Synthesis and tau RNA binding evaluation of ametantrone-containing ligands

Article abstract of DOI:10.1021/jo502661j

We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules.

Full text of DOI:10.1021/jo502661j

Article
pubs.acs.org/joc
Synthesis and Tau RNA Binding Evaluation of Ametantrone-
Containing Ligands
Gerard Artigas and Vicente Marchan*
́
̀ ̀
Departament de Química Organica and IBUB, Universitat de Barcelona, Martí i Franques 1-11, E-08028 Barcelona, Spain
S
* Supporting Information
ABSTRACT: We describe the synthesis and characterization of
ametantrone-containing RNA ligands based on the derivatization of
this intercalator with two neamine moieties (Amt-Nea,Nea) or with one
azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as
its combination with guanidinoneamine (Amt-NeaG4). Biophysical
studies revealed that guanidinylation of the parent ligand (Amt-Nea)
had a positive effect on the binding of the resulting compound for Tau
pre-mRNA target as well as on the stabilization upon complexation of
some of the mutated RNA sequences associated with the development
of tauopathies. Further studies by NMR revealed the existence of a
preferred binding site in the stem−loop structure, in which ametantrone
intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of
Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its
accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4.
Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double
derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity
for disease-causing RNA molecules.
at the exon 10-5′ intron junction of Tau pre-mRNA, which is
involved in the regulation of the alternative splicing of tau
protein.8 According to NMR structural data,7b this compound
intercalates between the two G:C pairs flanking the bulged
adenine in the stem−loop structure of Tau. In addition, the two
side arms of the anthraquinone moiety play an important role
in the recognition of this characteristic binding site.
Mitoxantrone also binds HIV-1 transactivation response
element (TAR) RNA with nM affinity, although nonspecific
binding was also found in competition assays with tRNA.9
Taking into account our previous results on Tau RNA
ligands based on the combination of acridines with the small
aminoglycoside neamine,10 we recently focused on a derivative
of mitoxantrone lacking the phenolic hydroxyl groups. This
heteroaromatic compound, known as ametantrone (Amt in
Scheme 1), was selected because of its proven reduced
cytotoxicity in eukaryotic cells compared with mitoxantrone.11
Dynamic combinatorial chemistry together with biophysical
experiments demonstrated that the combination of ametan-
trone with neamine led to ligands with high binding affinity
(nM scale for Tau RNA), particularly when both moieties were
conjugated through a short spacer (Amt-Nea in Scheme 1).12
NMR spectroscopic studies of the complex between this
compound and Tau RNA revealed the existence of a preferred
binding site in which ametantrone intercalates in the bulged
INTRODUCTION
■
The search for drugs based on small molecules to control RNA
function has been postulated in recent years as a promising
strategy to treat human diseases.1 This is supported by the great
biological relevance of both coding and noncoding RNA
molecules2 and by the fact that only a small number of the
proteins linked to genetic diseases can be targetable with drug-
like compounds.3 In addition, the folding of local secondary
structures of therapeutically relevant RNA targets into complex
three-dimensional architectures offers the possibility, like in
proteins, of using ligands for targeting specific binding sites in a
selective manner.4 However, despite the great potential of RNA
as a drug target, finding selective ligands for a specific sequence
and with drug-like properties still remains a challenge. The high
conformational dynamics of this biomolecule together with our
limited understanding of ligand-RNA recognition principles
complicate the de novo design of selective RNA ligands.5 Within
this scenario, the modification of known building blocks by
using fragment-based approaches6 offers an alternative for
developing compounds with improved RNA-binding properties
(e.g., affinity, sequence specificity, and cellular permeability) as
well as for gaining knowledge on RNA recognition mecha-
nisms.
Among the large number of RNA ligands described in the
literature so far, the anticancer drug mitoxantrone (Mtx in
Scheme 1) has received attention recently. This classical nucleic
acid intercalator was identified in a high-throughput screening
as a promising ligand7 of the RNA secondary structure located
Received: November 21, 2014
© XXXX American Chemical Society
A
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Scheme 1. (A) Structure of Mitoxantrone and Ametantrone and (B) Schematic Representation of Ligands Based on the
Derivatization of Ametantrone through One or the Two Side Chains of the Anthraquinone Ring
region of the stem−loop structure and the neamine moiety and
the Amt side chains interact with the major groove of the RNA.
Importantly, Amt-Nea was able to increase the thermodynamic
stability of Tau RNA mutated sequences involved in the onset
of frontotemporal dementia with parkinsonism linked to
chromosome 17 (FTDP-17)8 to a similar or even higher levels
than that of the wild-type sequence. The stabilization of Tau
RNA mutated sequences upon ligand complexation could be
used to modulate Tau pre-mRNA splicing and, for instance, to
counteract the negative effects of dementia-causing muta-
tions.7,8 Moreover, the presence of the characteristic bulged
adenine nucleotide flanked by G:C pairs in other therapeuti-
cally relevant RNA targets, such as the Rev response element
(RRE) RNA, opens the door to the development of selective
ligands for this RNA motif based on Mtx/Amt derivatives.13
On the basis of all these precedents, we wondered how
derivatization of the free side arm of the anthraquinone moiety
in Amt-Nea (Scheme 1) could affect its Tau RNA-binding
properties, particularly binding affinity and stabilizing ability
against FTDP-17-causing mutations, as well as to assess if the
preferred binding site at the bulged region is maintained. Here
we report the synthesis and Tau RNA binding studies of ligands
(Scheme 1) based on the derivatization of ametantrone with
two neamine moieties (Amt-Nea,Nea) or with one neamine
and one azaquinolone (Amt-Nea,Azq). The azaquinolone
moiety was selected because of the potential of this
heteroaromatic compound to recognize the bulged adenine in
the stem−loop via complementary hydrogen-bonding pairing.14
The guanidinylated analogue of Amt-Nea (named Amt-NeaG4)
was also included in this study, since guanidinylation has
proved to be a promising approach to improve RNA-binding
properties of aminoglycoside-containing compounds as well as
their cellular uptake.10b,15
Scheme 2. Synthesis of Boc-Protected Ametantrone
Derivatives 2 and 3
protected to afford intermediate 1.12 After Mitsunobu reaction
with DIAD, PPh3 and thiolacetic acid, the mono- (2, 36%) and
bis-thioacetyl (3, 59%) derivatives were isolated by silica
column chromatography and fully characterized by NMR and
MS.
