction was allowed to stir at room temperature for 2 hours. The reaction was quenched with H2O
(50 mL) and extracted with EtOAc (3 x 60 mL), the combined organic layers were then washed
with NaCl s.s., dried over anhydrous Na2SO4 and the solvent evaporated under reduced pressure.
The crude was purified by flash chromatography on silica gel (DCM/MeOH, 97:3; 0.5 % AcOH) to
1
give 6 (1.82 g, 87 %) as pale yellow solid. Rf = 0.45 (DCM/MeOH, 96:4; 0.5 % AcOH). H NMR
(400 MHz, CDCl3): δ 7.49 (d, J = 7.4 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 4.56 (s, 2H), 3.76 (s, 3H),
13
1.54 (s, 18H). C NMR (400 MHz, CDCl3): δ 167.81, 163.01, 154.90, 149.55, 148.12, 96.67,
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85.18, 52.92, 50.97, 27.80 ppm. HRMS: Calculated: 384.17708 for C17H26N3O7 [M+H+]. Found:
384.17624.
6
7
8
9
2-{4-[Bis(tert-butoxycarbonyl)amino]-2-oxopyrimidin-1(2H)-yl}acetic acid (7). Compound 6
(1.5 g, 3.91 mmol, 1 eq.) was dissolved in THF (16 mL) and placed on an ice bath under vigorous
stirring. Sodium hydroxide (188 mg, 4.69 mmol, 1.2 eq.) in H2O (2.35 mL, 2 N) was then added
dropwise and the reaction was allowed to stir at room temperature for 30 minutes. The mixture was
cooled to 0 °C, diluted with 70 mL of EtOAc and the pH neutralized by adding 0.78 mL of HCl aq.
1 N dropwise. The pH was finally adjusted to 2-3 by dropping HCl aq. 0.1 N and the aqueous layer
extracted with EtOAc (5 x 70 mL). The combined organic layers were then washed with a small
aliquot of saturated NaCl aq., dried with anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the desired compound 7 (1.37 mg, 95 %) as white solid. Rf = 0.35 (DCM/MeOH,
96:4; 0.5 % AcOH). 1H NMR (400 MHz, DMSO): 13.16 (s, 1H), 8.13 (d, J = 7.2 Hz, 1H), 6.83 (d,
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J = 7.2 Hz, 1H), 4.56 (s, 2H), 1.49 (s, 18H) ppm. C NMR (400 MHz, DMSO): δ 169.11, 162.07,
154.14, 151.16, 149.23, 95.44, 84.56, 50.83, 27.25 ppm. HRMS: Calculated: 370.16143 for
C16H23N3O7 [M+H+]. Found: 370.16046.
Methyl 2-{6-[Bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetate (9). Compound 8 (1.75 g,
5.22 mmol, 1 eq.)33 and dry Cs2CO3 (1.87 g, 5.74 mmol, 1.1 eq.) were suspended in dry CH3CN
(17 mL) and left to stir at 0 °C for 10 minutes under Ar atmosphere. A solution of BrCH2CO2Me
(0.543 mL, 5.74 mmol, 1.1 eq.) in dry CH3CN (3.5 mL) was added dropwise and the reaction was
allowed to stir at room temperature for 1 hour. The reaction was quenched with H2O (50 mL) and
extracted with EtOAc (3 x 50 mL), the combined organic layers were then washed with NaCl s.s.,
dried over anhydrous Na2SO4 and the solvent evaporated under reduced pressure. The crude was
purified by flash chromatography on silica gel (EtOAc/hexane, 75:25) to give 9 (1.77 g, 83 %) as
1
off-white foam. Rf = 0.30 (DCM/MeOH, 98:2). H NMR (400 MHz, CDCl3): δ 8.84 (s, 1 H), 8.13
13
(s, 1 H), 5.06 (s, 2 H), 3.80 (s, 3 H), 1.43 (s, 18 H) ppm. C NMR (400 MHz, CDCl3): δ 167.28,
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153.50, 152.43, 150.61, 150.42, 145.06, 128.44, 83.92, 53.22, 44.32, 27.88 ppm. HRMS:
Calculated: 408.18831 for C18H26N5O6 [M+H+]. Found: 408.18732.
6
7
8
9
2-{6-[Bis(tert-butoxycarbonyl)amino]-9H-purin-9-yl}acetic acid (10). Compound 9 (1.5 g,
3.68 mmol, 1 eq.) was dissolved in THF (15 mL) and placed in an ice bath under vigorous stirring.
