Targeting loop adenines in G-quadruplex by a selective oxirane

Article abstract of DOI:10.1002/chem.201203097

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

Full text of DOI:10.1002/chem.201203097

DOI: 10.1002/chem.201203097
Targeting Loop Adenines in G-Quadruplex by a Selective Oxirane
Filippo Doria,[a] Matteo Nadai,[b] Marco Folini,[c] Matteo Scalabrin,[d] Luca Germani,[a]
Giovanna Sattin,[b] Mariella Mella,[a] Manlio Palumbo,[e] Nadia Zaffaroni,[c]
Daniele Fabris,[d] Mauro Freccero,*[a] and Sara N. Richter*[b]
G-quadruplexes (G4s) are DNA or RNA four-stranded
supramolecular architectures that can form in G-rich re-
gions. G4s have been found in biologically significant re-
gions of the genome, such as telomeres, gene promoters, and
in sequences associated with human disease.[1] Due to their
critical role in key biological processes, G4s have been the
object of intense study for their potential as therapeutic tar-
gets.[2] To date, a broad range of compounds have been iden-
tified as G4 ligands,[3] both in vitro and in vivo, with encour-
aging results in clinical trials.[2a] Among them, numerous tri-
and tetra-substituted naphthalene diimides (NDIs) have
shown high affinity for telomeric G4s and good antiprolifer-
ative activity.[4] In this context, we recently began the devel-
opment of hybrid ligand-alkylating NDIs that possess a
binding core tethered to an electrophile precursor, such as a
quinone methide (QM, Scheme 1),[5] which can interact co-
valently with G4 structures.[4d–f] Covalent G4 targeting was
also explored by using PtII–terpyridine complexes.[6] The
strategy highlighted in Scheme 1 affords the possibility of
triggering the alkylating activity under well-defined environ-
mental conditions (e.g., light or mild digestion at 408C),
which would help minimize typical off-target reactivity
before the site of attack is reached.[7] Unfortunately, the
QMs tested to date have yielded rather reversible DNA ad-
ducts, which eluded structural characterization.[4e,f] In the at-
tempt to overcome such a drawback we have developed
NDIs tethering the reversible ligands 1–4 (Scheme 1), to
both a QM precursor (5 and 6) and an intrinsically reactive
oxirane (7). The reversible binding of 1–4 and the alkylating
properties of 5–7 were evaluated against a model G4 struc-
ture corresponding to the human telomere. The superior re-
activity and selectivity of the alkylating oxirane 7 were un-
ambiguously and unprecedentedly assessed by different
mass spectrometric approaches.
The NDIs 1–6 were synthesized by a nucleophilic aromat-
ic substitution (SNAr), on the dibromo-NDI 8,[8] in the pres-
ence of 4-(2-aminoethyl) phenol and 4-ethenyl benzene-
ACHUTNGERNmNUG ethAHCTUNGTRNENaUGN namine for 1–6 and 7, respectively (Supporting Infor-
mation, Scheme S1). The desired side chains and the alkylat-
ing moieties were introduced only subsequently to the SNAr,
in order to preserve their structural integrity. In this way,
the multistep synthesis for 1–4 involved an SNAr and a Man-
nich reaction, followed by an exhaustive methylation of the
amines 3 and 4, yielding the quaternary ammonium salts (5,
6). The oxirane 7 was obtained as a racemic mixture by
SNAr with the use of 4-vinylbenzylamine and subsequent ep-
oxidation with dimethyldioxirane (DMD) or meta-chloro-
perbenzoic acid (MCPBA, yield >90%).
The activity of reversible ligands 3 and 4 was initially as-
sayed by mixing them with a labeled oligonucleotide that re-
produced the human telomeric DNA sequence (F21T; Sup-
porting Information, Figure S1) and by using FRET to
measure the melting of the structure.[9] Addition of 1.0 mm
NDIs to a 0.25 mm solution of F21T in the presence of K+
[a] Dr. F. Doria,+ Dr. L. Germani, Prof. M. Mella, Prof. M. Freccero
Dipartimento di Chimica, Universitꢀ di Pavia
V.le Taramelli 10, 27100 Pavia (Italy)
E-mail: mauro.freccero@unipv.it
[b] Dr. M. Nadai,+ Dr. G. Sattin, Dr. S. N. Richter
induced melting temperature increases (DTm) of 10
(Æ1.0)8C
and 11
ACHTUNGTRENNUNG
Dipartimento di Medicina Molecolare
variations were observed upon incubation with labeled
double-stranded DNA. Possible structural perturbations
were also monitored by circular dichroism (CD).[10] In this
case, minor spectral variations were detected when the
NDIs were added to a fully-folded hTel (unlabelled human
telomeric sequence), which displayed the typical G4 finger-
print with a maximum at 290 nm. When the compounds
were added before folding, an additional maximum was
noted at 260 nm (Supporting Information, Figure S2). This
behavior indicates that the NDIs may partially direct the
folding towards a parallel-like conformation, as described
for other NDIs.[4e,f] The continuous variation method (Job
plot)[11] was employed to evaluate the 1:1 binding stoichiom-
etry (Supporting Information, Figure S2).
Universitꢀ di Padova, via Gabelli 63, 35121 Padua (Italy)
E-mail: sara.richter@unipd.it
[c] Dr. M. Folini, Dr. N. Zaffaroni
Dipartimento di Oncologia Sperimentale e Medicina Molecolare
Fondazione IRCCS Istituto Nazionale dei Tumori
Via G. Amadeo 42, 20133 Milano (Italy)
[d] Dr. M. Scalabrin, Dr. D. Fabris
The RNA Institute, University at Albany
1400 Washington Ave., Albany, NY 12222, USA
[e] Prof. M. Palumbo
Department of Pharmaceutical and Pharmacological Sciences
Universitꢀ di Padova, Via Marzolo 5, 35131 Padova (Italy)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203097.
78
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Chem. Eur. J. 2013, 19, 78 – 81