For the synthesis of the guanidinylated analogue of Amt-Nea,
we used N,N′-di-Boc-N″-triflylguanidine, since we and others
have demonstrated previously the utility of this reagent to
transform amino groups into guanidinium in compounds
incorporating aminoglycoside fragments such as neamine or
neomycin.10b,15,17 To avoid possible side-reactions with the two
alkyl secondary amino functions of the anthraquinone moiety
during guanidinylation, we decided to keep them masked with
the Boc group (Scheme 3). First, the thiol-containing neamine
derivative 410 was activated with DTNP under acidic conditions
overnight under an Ar atmosphere. Reaction of intermediate 5
with the thiol derivative of bis-Boc-protected ametantrone 6,12
which was easily prepared by hydrolysis of the thioester group
of 2 with sodium methoxide, afforded the expected disulfide
intermediate 7 after purification by reversed-phase HPLC.
Finally, reaction of 7 with N,N′-di-Boc-N″-triflylguanidine in
the presence of triethylamine followed by acidic deprotection
with a TFA cocktail containing triisopropylsilane as cation
scavenger afforded the trifluoroacetate salt of the desired
compound as a dark-blue solid after purification by HPLC and
lyophilization (yield 64%). Amt-NeaG4 was fully characterized
RESULTS AND DISCUSSION
■
Synthesis of Amt-Containing Ligands. The synthesis of
second-generation ametantrone-containing ligands (Amt-
NeaG4, Amt-Nea,Nea, and Amt-Nea,Azq; Scheme 1) was
planned in solution by using thiol−disulfide exchange reactions
mediated by 2,2′-dithiobis(5-nitropyridine) (DTNP).12,15,16
For this purpose, two ametantrone derivatives were prepared
in which one or the two hydroxyl groups of the side chains of
the anthraquinone heterocycle were replaced by thiol functions
masked as thioacetyl (2 and 3, respectively, see Scheme 2).
First, reaction of 1,4-difluoroanthraquinone with an excess of
N-(2-hydroxyethyl)ethylenediamine afforded ametantrone,
whose secondary alkyl amino functions were selectively Boc
1
by NMR (1D H, COSY, TOCSY) and high-resolution ESI
mass spectrometry. Despite the prolonged reaction time (14
days) and the use of a large excess of the guanidinylating
B
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Scheme 3. Synthesis of the Guanidinylated Ligand Amt-NeaG4
Scheme 4. Synthesis of the Doubly-Functionalized Ligands, (A) Amt-Nea,Nea and (B) Amt-Nea,Azq
reagent (up to 360-fold molar equivalent), the triguanidinylated
derivative was also identified in the reaction crude (see Figure
S1 in the Supporting Information), which can be attributed to
the steric hindrance caused by the Boc groups in the
anthraquinone moiety.
As shown in Scheme 4, a similar approach was used for the
synthesis of doubly-functionalized amentantrone-containing
ligands. In this case, the Boc-protected derivative of
ametantrone containing one thioacetyl group at each side
chain was used (3). After hydrolysis of the two thioester groups
with sodium methoxide, the thiol derivative 8 was reacted with
an excess of the activated neamine 4 under slightly acidic
conditions during 5 days at room temperature. The Boc-
protected intermediate, Amt(Boc)2-Nea,Nea was treated with
TFA/TIS/H2O 95:2.5:2.5 for 1 h at room temperature, and the
expected ligand, Amt-Nea,Nea, was isolated by HPLC with an
overall 6% yield. Regarding the synthesis of the compound
containing three different RNA-binding moieties, the thiol-
containing ametantrone derivative 8 was reacted simultaneously
with activated neamine (5) and activated azaquinolone (9)12
derivatives. After TFA treatment, Amt-Nea,Azq was isolated by
HPLC and characterized by MS and NMR (overall yield 8%).
The formation of several side-products (e.g., Amt-Nea,Nea and
Amt-Azq,Azq during the synthesis of Amt-Nea,Azq) due to lack
of selectivity during the formation of disulfide bonds accounts
for the low yield in both cases (see HPLC traces of the crudes
in Figure S1 in the Supporting Information).
Biophysical Studies on the Interaction of Amt-
Containing Ligands with Tau RNA. Our next objective
was to study the interaction of the new ametantrone-containing
ligands (Amt-NeaG4, Amt-Nea,Nea, and Amt-Nea,Azq) with
Tau RNA and in particular to assess how the modifications
introduced in the parent Amt-Nea ligand (e.g., guanidinylation
or double functionalization) influence RNA binding affinity as
C
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Figure 1. (A) Sequences and secondary structure of wild-type (wt) and of some mutated Tau stem−loop RNAs (+3, +13, +14, and +16). Exonic
sequences are shown in capital letters and intronic sequences in lower case. Nucleotides involved in base pairs, previously identified by NMR, are
connected by a dash.7b,8b An asterisk denotes 2′-O-methyl modification. In fluorescence binding experiments, fluorescein derivatization was
performed at the 5′ end. (B) Fluorescence quenching of wt Tau RNA labeled with fluorescein upon addition of increasing concentrations of Amt-
NeaG4. Experimental conditions: [RNA] = 25 nM and [ligand] = 0−614 nM, in 10 mM sodium phosphate buffer pH 6.8, 100 mM NaCl, and 0.1
mM Na2EDTA. (C) Plot of the normalized fluorescence signal at 517 nm against the log of the ligand concentration. The normalized fluorescence
was calculated by dividing the difference between the observed fluorescence, F′, and the final fluorescence, Ff, by the difference between the initial
fluorescence, F0, and the final fluorescence, Ff.