Sodium hydroxide (177 mg, 4.42 mmol, 1.2 eq.) in H2O (2.2 mL, 2 N) was then added dropwise
and the reaction was allowed to stir at room temperature for 30 minutes. The mixture was cooled to
0 °C, diluted with 80 mL of EtOAc and the pH neutralized by adding 0.74 mL of HCl aq. 1 N
dropwise. The pH was finally adjusted to 2-3 by dropping HCl aq. 0.1 N and the aqueous layer
extracted with EtOAc (6 x 80 mL). The combined organic layers were then washed with a small
aliquot of saturated NaCl aq., dried with anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the desired compound 10 (1.38 mg, 95 %) as white and fluffy solid. Rf = 0.38
10
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1
(DCM/MeOH, 88:12; 0.5% AcOH). H NMR (400 MHz, DMSO): δ 13.43 (s, 1H), 8.84 (s, 1H),
13
8.61 (s, 1H), 5.15 (s, 2H), 1.38 (s, 18H) ppm. C NMR (400 MHz, DMSO): δ 168.86, 153.30,
151.59, 150.01, 149.04, 147.36, 127.40, 83.33, 44.52, 27.28 ppm. HRMS: Calculated: 394.17266
for C17H23N5O6 [M+H+]. Found: 394.17172.
Methyl 2-{2-[Bis(tert-butoxycarbonyl)amino]-6-chloro-9H-purin-9-yl}acetate (12). Compound
33
11 (1.9 g, 5.14 mmol, 1 eq.) and dry Cs2CO3 (1.84 g, 5.65 mmol, 1.1 eq.) were suspended in dry
CH3CN (20 mL) and left to stir at 0 °C for 10 minutes under Ar atmosphere. A solution of
BrCH2CO2Me (0.535 mL, 5.65 mmol, 1.1 eq.) in dry CH3CN (4 mL) was added dropwise and the
reaction was allowed to stir at room temperature for 1 hour. The reaction was quenched with H2O
(60 mL) and extracted with EtOAc (3 x 60 mL), the combined organic layers were then washed
with NaCl s.s., dried over anhydrous Na2SO4 and the volatiles evaporated to dryness in vacuo. The
crude was purified by flash chromatography on silica gel (DCM/MeOH, 100:1; 0.5% AcOH) to
1
give 12 (2.04 g, 85 %) as white solid. Rf = 0.25 (DCM/MeOH, 100:1; 0.5% AcOH). H NMR
(400 MHz, CDCl3): δ 8.21 (s, 1H), 5.04 (s, 2H), 3.79 (s, 3H), 1.41 (s, 18H) ppm. 13C NMR
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(400 MHz, CDCl3): δ 166.87, 152.91, 152.26, 151.44, 150.56, 146.46, 129.68, 83.85, 53.26, 44.57,
5
6
27.92 ppm. HRMS: Calculated: 442.14934 for C18H24ClN5O6 [M+H+]. Found: 442.14832.
7
8
9
Methyl
2-{2-[Bis(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-
9(6H)-yl}acetate
(13).
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Compound 12 (1.35 g, 3.06 mmol, 1 eq.) and CH3COOK (901 mg, 9.18 mmol, 3 eq.) were
suspended in 1,4-dioxane (40 mL) and Et3N (1,28 mL, 9.18 mmol, 3 eq.) was added. The so
obtained suspension was sonicated for 30 minutes at room temperature then DABCO (34 mg,
0.306 mmol, 0.1 eq) was added portionwise while vigorously stirring and the mixture allowed to
stir at room temperature overnight. Saturated NaHCO3 aq. (30 mL) was added and the mixture
vigorously stirred for other 30 minutes. The suspension was poured in a separatory funnel and
washed with EtOAc (50 mL), the organic phase was back-extracted with further saturated NaHCO3
aq. aliquots (2 x 15 mL). The combined aqueous layers were then placed in ice bath, neutralized
dropwise with HCl 1N and then adjusted to pH 2-3 by dropping HCl 0.1 N. The solution was
extracted with EtOAc (4 x 100 mL) and the combined organic phases dried with anhydrous Na2SO4,
filtered and concentrated in vacuo to give a white solid which was triturated in a mixture of
hexane/ethylic ether 8:2 (20 mL). The suspension was filtered and the desired product 13 was
1
obtained as white solid (1.24 g, 93 %). Rf = 0.33 (hexane/EtOAc, 1:9). H NMR (400 MHz,
13
CDCl3): δ 11.39 (s, 1H), 7.72 (s, 1H), 4.79 (s, 2H), 3.75 (s, 3H), 1.53 (s, 18H) ppm. C NMR
(400 MHz, CDCl3): δ 167.17, 155.96, 149.10, 148.56, 146.49, 139.53, 120.96, 86.28, 53.01, 44.50,
27.70 ppm. HRMS: Calculated: 424.18332 for C18H26N5O7 [M+H+]. Found: 424.18222.