COMMUNICATION
in up to 1.0m KCl solution
(Supporting Information, Fig-
ure S4). By comparison, these
conditions were found suffi-
cient to partially revert adducts
formed by the activatable QM
compound 6 (Supporting Infor-
mation, Figure S5). The higher
efficiency in the modification
of hTel by 7 in comparison to 6
(Figure 1A vs. Figure 1C) and
the increased stability of the
resulting 7–hTel (Supporting
Information, Figure S4) in
comparison to 6–hTel (Sup-
porting
Information,
Fig-
ure S5), prompted us to further
study the nature of the former.
Therefore, the structure of the
7–hTel adduct was investigated
by electrospray ionization mass
spectrometry (ESI-MS), which
was performed on both un-
Scheme 1. NDIs as reversible (1–4) and alkylating G4 binders (5–7).
reacted and alkylated species
after isolation by PAGE. The
The alkylating properties of 5 and 6 were assayed by incu-
bating them with selected substrates at 408C and by moni-
toring adduct formation by denaturing polyacrylamide gel
electrophoresis (PAGE). Reactivity was tested against the
folded hTel, single-stranded (ss) and double-stranded (ds)
scrambled hTel sequences (Supporting Information, Fig-
ure S1). Adduct formation was observed at a molar ratio of
0.8:1 NDI/hTel, and yields increased to approximately 10%
adduct at a ratio of 12:1 (Figure 1A and B). In contrast, the
same amount of ss substrate failed to produce detectable ad-
ducts even when treated with up to 50:1 NDI/ss (Figure 1B).
Similarly, the ds substrate produced only a modest, approxi-
mately 2%, adduct when the analogue concentrations were
increased to 200:1 NDI/ds (Figure 1B, and Supporting Infor-
mation, Figure S3). Under the same conditions, the oxirane
7 displayed greater reactivity than the corresponding QM
activatable counterparts (Figure 1C). In this case, up to ap-
proximately 16% adduct yield was obtained at 12:1 NDI/
hTel (Figure 1D). Conversely, alkylation of ss and ds sub-
strates was modest (<2%, when treated with up to 200:1
NDI/substrate). The negligible reactivity manifested by sub-
strates that do not fold into G4 suggests a mechanism in-
volving specific substrate–ligand recognition before the
actual alkylation can take place. Substrates that are unable
to sustain NDI binding appear incapable of undergoing sig-
nificant alkylation, even though they still contain putative
reactive sites of A and G.
observed mass of 7464.33 Da matched very closely the mass
of 7464.34 Da calculated for a 1:1 adduct by adding the
masses of the initial G4 construct and 7 (Supporting Infor-
mation, Figure S6). These data suggest that the putative
adduct was produced by the opening of the oxirane induced
by hTel. When submitted to tandem mass spectrometry
(MS/MS), the alkylated product provided typical fragment
ions that matched the sequence of hTel. As typically ob-
served for covalent adducts of nucleic acids,[13] the fragments
included a characteristic mass shift corresponding to ana-
logue 7 (i.e., 501 Da incremental mass), which enabled us to
locate its putative position at A1, A7, A13, and A19
(Figure 2). The detection of a fragment corresponding to the
adduct 7 bound to an A nucleobase (i.e., m/z 637.2, Sup-
porting Information, Figure S7A) was consistent with the
mechanism of DNA fragmentation, which is prompted by
initial base loss.[13] A similar alkylation selectivity has been
reported for PtII–terpyridines, which coordinate exclusively
the adenine nucleobases present in the G4 loops.[6] Consid-
ering the well-known styrene oxide reactivity towards all
four deoxyribonucleosides dG>dC>dA@dT[14] it was sur-
prising to observe that A exhibited greater susceptibility
than G. In contrast, the alkylated adduct of 7 with G nucleo-
base was detected when the substrate considered was the ss
construct with scrambled sequence. Indeed, a fragment cor-
responding to alkylated G was recognizable in the MS/MS
spectrum of the ss-DNA/oxirane adduct (Supporting Infor-
mation, Figure S7B). At the same time, no product of A al-
kylation could be detected; this suggests that adduct forma-
tion was profoundly affected by the structural context.
In conclusion, we have developed two types of G4 alkylat-
ing agents. Both alkylation efficiency and selectivity were
These data indicate a selective alkylation of the G4 struc-
ture over unstructured or double helix DNA. The possible
reversible character of the alkylated G4 product was ex-
plored as a function of temperature and salt concentration.
The adduct was stable after 10 min incubation at 958C and
Chem. Eur. J. 2013, 19, 78 – 81
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79