well as their ability to stabilize the disease-causing mutated
sequences.8 First, quantitative binding studies were carried out
by fluorescence titration experiments by using 5′-end
fluorescein-labeled wt RNA. As shown in Figure 1, the
oligoribonucleotide fluorescence was quenched upon addition
of increasing concentrations of the ligands, which reproduce the
tendency previously found with other Tau RNA ligands
containing heteroaromatic moieties.7b,10,12 A characteristic
dose-dependent curve was obtained when the normalized
fluorescence was plotted in front of the compound concen-
tration. The inherent fluorescence of the ligands was always
subtracted from that of the labeled RNA by repeating the full
titration in the absence of RNA. This approach allows the
determination of EC50 values (the effective ligand concen-
tration required for 50% RNA response) by fitting the data to a
sigmoidal dose−response curve (Figure 1).
comparison purposes.12 Consistent with our previous re-
sults,10b,15 the guanidinylation of the neamine moiety had a
positive effect on the binding affinity of Amt-Nea. Indeed, the
EC50 value for Amt-NeaG4 was about 1.2 times lower than for
the parent amino ligand (e.g., EC50 = 57.2 2.0 nM for Amt-
NeaG4 vs EC50 = 70.6 7.2 nM for Amt-Nea). The opposite
tendency was found when a second fragment (neamine or
azaquinolone) was incorporated in the free side chain of
ametantrone in Amt-Nea: the EC50 values for Amt-Nea,Nea
and for Amt-Nea,Azq were 1.1 and 1.2 times higher than for
Amt-Nea, respectively. However, it is worth noting that the
affinity of both doubly-functionalized ligands is still higher than
that of ametantrone, thereby suggesting that, upon ligand
complexation, the two RNA binding moieties attached to the
anthraquinone intercalator might participate in the interaction
with the RNA target.
Fluorescence binding assays were repeated in the presence of
a biologically relevant competitor (a tRNAmix from baker’s
yeast) to get some insights on their specificity for Tau RNA. As
shown in Table 1, the specificity of the ligands was highly
dependent on the nature of the modification introduced in the
parent ligand. In the presence of the competitor, the EC50 value
of Amt-NeaG4 for Tau RNA was increased by 10-fold, whereas
those of Amt-Nea,Nea and Amt-Nea,Azq were increased by
about 7-fold. Hence, guanidinylation of the neamine moiety in
Amt-Nea seems to have a negative effect on the specificity
compared with the introduction of a second RNA binding
moiety. The fact that all ametantrone ligands containing one or
two neamine moieties or guanidinoneamine are less specific
than the parent anthraquinone building blocks (Mtx or Amt) or
Amt-Azq is in agreement with the known promiscuity of
aminoglycosides and their derivatives.
The EC50 values of the new ametantrone-containing ligands
together with those of ametantrone, mitoxantrone, neamine,
and guanidinoneamine are shown in Table 1. EC50 values of the
Amt-Nea and Amt-Azq ligands have also been included for
Table 1. Binding of the Ligands to wt RNA in the Absence or
Presence of a tRNA Competitor
EC50 (nM) +
EC50 + tRNA/
EC50
a
b
ligand
neamine
EC50 (nM)
3.1 × 106
tRNA
nd
nd
nd
guanidinoneamine
mitoxantrone
830
nd
168.8 6.2
231.8 8.0
162.5 5.7
70.6 7.2
57.2 2.0
76.2 4.1
84.5 3.8
803.3
675.6
570.7
569.8
580.1
519.7
558.6
4.8
2.9
3.5
8.1
10.2
6.8
6.6
ametantrone
c
Amt-Azq
c
Amt-Nea
Amt-NeaG4
Amt-Nea,Nea
Amt-Nea,Azq
Next, we investigated the ability of second-generation Amt-
containing ligands to stabilize Tau RNA, in particular some of
the mutated sequences found in patients suffering from
tauopathies like FTDP-17. These intronic mutations (denoted
as +3, +13, +14, and +16 in Figure 1) are known to decrease
the thermodynamic stability of the stem−loop structure located
at the exon 10-5′ intron junction of Tau pre-mRNA.8 As
previously stated, reversing the destabilization of these mutated
a
All fluorescence measurements were performed in 10 mM sodium
phosphate buffer pH 6.8, 100 mM NaCl, and 0.1 mM Na2EDTA.
b
Measured in the presence of a 30-fold nucleotide excess of a mixture
of tRNA (tRNAmix). EC50 values of these ligands12 have been
c
included in the table for comparison purposes.
D
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a
Table 2. Melting Temperatures (Tm) for the Complexation of the Ligands with Tau RNAs
b
b
b
b
Tm wt
ΔTm
Tm +3
ΔTm
Tm +14
ΔTm
Tm +16
ΔTm
no ligand
66.5
66.9
67.7
69.2
67.1
67.8
75.4
78.5
79.5
71.2
−
51.4
51.5
52.8
57.1
54.2
56.1
64.3
63.0
67.0
58.2
−
+0.1
53.5
54.0
54.5
58.1
55.7
57.3
63.9
66.3
67.9
61.1
−
+0.5
59.6
60.1
61.0
63.4
61.6
61.8
69.4
70.1
70.8
66.3
−
neamine
+0.4
+1.2
+2.7
+0.5
+1.3
+8.9
+12.0
+13.0
+4.7
+0.5
+1.4
+3.8
+2.0
+2.2
+9.8
+10.5
+11.2
+6.7
guanidinoneamine
mitoxantrone
ametantrone
Amt-Azq
+1.4
+1.0
+5.7
+4.6
+2.8
+2.2
+4.7
+3.8
Amt-Nea
+12.9
+11.6
+15.6
+6.8
+10.4
+12.8
+14.4
+7.6
Amt-NeaG4
Amt-Nea,Nea
Amt-Nea,Azq
a
b
1 μM both in RNA and in ligands in 10 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl and 0.1 mM Na2EDTA. ΔTm = (Tm of the RNA in
the presence of ligand) − (Tm of RNA alone).
