Communication
[
18b,21]
at 410 nm, with a broad and weak absorption at 600 nm.
Qualitative analysis by matrix-assisted laser desorption/ionisa-
tion time-of-flight (MALDI-TOF) mass spectrometry of a typical
reaction mixture using a 12.5 mm concentration of 2 displayed
a signal corresponding to unreactive Tel22 (6966 Da), together
with an additional signal detected for a species with 7568 Da
(Figure S6 in the Supporting Information), in agreement with
a [M+1] adduct of a Tel22 construct with 2. The possible rever-
sible character of the 2–Tel22 adduct was evaluated as a func-
tion of temperature and salt concentration. The adduct was
stable after 10 min of incubation at 958C (Figure S7a in the
Supporting Information) and in a 0.5m KCl solution (Figure S7b
in the Supporting Information). Therefore, the bands with
lower mobility were attributed to a covalently modified Tel22,
as the photoadducts provide additional mass and positive
charges. To directly identify the specific base involved in the
photoreaction, we constructed three telomere sequences
Tel22-Cn (n=1, 2, 3), in which the first (n=1) or second (n=2)
T residues or third base A (n=3) in the three TTA loops were
simultaneously replaced with C residues. A fourth telomere se-
quence, Tel22-T3, in which the A residues of the loops were re-
placed with T residues was investigated as well (Figure 4).
Therefore, our LFP data on the bimolecular model suggest that
NDI 2 should be able to generate a phenoxyl radical upon
green light irradiation. Following the notion that phenoxyl rad-
icals react as carbon radicals at the ortho position with thymine
[
22]
and thymidine, we investigated the ability of NDI 2 to deliv-
er reactive phenoxyl radicals, generated by intramolecular PeT,
onto the thymine residues of the Tel22 loops. The photoreac-
tivity of 2 in the presence of the G4 folded Tel22 or scrambled
(
scr, same base composition in a sequence unable to fold into
G4) ss and ds Tel22 was initially assessed. Increasing amounts
of compound 2 (12 nm–50 mm) were irradiated with folded or
3
2
scr P-labelled Tel22 templates (0.25 mm), at 208C for 24 h,
using visible light (one lamp, 15 W, 400 nm<l<650 nm). Iden-
tical samples were incubated in the dark.
The reaction samples were analysed by denaturing poly-
acrylamide gel electrophoresis (PAGE). A concentration-depen-
dent lower-mobility band was observed in the presence of the
G4 folded Tel22, from 50 nm to 12.5 mm (0.2:1 to 50:1 2/Tel22
ratio). The adduct yield was 64% at 12.5 mm (Figure 3). To our
knowledge, this is the highest adduct efficiency ever achieved
for G4 covalent targeting.
Figure 4. PAGE (denaturing conditions) of Tel22 and mutant Tel22 oligonu-
cleotides (0.25 mm) in the presence of 2 and visible light.
Figure 3. PAGE under denaturing conditions of increasing amounts of 2 in
the presence of the G4 folded Tel22 and scrambled ss and ds Tel22
Mutant Tel22-T3 and Tel22-C3 generated gel bands upon ir-
radiation, with similar mobility and only slightly lower efficien-
cy to that achieved with the unmodified Tel22. Therefore, loop
adenines appear not to be the main target sites. On the con-
trary, the efficiency of the covalent adduct generation was
strongly affected by mutations replacing T residues with C resi-
dues. In fact, Tel22-C2 was fully unreactive, as no covalent
adduct was detected by irradiation in the presence of 2
(Figure 4). Likewise, Tel22-C1 was alkylated to a very low extent
(3.5% yield). The dichroic behaviour of the four modified oligo-
nucleotides in the presence of NDI 2 was similar, but not su-
perimposable (Figure 5). In particular, among the modified oli-
gonucleotides used, oligonucleotide Tel22-T3, with an addition-
al shoulder at 260 nm, was the least similar to Tel22.
Nevertheless, such a topological difference affected the effi-
ciency of the covalent modification only marginally. In addi-
tion, Tel22-C1, the dichroic behaviour of which is the closest to
that of Tel22, was the second-least-alkylated oligonucleotide.
Therefore, the striking selective photoreactivity between Tel22
and Tel22-C1 or Tel22-C2 is likely caused by the different base
composition of the loops, rather than the G4 topology; in par-
ticular, by the presence of accessible T residues in the middle
of the loop, such as T6, T12 and T18. The evaluation of the al-
kylation selectivity on three additional mutant Tel22, namely,
C2a, C2b and C3c (see Figure 6 for the composition and Fig-
(0.25 mm), upon visible-light irradiation. Control samples were either irradiat-
ed with visible light in the absence of 2 or incubated in the dark in the pres-
ence of 2 at 50 mm.
In contrast, the same band was barely detectable in the
scrambled ss Tel22 substrate and was absent in the scrambled
ds oligonucleotide, even when treated with a 200:1 2/Tel22
ratio (Figure 3). These data indicate a remarkable selective in-
teraction of the G4 structure over unstructured or double-helix
DNA by 2. Irradiation of 1 with the G4 folded Tel22 under iden-
tical conditions produced at the highest concentrations only
a faint band with similar gel mobility (Figure S5 in the Support-
ing Information). The lack of reactivity of 1 is consistent with
LFP data, as the phenol moiety is more difficult to oxidise by
the triplet-excited state of the NDI core than its Mannich base
ꢀ
5
analogue. The behaviour of NDI 2 (3ꢁ10 m) was further in-
vestigated by LFP, in the presence of Tel22 (1 equiv; potassium
phosphate buffer, 20 mm in K PO , pH 7.0). Unfortunately, we
3
4
were unable to detect any transient species. It is likely that the
fast intramolecular deactivation of the photogenerated radical
ions by back electron transfer competes favourably with the
generation of the phenoxyl radical (Scheme S1 in the Support-
ing Information). Such a hypothesis is consistent with the long
irradiation time required to reach the high conversion of Tel22.
Chem. Eur. J. 2015, 21, 2330 – 2334
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