M. Freccero, S. N. Richter et al.
Figure 2. MS/MS spectrum of 7–hTel adduct obtained in negative ion
mode. Characteristic ion series are labeled according to standard nomen-
clature;[12] [MÀ5H]5À indicates the precursor ion at m/z 1492.46. Ions
marked with an asterisk included a 501 Da mass shift from the corre-
sponding unmodified fragment, in agreement with the presence of one
unit of 7. All fragments are summarized on the hTel sequence to enable
a direct comparison of ion series with (marked with an asterisk) and
without (unmarked) adduct.
Figure 1. Alkylation of 6 and 7 analyzed by PAGE. A) 32P-labeled hTel
alone (lane 1) and digested with 6 at 408C for 24 h (lanes 2–9) or at 48C
for 24 h (lane 10). B) Quantification of adduct bands obtained by treating
Keywords: alkylation · DNA damage · G-quadruplexes ·
oxirane · quinone methides
!
*
*
hTel ( ), ss- ( ) and ds-scrambled hTel ( ) with compound 6. C) Com-
pound 7 with 32P-labeled hTel, ss- and ds-scrambled hTel. D) Quantifica-
*
*
tion of adduct bands obtained by treating hTel ( ), ss- ( ) and ds-scram-
!
bled hTel ( ) with compound 7.
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Acknowledgements
Financial support from MIUR, Rome (FIRB-IdeasRBID082ATK, and
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National Institutes of Health (GM064328-12 to D.F.) is gratefully ac-
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Targeting Loop Adenines in G-Quadruplex
COMMUNICATION
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Received: August 31, 2012
Published online: December 4, 2012
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