Figure 2. Fluorescence emission spectra of (A) Amt-NeaG4, (B) Amt-Nea,Nea, and (C) Amt-Nea,Azq in the absence and in the presence of
increasing amounts of wt RNA in a 10 mM sodium phosphate buffer, pH 6.8, containing 100 mM NaCl and 0.1 mM Na2EDTA. The concentration
of the ligands was 2 μM in fluorescence titration experiments. The emission spectra were recorded from 600−850 nm with λex = 547 nm.
sequences upon ligand complexation has been postulated as a
therapeutic tool to restore the physiological balance of tau
isoforms generated upon abnormal alternative splicing and, for
instance, as a potential treatment for such neurodegenerative
diseases.
UV melting experiments were carried out by monitoring the
absorbance as a function of temperature. The midpoint of the
transition (see Figures S2−S5 in the Supporting Information)
is referred to as the melting temperature (Tm) and provides an
indicative of the thermal stability of the RNA secondary
structure in the presence of a given ligand. As shown in Table 2,
guanidinylation of Amt-Nea had a positive effect on the thermal
stability of wt RNA upon complexation (ΔTmG = +3.1 °C;
ΔTmG indicates the effect of guanidinylation compared with
that of the parent nonguanidinylated ligand, Amt-Nea). This
effect was similar to the +14 mutated sequence (ΔTmG = +2.4
°C) but slightly lower with the +16 mutant (ΔTmG = +0.7 °C).
However, the stabilizing effect of Amt-NeaG4 upon complex-
ation with the +3 mutated RNA (ΔTmG = −1.3 °C) was lower
than in the presence of Amt-Nea, but still substantially higher
(ΔTm = +11.6 °C) compared with that of Amt (ΔTm = +2.8
°C) or Mtx (ΔTm = +5.7 °C) with this mutated sequence.
Regarding doubly-functionalized ligands, the tendency was
found to be the opposite depending on the nature of the
E
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Figure 3. Secondary structure of wt Tau RNA sequence showing the labeling of the characteristic imino signals (A) and imino region of the NMR
spectra of the oligoribonucleotide alone and in the presence of increasing amounts of the ligands, (B) Amt-NeaG4, (C) Amt-Nea,Azq, or (D) Amt-
Nea,Nea. From bottom to top: ligand/RNA ratio = 0.0, 0.5, 1.0, and 1.5. Assignments for the RNA alone are labeled according to previously
reported works.7b,8b,12 Imino signals at RNA/ligand ratio 1:1 are labeled according to the complex between Tau RNA and Amt-Nea.12 Experimental
conditions: [RNA] = 70 μM, 10 mM phosphate buffer in H2O/D2O 90:10, pH = 6.8, T = 5 °C.
second moiety attached to the anthraquinone fragment in the
Amt-Nea ligand, neamine or azaquinolone. As shown in Table
2, the effect of Amt-Nea,Nea on the thermal stability of all RNA
sequences was always higher than that of the parent ligand.
This increase was particularly high for the wt (ΔTm = +8.9 °C
with Amt-Nea vs ΔTm = +13 °C with Amt-Nea,Nea) and the
+14 mutant (ΔTm = +10.4 °C with Amt-Nea vs ΔTm = +14.4
°C with Amt-Nea,Nea). However, the stabilization induced by
Amt-Nea,Azq was considerably lower than that of Amt-Nea.
Such differences were particularly important with wt RNA
(ΔTm = +8.9 °C with Amt-Nea vs ΔTm = +4.7 °C with Amt-
Nea,Azq) and with the +3 mutated sequence (ΔTm = +12.9 °C
with Amt-Nea vs ΔTm = +6.8 °C with Amt-Nea,Azq). These
results are quite surprising because EC50 values of Amt-
Nea,Nea and Amt-Nea,Azq point to similar binding affinities
for Tau RNA. Despite this reduced stabilizing ability, the
neamine moiety in Amt-Nea,Azq still seems to have a positive
effect on the thermal stabilization of the RNAs upon
complexation, as inferred from the higher Tm values of its
complexes compared with those obtained in the presence of
Amt-Azq.
Spectroscopic Studies on the Interaction of Amt-
Containing Ligands with Tau RNA. The overall biophysical
studies indicated that both binding affinity and stabilizing ability
of the parent Amt-Nea ligand are influenced either by
guanidinylation of the aminoglycoside moiety or by function-
alization of the second arm of ametantrone. Hence, our next
objective was focused on investigating the effect of these ligands
(Amt-NeaG4, Amt-Nea,Nea, and Amt-Nea,Azq) on the
structure of Tau RNA upon complexation and in particular
to determine if they also have a preferred binding site. As
previously stated, intercalation of the anthraquinone moiety in
the bulged region of the stem−loop structure of Tau RNA
together with specific contacts between the positively charged
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side chains and the neamine moiety with the RNA accounts for
the high binding affinity and stabilizing ability of Amt-Nea.12
First, fluorescence emission spectra (upon excitation at 547
nm) were recorded for the three compounds in the absence
and presence of increasing amounts of wt Tau RNA. As shown
in Figure 2, hypochromic and bathochromic effects were
observed in the fluorescence spectra of the ligands during the
titration with RNA, although those effects were more
pronounced in the case of Amt-NeaG4 and Amt-Nea,Nea,
which is consistent with a higher binding affinity. Interestingly,
a strong hypochromism was observed in the fluorescence
spectra of these ligands in the first stage of the titration (0.07
mol equiv of RNA for Amt-NeaG4 and 0.17 mol equiv of RNA
for Amt-Nea,Nea). Then, in the second stage of the titration,
the intensity of the fluorescence emission band increased
gradually until saturation was reached and then decreased again.