2-{2-[Bis(tert-butoxycarbonyl)amino]-6-oxo-1H-purin-9(6H)- yl}acetic acid (14). Compound 13
(1.1 g, 2.60 mmol, 1 eq.) was dissolved in THF (12 mL) and placed in an ice bath under a vigorous
stirring. Sodium hydroxide (229 mg, 5.72 mmol, 2.2 eq.) in H2O (2.9 mL, 2 N) was then added
dropwise and the reaction was allowed to stir at room temperature for 1 hour. The mixture was
cooled to 0 °C, diluted with 80 mL of EtOAc and the pH neutralized by adding 3.2 mL of HCl aq.
1 N dropwise. The pH was finally adjusted to 2-3 by dropping HCl aq. 0.1 N and the aqueous layer
extracted with EtOAc (6 x 80 mL). The combined organic layers were then washed with a small
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aliquot of saturated NaCl aq., dried with anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the desired compound 14 (926 mg, 87 %) as white solid. Rf = 0.40 (DCM/MeOH,
9:1, 1.5 % AcOH). 1H NMR (400 MHz, DMSO): δ 12.83 (s, 1H), 8.08 (s, 1H), 4.92 (s, 2H), 1.37 (s,
18H) ppm. 13C NMR (400 MHz, DMSO): δ 168.90, 156.96, 148.75, 148.20, 145.15, 141.91,
122.23, 83.79, 44.76, 27.36 ppm. HRMS: Calculated: 410.2 for C17H24N5O7 [M+H+]. Found:
410.166782; Calculated: 410.16757.
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Nucleopeptides 21-23. A Rink Amide AM-PS resin (50 mg, 0.48 mmol/g) was swelled in a
mixture of DCM/DMF 1:1 over 30 min and washed with DCM and DMF three times. Fmoc
protecting group was removed treating with 20% (v/v) piperidine solution in DMF (1 x 5 min; 1 x
25 min). The following amide couplings were carried out using 4 equivalents (according to the
initial loading of the resin) of the Fmoc-protected L-amino acids. To a pre-stirred solution of Fmoc-
L-Arg(Pbf)-OH (for compounds 21 and 23) (62.3 mg, 4 eq.) or Fmoc-L-Lys(Boc)-OH (44.98 mg, 4
eq.) (for peptide 22) (45.0 mg, 4 eq.) with HBTU (36.4 mg, 4 eq.) and HOBT·H2O (14.7 mg, 4 eq.)
in DMF (1.0 mL), DIPEA (33.4 µL, 8 eq.) was added and the solution poured to the pre-swollen
resin. The mixture was gently shaken for 2 h at rt and then washed with DMF (3 x 1 min) and DCM
(3 x 1 min). Subsequently, to cap the remaining free amines the resin was treated with a solution of
Ac2O (4.5 µL, 2 eq.) and DIPEA (8.4 µL, 2 eq.) in DMF (1 mL) and then shaken for 10 min.
Elongation of the linear peptide sequence was obtained by iterative cycles of the aforementioned
Fmoc deprotections and coupling reactions with Fmoc-L-Lys(oNBS)-OH and Fmoc-L-Arg(Pbf)-
OH. Completion of these steps were qualitatively monitored by Kaiser ninhydrine and TNBS test
and quantitatively ascertained by Fmoc UV spectroscopic measurements. After the last Fmoc
removal, N-terminal was acetylated by treatment with a mixture of Ac2O (4.5 µL, 2 eq.) and DIPEA
(8.4 µL, 2 eq.) in DMF (1 mL) over 30 min. One-pot o-Nitrobenzenesulfonyl- (oNBS) removal on
lysine side chains was obtained adding 1.5 mL of a centrifuged 5% (v/v) solution of thiophenol in
DMF, previously saturated with 2 eq. of K2CO3 (relative to thiophenol) to the resin (3 x 10 min).
Nucleopeptide 21-22. Final coupling was performed using 3 equivalents of nucleobase derivative,
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HBTU and HOBt, and 6 equivalents of DIPEA relative to each lysine ε amino group. Thymin-1-yl-
acetic acid (compound 5, 79.6 mg, 3 eq.), HBTU (163.7 mg, 3 eq.) and HOBt (66.2 mg, 3 eq.) were
dissolved in DMF (1.5 mL) then DIPEA (150.5 µL, 6 eq.) was added and the resulting solution
poured to the resin. The mixture was shaken at rt for 8 h. The unreacted free amines were capped
with a solution of Ac2O (27.2 µL, 2 eq. relative to each free amine) and DIPEA (50.2 µL, 2 eq.) in
DMF (1 mL) over 10 min.
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Nucleopeptide 23: acetylation of was achieved using 2 equivalents of Ac2O and DIPEA relative to
each lysine ε amino group. A pre-stirred solution of Ac2O (27.2 µL, 2 eq.) and DIPEA (50.2 µL, 2
eq.) in DMF (1 mL) was added and the resin shaked for 30 min.