In both cases, an 8 nm shift to higher wavelengths was observed
(from the initial 656 nm band in the free ligand). These results
reproduced those previously observed for mitoxantrone8b and
for the parent Amt-Nea ligand12 and suggest that neither
guanidinylation nor double functionalization of ametantrone
with two neamine moieties seem to interfere with the
intercalation of the anthraquinone fragment. Although this
two-stage binding mode was not completely reproduced in the
case of Amt−Nea,Azq, which could be attributed to a lower
binding affinity compared with that of the Amt-NeaG4 or Amt-
Nea,Nea, the quenching of the fluorescence intensity points
also to the intercalation of the ametantrone fragment.
effect of Amt-Nea,Azq on the imino signal of U0 was much
higher than that provoked by Amt-NeaG4 (+0.35 and +0.13
ppm, respectively), and in both cases imino signals of G+1, U
+2, and G+3 were not significantly affected in the complexes.
As previously found with Amt-Nea, appearance of an imino
signal which was assigned to G+17 confirms the allocation of
both ligands at the bulged region of the stem−loop structure,
particularly of the amentatrone fragment which would protect
the C-3:G+17 base pair from the solvent in the complex. In
addition, the two signals in the exchangeable proton region (δ
≈ 9.9 and 9.5 ppm), corresponding to the aliphatic amino
protons of the two side chains of the ametantrone moiety of the
ligand, were clearly observed upon complexation with Amt-
NeaG4 or Amt-Nea,Azq, suggesting again a similar binding
mode to that described for Amt-Nea.12
To our surprise, addition of Amt-Nea,Nea caused a general
line broadening of all signals together with major changes in the
chemical shifts of the imino protons of the RNA (see Figure 3).
The fact that two sets of signals corresponding to the aliphatic
amino protons of the ametantrone moiety of the ligand were
observed upon complexation (δ ≈ 9.9 and 9.4 ppm, and δ ≈
10.5 and 10.6 ppm) suggests the presence of two binding sites
for this ligand in Tau RNA. Hence, besides allocation at the
bulged region of Tau RNA, the presence of two neamine
fragments seems to direct the binding of Amt-Nea,Nea to
another region of the stem−loop structure, probably at the loop
or at the major groove. In addition, the large number of signals
at the imino proton region also suggests weak aggregation as a
consequence of the complexation of Tau RNA with Amt-
Nea,Nea.
Although qualitative information on the interaction of
heteroaromatic compounds with nucleic acids can be obtained
from UV−vis or fluorescence spectroscopy,18 these techniques
do not provide detailed structural information on their binding
mode, which is necessary to rationalize their RNA-binding
properties and, more importantly, to help in the design of more
selective ligands with improved ones. In this context, NMR
spectroscopy is a powerful technique to detect RNA-ligand
interactions, particularly in the case of ligands based on the
combination of fragments.19
CONCLUSIONS
■
In summary, in this work we have described two new Tau RNA
ligands based on the derivatization of ametantrone with two
neamine moieties (Amt-Nea,Nea) or with one azaquinolone
heterocycle and one neamine moiety (Amt-Nea,Azq), with the
aim of exploring how functionalization of the two side chains of
this anthraquinone intercalator affects RNA-binding properties.
A guanidinylated ligand based on the derivatization of
ametantrone with a single guanidinoneamine moiety (Amt-
NeaG4) was also synthesized. Biophysical studies have
demonstrated that these compounds bind Tau RNA with
Titration experiments of Tau RNA with ametantrone-
containing ligands (Amt-NeaG4, Amt-Nea,Nea and Amt-
Nea,Azq) were monitored by NMR spectroscopy (see Figure
3 and Figure S6 in the Supporting Information) to get more
detailed information on their mode of interaction. Interestingly,
a similar behavior to that previously found with the parent
ligand Amt-Nea12 was observed upon addition of either Amt-
NeaG4 or Amt-Nea,Azq (see the exchangeable proton region
of the NMR spectra in Figure 3), including little line
broadening and changes in the chemical shifts of some imino
protons of the RNA close to the bulged region. The coexistence
of imino signals arising from the free and bound RNA at a
RNA:ligand ratio of 1:0.5 indicates that both species are in slow
equilibrium in the NMR time scale. Moreover, the fact that
only one set of signals at a 1:1 ratio was observed suggests that
Amt-NeaG4 and Amt-Nea,Azq bind Tau RNA in a well-defined
binding site. As shown in Figure 3, imino signals of the complex
between Tau RNA and the ligands at a 1:1 ratio were labeled
according to previously reported data from the complex Tau-
Amt-Nea due to their similar behavior during the titration.12 In
both cases, the most pronounced changes in chemical shifts
involved the imino proton of G-1 (see Figure 3 for the labeling
of the imino protons), which was shifted by about −0.85 ppm
upon complexation with Amt-NeG4 or Amt-Nea,Azq,
compared with their values in the free RNA. In contrast, the
high affinity, particularly the guanidinylated derivative (EC50
=
57.2 nM), and that the double functionalization of ametantrone
does not conduce to a significant decrease in their binding
affinity (only about 1.1−1.2 fold) when compared with the
parent ligand (Amt-Nea). In addition, all the compounds cause
a significant increase in the thermal stability of Tau RNA,
particularly Amt-NeaG4 and Amt-Nea,Nea (ΔTm = +12 and
+13 °C, respectively). Interestingly, these compounds are able
to restore the thermodynamic stability of some of the mutated
sequences associated with the development of FTDP-17 disease
to a higher level than that of wt RNA, which is of high relevance
for developing potential modulators of Tau pre-mRNA splicing.
For example, the Tm value of the +16 mutated sequence when
complexed with these ligands was about 4 °C higher than that
of wt RNA alone. Finally, NMR titration experiments revealed
that Amt-NeaG4 and Amt-Nea,Azq have a preferred binding
site in the stem−loop structure of Tau RNA, in which
ametantrone intercalates in the bulged region, thereby
indicating that conjugation with guanidinoneamine or with
neamine and azaquinolone does not seem to modify the
preference of this anthraquinone derivative for this region.
G
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The Journal of Organic Chemistry
Article
AcOEt to remove the excess 2,2′-dithiobis(5-nitropyridine) (typically
6 × 2 mL or until no yellow color was detected in the organic phase)
and lyophilized to afford activated neamine 5. Second, to a stirred
solution of thioacetyl derivative 2 (2.4 mg, 3.6 μmol) in MeOH (1
mL) was added sodium methoxide (24 μL, 1 M in MeOH, 24 μmol),
and the mixture stirred for 5 min at RT under argon. After evaporation
in vacuo, the crude containing thiol amentantrone derivative 6 was
dissolved in H2O/ACN 8:2 (v/v) containing 0.1% TFA (8 mL) and
added over the activated neamine 5, and the mixture was stirred
overnight under argon at RT. After purification by semipreparative
reversed-phase HPLC (linear gradient from 0 to 80% B in 35 min; A,
0.1% TFA in H2O; B, 0.1% TFA in ACN; Rt = 21 min) and
lyophilization, the TFA salt of Amt(Boc)2-Nea (7) was obtained as a
blue solid (0.48 mg, yield 11%).
Although attachment of two aminoglycoside moieties conduces
to a ligand with promising RNA-binding properties, the lack of
a preferred binding site in the RNA target discards Amt-
Nea,Nea as a lead compound for further developing RNA
ligands for regulating the alternative splicing of Tau pre-mRNA.
All together the present work reported here provides new
insights for designing ligands based on amentantrone with
improved RNA-binding properties. Current efforts are aimed at
exploring the use of these and other amentantrone-containing
derivatives as ligands of other therapeutically relevant RNA
secondary structures involved in the onset of human diseases.
EXPERIMENTAL SECTION
C o m p o u n d
7
( 4 0 0 n m o l ) a n d 1 , 3 - d i - B o c - 2 -
■
trifluoromethylsulfonyl)guanidine (12.5 mg, 32 μmol) were dissolved
in a 5:3 (v/v) mixture of MeOH and CHCl3 (2 mL) under argon.
Then, triethylamine (27 μL, 192 μmol) was added, and the reaction
mixture was stirred for 14 days at RT under Ar. A total of 360 mol
equiv of guanidinylating reagent and of 1680 mol equiv of
triethylamine was added over the entire reaction period. After
evaporation in vacuo, the crude was diluted with DCM (5 mL) and
washed with a 10% aqueous solution of citric acid (3 × 1 mL) and
with brine (3 × 1 mL). The organic phase was taken up and dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to dryness. The
crude product was dissolved in a 1:1 (v/v) mixture of TFA and DCM
containing 2.5% of TIS (2 mL). After it was stirred at RT for 2 h under
Ar, the mixture was diluted with toluene (2 mL) and evaporated in
vacuo. After several co-evaporations from toluene, the residue was
dissolved in Milli-Q H2O (5 mL) and lyophilized to provide a blue
solid. Purification was carried out by analytical reversed-phase HPLC
(linear gradient from 0 to 30% B in 35 min; A, 0.045% TFA in H2O; B,
0.036% TFA in ACN), and the TFA salt of the desired product was
Materials and Methods. Unless otherwise stated, common
chemicals and solvents (HPLC grade or reagent grade quality) were
purchased from commercial sources and used without further
purification. Aluminum plates coated with a 0.2 mm thick layer of
silica gel 60 F254 were used for thin-layer chromatography analyses
(TLC), whereas flash column chromatography purification was carried
out using silica gel 60 (230−400 mesh).
Reversed-phase high-performance liquid chromatography (HPLC)
analyses of the ligands and their precursors were carried out on a
Jupiter Proteo C18 column (250 × 4.6 mm, 90 Å 4 μm, flow rate: 1
mL/min) using linear gradients of 0.045% TFA in H2O (A) and
0.036% TFA in ACN (B). In some cases, purification was carried out
using the same analytical column. A semipreparative Jupiter Proteo
column was used for the purification of some compounds (250 × 10
mm, 90 Å 10 μm, flow rate: 3 mL/min), using linear gradients of 0.1%
TFA in H2O (A) and 0.1% TFA in ACN (B).