Nucleopeptides 26-27: A Rink Amide AM-PS resin (50 mg, 0.48 mmol/g) was swelled in a
mixture of DCM/DMF 1:1 over 30 min and washed with DCM and DMF three times. Fmoc
protecting group was removed treating with 20% (v/v) piperidine solution in DMF (1 x 5 min; 1 x
25 min). The following amide couplings were carried out using 4 equivalents (according to the
initial loading of the resin) of the Fmoc-protected L-amino acids. To a pre-stirred solution of Fmoc-
L-Arg(Pbf)-OH (for compound 26) (62.3 mg, 4 eq.) or Fmoc-L-Gln(Trt)-OH (58.6 mg, 4 eq.) (for
compound 27) with HBTU (36.4 mg, 4 eq.) and HOBt*H2O (14.7 mg, 4 eq.) in DMF (1.0 mL),
DIPEA (33.4 µL, 8 eq.) was added and the solution poured to the pre-swollen resin. The mixture
was gently shaken for 2 h at rt and then washed with DMF (3 x 1 min) and DCM (3 x 1 min).
Subsequently, to cap the remaining free amines the resin was treated with a solution of Ac2O
(4.5 µL, 2 eq.) and DIPEA (8.4 µL, 2 eq.) in DMF (1 mL) and then shaken for 10 min. Elongation
of the linear peptide sequence was obtained by iterative cycles of the aforementioned Fmoc
deprotections and coupling reactions with Fmoc-L-Lys(oNBS)-OH and Fmoc-L-Arg(Pbf)-OH.
Completion of these steps were qualitatively monitored by Kaiser ninhydrine and TNBS test and
quantitatively ascertained by Fmoc UV spectroscopic measurements. After Fmoc removal, the N-
terminal amino group was coupled with Aloc-Ahx-OH (mg, 4 eq.) in presence of HBTU (36.4 mg,
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4 eq.) and HOBt*H2O (14.7 mg, 4 eq.) in DMF (1.0 mL) and DIPEA (33.4 µL, 8 eq.) as base. The
mixture was gently shaken for 2 h at rt and then washed with DMF (3 x 1 min) and DCM (3 x 1
min). Subsequently, to cap the remaining free amines the resin was treated with a solution of Ac2O
(4.5 µL, 2 eq.) and DIPEA (8.4 µL, 2 eq.) in DMF (1 mL) and then shaken for 10 min. One-pot o-
Nitrobenzenesulfonyl- (oNBS) removal on lysine side chains was obtained adding 1.5 mL of a
centrifuged 5% (v/v) solution of thiophenol in DMF, previously saturated with 2 eq. of K2CO3
(relative to thiophenol) to the resin (3 x 10 min). The coupling of thimine residues was performed
using 3 equivalents of nucleobase derivative, HBTU and HOBt, and 6 equivalents of DIPEA
relative to each lysine ε amino group. Thymin-1-yl-acetic acid (compound 5, 79.6 mg, 3 eq.),
HBTU (163.7 mg, 3 eq.) and HOBt (66.2 mg, 3 eq.) were dissolved in DMF (1.5 mL) then DIPEA
(150.5 µL, 6 eq.) was added and the resulting solution poured to the resin. The mixture was shaken
at rt for 3 h. The unreacted free amines were capped with a solution of Ac2O (27.2 µL, 2 eq. relative
to each free amine) and DIPEA (50.2 µL, 2 eq.) in DMF (1 mL) over 10 min. At this step the
allyloxy protecting group of Ahx was removed by treating the resin with
tetrakis(triphenyphosphine)-palladium(0) (12.0 mg, 0.1 equiv) and N,N’-dimethylbarbituric acid
(156.1 mg, 10 equiv) in DCM/DMF (3:1, 3 mL) for 1 h at rt under nitrogen atmosphere, and the
reaction was repeated again. After the resin was washed with DMF (3 × 1 min) and DCM (3 x 1
min), fluorescein-5- isothiocyanate (FITC) (233.6 mg, 6 equiv) and DIEA (209 µL, 12 equiv) in
DMF (2 ml) were added and the reaction mixture was shaken 12 h in the dark. The unreacted free
amines were capped with a solution of Ac2O (27.2 µL, 2 eq. relative to each free amine) and DIPEA
(50.2 µL, 2 eq.) in DMF (1 mL) over 10 min.
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Cleavage, Purification and Characterization of 21-27. The resulting resin-bound nucleopeptides
21-27 were washed exhaustively with DMF (5 x 1 min), DCM (5 x 1 min), Et2O (2 x 1 min) and
then dried. Finally, 21-27 were cleaved from the resin by treatment with TFA/TIS/H2O (95:2.5:2.5,
32 mL) for 3 h at rt (in the dark for 26 and 27). The resin was filtered and the crude nucleopeptides
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