NMR spectra were recorded at 25 °C in a 600 MHz spectrometer
using deuterated solvents. The residual signal of the solvent was used
1
1
obtained after lyophilization (0.43 mg, yield 64% from 7). H NMR
as a reference in H spectra. Chemical shifts are reported in parts per
(600 MHz, D2O) δ (ppm): 8.31 (2H, H6 + H7 Amt, m), 7.86 (2H, H5
+ H8 Amt, m), 7.58 (2H, H2 + H3 Amt, m), 5.58 (1H, H1′, d, J = 3.6
Hz), 4.08 (1H, −S−CH2−CH2−O−, m), 3.92 (2H, −NH−CH2−
CH2−OH, t, J = 6.4 Hz), 3.84 (4H, −NH−CH2−CH2−NH−CH2−, t,
J = 5.3 Hz), 3.81 (1H, H5′, m), 3.77 (1H, −S−CH2−CH2−O−, m),
3.70 (2H, −NH−CH2−CH2−S−, m), 3.68−3.58 (5H, H2′, H4′, H4,
H5, H6), 3.50−3.43 (5H, H1, H3, H3′, −NH−CH2−CH2−S−, m), 3.40
(2H, −NH−CH2-CH2−OH, m), 3.20 (4H, −NH−CH2−CH2−NH−
CH2−, m), 2.89 (2H, H6′, m), 2.77 (2H, −S−CH2−CH2−O−, m),
2.21 (1H, H2,eq, m), 1.62 (1H, H2,ax, m); HR-ESI MS, positive mode:
m/z 977.4553 (calcd mass for C40H65N16O9S2 [M + H]+: 977.4562),
m/z 489.2313 (calcd mass for C40H66N16O9S2 [M + 2H]2+: 489.2320),
m/z 326.4905 (calcd mass for C40H67N16O9S2 [M + 3H]3+: 326.4906);
analytical HPLC (linear gradient from 0 to 30% B in 30 min): Rt =
24.4 min.
million (ppm) in the δ scale, coupling constants in Hz, and multiplicity
as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), qt
(quintuplet), m (multiplet), dd (doublet of doublets), td (doublet of
triplets), ddd (doublet of doublet of doublets), and br (broad signal).
High-resolution matrix-assisted laser desorption ionization time-of-
flight (MALDI-TOF) mass spectra were recorded both in positive
(2,4-dihydroxybenzoic acid matrix) or negative mode (2,4,6-
trihydroxyacetophenone matrix with ammonium citrate as an
additive). Electrospray ionization mass spectra (ESI-MS) were
recorded on an instrument equipped with single quadrupole detector
coupled to an HPLC, and high-resolution (HR) ESI-MS on LC/MS-
TOF instrument.
Oligoribonucleotides were synthesized on a DNA automatic
synthesizer (1 μmol scale) using 2′-O-tert-butyldimethylsilyl protec-
tion and following standard procedures (phosphite triester approach).
RNA and fluorescein phosphoramidites, solid-supports, reagents, and
solvents for oligoribonucleotide synthesis were purchased from a
chemical supplier. RNase-free reagents, solutions, and materials were
used when manipulating oligoribonucleotides. RNase-free water was
obtained directly from a Milli-Q system equipped with a 5000 Da
ultrafiltration cartridge.
Reversed-phase HPLC was used for the analysis and purification of
the oligoribonucleotides using linear gradients of 0.1 M aqueous
NH4HCO3 and a 1:1 mixture of 0.1 M aqueous NH4HCO3 and ACN.
A Kromasil C18 column (250 × 4.6 mm, 10 μm, flow rate: 1 mL/min)
was used for RNA analysis, whereas a semipreparative Jupiter C18
column (250 × 10 mm, 300 Å 10 μm, flow rate: 3 mL/min) was used
for purification at large scale. Characterization was carried out by high
resolution MALDI-TOF MS (negative mode, THAP matrix with
ammonium citrate).
Synthesis of Ametantrone-Containing Ligands. Amt-NeaG4.
First, 2,2′-dithiobis(5-nitropyridine) (11.6 mg, 37.5 μmol) and the
neamine thiol monomer 4 (3 μmol) were reacted in a 2:1 (v/v)
mixture of THF/aqueous 0.1% TFA (2 mL) under argon at RT. After
17 h, THF was evaporated in vacuo, and the remaining yellow solution
was diluted with H2O (2 mL). The aqueous phase was washed with
Amt-Nea,Nea. First, the thiol function of the neamine derivative 4
(7 μmol) was activated by reaction with DTNP (27.2 mg, 87.5 μmol)
in a 2:1 (v/v) mixture of THF/aqueous 0.1% TFA (8 mL) under
argon at RT for 17 h. After evaporation in vacuo, excess of DTNP was
eliminated, and the crude containing 5 was lyophilized. Second, to a
stirred solution of the bis-thioester derivative 3 (1.4 mg, 1.9 μmol) in
MeOH (1 mL) was added sodium methoxide (190 μL, 0.1 M in
MeOH, 19 μmol), and the mixture stirred for 5 min at RT under
argon. After evaporation in vacuo, the crude containing bis-thiol
amentantrone derivative 8 was dissolved in H2O/ACN 8:2 (v/v)
containing 0.1% TFA (10 mL) and added over the activated neamine
5, and the mixture was stirred for 5 days under argon at RT. After
purification by analytical reversed-phase HPLC (linear gradient from 0
to 80% B in 35 min; A, 0.045% TFA in H2O; B, 0.036% TFA in ACN;
Rt = 18 min) and lyophilization, the TFA salt of Amt(Boc)2-Nea,Nea
was obtained. Finally, treatment with TFA/TIS/H2O 95:2.5:2.5 (1
mL) for 1 h at RT afforded the TFA salt of Amt-Nea,Nea as a blue
1
solid after HPLC purification (0.27 mg, yield 6%). H NMR (600
MHz, D2O) δ (ppm): 8.32 (2H, H6 + H7 Amt, m), 7.89 (2H, H5 + H8
Amt, m), 7.56 (2H, H2 + H3 Amt, m), 5.62 (2H, H1′, m), 4.15 (2H,
−S−CH2−CH2−O−, m), 3.98 (6H, H5′, H6, −S−CH2−CH2−O−,
H
DOI: 10.1021/jo502661j
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The Journal of Organic Chemistry
Article
m), 3.93 (4H, −NH−CH2−CH2−NH−CH2−, t, J = 6.0 Hz), 3.80
(2H, H3′), 3.70 (4H, H4, H5, m), 3.61 (2H, H4′, m), 3.46 (8H, −NH−
CH2−CH2−NH−CH2−, m), 3.40 (4H, H6′, m), 3.20 (4H, H1, H3, m),
3.12 (2H, H2′, m), 3.02 (4H, NH−CH2−CH2−S−, t, J = 6.8 Hz), 2.92
(4H, −S−CH2−CH2−O−, t, J = 6.3 Hz), 2.23 (2H, H2,eq, m), 1.56
(2H, H2,ax, m); HR-ESI MS, positive mode: m/z 1205.5241 (calcd
mass for C50H85N12O14S4 [M + H]+: 1205.5191), m/z 603.2642 (calcd
mass for C50H86N12O14S4 [M + 2H]2+: 603.2635), m/z 402.5122
(calcd mass for C50H87N12O14S4 [M + 3H]3+: 402.5116); analytical
HPLC (linear gradient from 0 to 30% B in 30 min): Rt = 22.7 min.
Amt-Nea,Azq. First, the thiol functions of neamine (1.5 μmol) and
of azaquinolone monomers10 (3 μmol) were activated with DTNP
(5.8 mg, 18.8 μmol and 11.6 mg, 37.5 μmol, respectively) in a 2:1 (v/
v) mixture of THF/aqueous 0.1% TFA (1.5 and 2 mL, respectively)
under argon at RT for 17 h. After evaporation in vacuo, excess of
DTNP was eliminated, and both activated derivatives were isolated by
semipreparative reversed-phase HPLC (linear gradient from 0 to 80%
B in 30 min; A, 0.1% TFA in H2O; B, 0.1% TFA in ACN; Rt = 14.1
min for 5 and Rt = 17.9 min for 9). Second, to a stirred solution of 3 (1
mg, 1.4 μmol) in MeOH (1 mL) was added sodium methoxide (275
μL, 0.1 M in MeOH, 27.5 μmol), and the mixture stirred for 5 min at
RT under argon. After evaporation in vacuo, the crude containing
derivative 8 was dissolved in H2O/ACN 8:2 (v/v) containing 0.1%
TFA (12 mL) and added over activated monomers 5 and 9, and the
mixture was stirred for 6 days under argon at RT. After purification by
analytical reversed-phase HPLC (linear gradient from 0 to 80% B in 35
min; A, 0.045% TFA in H2O; B, 0.036% TFA in ACN; Rt = 20.5 min)
and lyophilization, the TFA salt of Amt(Boc)2-Nea,Azq was obtained.
Finally, treatment with TFA/TIS/H2O 95:2.5:2.5 (1 mL) for 1 h at
RT afforded the TFA salt of Amt-Nea,Azq as a blue solid after HPLC
purification (0.21 mg, yield 8%). 1H NMR (600 MHz, D2O) δ (ppm):
7.95 (2H, H6 + H7 Amt, m), 7.65 (2H, H5 + H8 Amt, m), 7.57 (1H,
H5 Azq, d, J = 7.8 Hz), 7.53 (1H, H4 Azq, d, J = 9.5 Hz), 7.33 (2H, H2
+ H3 Amt, m), 6.80 (1H, H6 Azq, d, J = 7.8 Hz), 6.30 (1H, H3 Azq, d, J
= 9.5 Hz), 5.35 (1H, H1′, m), 4.01 (2H, CH2 Azq, s), 3.88−3.72 (7H,
−S−CH2−CH2−O−, −NH−CH2−CH2−NH−, H5′, m), 3.43−3.10
(16H, H4, H5, H6, H2′, H3′, H4′, −CO−NH−CH2−CH2−CH2−NH−,
−NH−CH2−CH2−S−, m), 3.08−2.97 (4H, H1, H3, H6′, m), 2.95 (2H,
−CO−NH−CH2−CH2−CH2−NH−, t, J = 7.9 Hz), 2.90 (2H, −NH−
CH2−CH2−S−, t, J = 6.3 Hz), 2.85 (2H, −NH−CH2−CH2−S−, t, J =
6.3 Hz), 2.81 (2H, −S−CH2−CH2−CO−NH−, t, J = 6.4 Hz), 2.72
(2H, −S−CH2−CH2−O−, m), 2.52 (2H, −S−CH2−CH2−CO−
NH−, t, J = 6.4 Hz), 2.02 (1H, H2,eq, m), 1.79 (2H, −CO−NH−
CH2−CH2−CH2−NH−, m), 1.38 (1H, H2,ax, m); HR-ESI MS,
positive mode: m/z 1143.4599 (calcd mass for C51H75N12O10S4 [M +
H]+: 1143.4612), m/z 572.2334 (calcd mass for C51H76N12O10S4 [M +
2H]2+: 572.2345), m/z 381.8250 (calcd mass for C51H77N12O10S4 [M
+ 3H]3+: 381.8256); analytical HPLC (linear gradient from 5 to 35% B
in 35 min): Rt = 22.8 min.
Fernan
́
dez and L. Ortiz from the facilities of the Servei
d’Espectrometria de Masses of the University of Barcelona for
MS support. This work was supported by funds from the
Spanish Ministerio de Ciencia e Innovacion (grant CTQ2010-
́
21567-C02-01-02 and the RNAREG project, grant CSD2009-
00080), the Generalitat de Catalunya (2009SGR-208 and the
Xarxa de Referen
d’Intensificacio de la Recerca (Universitat de Barcelona). G.A.
received a fellowship from the Universitat de Barcelona.
̀
cia de Biotecnologia), and the Programa
́
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures for the study of the interaction of the
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1
ligands with RNA. Reversed-phase HPLC traces and H NMR
spectra of the ligands. Representative UV melting curves of
RNA-ligand complexes. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: vmarchan@ub.edu. Fax: + (34) 93 339 7878
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors acknowledge Dr. M. Gairi from the Barcelona
Scientific Park for NMR technical support, and Dr. I.
■
́
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DOI: 10.1021/jo502661j
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The Journal of Organic Chemistry
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