Visible-light-induced cleavage of 4-α-amino acid substituted naphthalimides and its application in DNA photocleavage

Article abstract of DOI:10.1039/c5ob00302d

A new kind of visible-light photocleavable molecule, 4-α-amino acid substituted naphthalimide, is reported. The cleavage occurred at the C-N bond between the 4-amino and the amino acid residue and released a 4-aminonaphthalimide. A lysine substituted naphthalimide exhibited a strong DNA photocleavage activity when irradiated with a blue light LED. This journal is

Full text of DOI:10.1039/c5ob00302d

Organic &
Biomolecular Chemistry
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Visible-light-induced cleavage of 4-α-amino acid
substituted naphthalimides and its application in
DNA photocleavage†
Cite this: Org. Biomol. Chem., 2015,
13, 3931
Jin Zhou,‡ Canliang Fang,‡ Ying Liu, Yao Zhao, Nan Zhang, Xiangjun Liu, Fuyi Wang
and Dihua Shangguan*
Received 12th February 2015,
Accepted 12th February 2015
A new kind of visible-light photocleavable molecule, 4-α-amino acid substituted naphthalimide, is
reported. The cleavage occurred at the C–N bond between the 4-amino and the amino acid residue and
released a 4-aminonaphthalimide. A lysine substituted naphthalimide exhibited a strong DNA photo-
cleavage activity when irradiated with a blue light LED.
DOI: 10.1039/c5ob00302d
www.rsc.org/obc
Organic molecules capable of releasing active moieties after knowledge, no photocleavable naphthalimide has been
light irradiation are termed photocages or photocleavable reported. Here, we report the photocleavage property of
(photolabile, photoreleasable, photoremovable or photoactivat- 4-α-amino acid substituted naphthalimides under visible light
able) molecules. These molecules are extensively explored in irradiation.
A designed lysine substituted naphthalimide
biological applications, organic synthesis, and photolitho- (DNLys) was demonstrated to have a strong DNA photocleavage
graphic techniques as tools for spatial and temporal control.1 activity when irradiated with a blue light LED (465–470 nm).
However, most of the photocleavable molecules require
In our research, we observed that a lysine substituted
irradiation with short wavelength light (<400 nm),1b which naphthalimide, 1, could be gradually cleaved under room
limits their applications in living biosystems. Therefore, great light. Because 4-aminonaphthalimides are very stable under
efforts have been made to develop photocleavable chromo- room light, we suspected that the photocleavage of 1 might
phores that absorb visible light or near-IR light,2 especially the relate to the α-carboxyl group of lysine. Therefore we syn-
photocleavable fluorophores that have the ability to act both thesized seven 4-α-amino acid substituted naphthalimides
as a “phototrigger” for active molecule release and a “fluoro- (compounds 1–7) and eight 4-amino substituted naphthal-
phore” for active molecule visualization. So far, only a few imides (without an α-carboxyl group) (compounds 8–15)
fluorophores have been modified as photocleavable groups (Fig. 1). These 4-aminonaphthalimides were dissolved in
under visible light, such as coumarin,3 xanthene,4 perylene5 ethanol, ethanol containing 1% trifluoroacetic acid (TFA) or
and organic–inorganic hybrid ruthenium compounds.6
ethanol containing 1% ammonia, put in glass bottles and
Naphthalimide derivatives have been used in a wide variety then exposed to daylight for 2 h. The thin layer chromato-
of fields. As a class of DNA-intercalating agents, they have graphy (TLC) assay showed that all the seven α-amino acid sub-
been extensively explored as antitumor agents.7 Due to the stituted naphthalimides were photocleaved (compared with
electronic push–pull structure, 3- and 4-aminonaphthalimides lane a) in all the three solutions, however other 4-amino sub-
show strong absorption and emission with a large Stokes shift stituted naphthalimides were not changed except for com-
in the visible region, and have been widely developed as fluore- pound 13 in 1% TFA (Fig. 1). It is worth noting that 13 (which
scent and colorimetric probes for detection of ions and bio- contains a γ-carboxyl group) was photocleaved in a 1% TFA
active molecules,8 as well as for cellular imaging.9 These solution, but not under neutral and basic conditions (1%
probes are considered to have high photostability when ammonia). This set of results suggests that 4-α-amino acid
irradiated at their absorption maxima.9a,10 To the best of our substituted naphthalimides are highly photolabile.
To understand the mechanism of the photocleavage of
4-α-amino acid substituted naphthalimides, we compared the
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
photocleaved fluorescent products of 1 and 2 after irradiation
in an ethanol solution by HPLC with detection at 430 nm
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing, 100190, China. E-mail: sgdh@iccas.ac.cn; Fax: +86-10-62528509
(Fig. S1†). The HPLC results showed that the photocleaved
†Electronic supplementary information (ESI) available: Experimental infor-
mation and figures. See DOI: 10.1039/c5ob00302d
fluorescent products of both compounds were identical. The
1H NMR and 13C-NMR spectra of the photocleaved fluorescent
‡Primary authors contributed equally to this work.
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Fig. 2 The
photocleavage
of
4-α-amino
acid
substituted
naphthalimides.
why other 4-amino-1,8-naphthalimides (without an α-carboxyl
group) cannot be photocleaved (Fig. 2). 4-Amino-1,8-naphthal-
imides are known to be intramolecular charge transfer (ICT)
fluorophores with “push–pull” substituent pairs (electron
donor/acceptor).8 The photochemical excitation leads to the
ICT from the 4-amino group to the excited imide, resulting in
the cleavage of the C–N bond of the α-amino acid and the
intramolecular hydrogen transfer from the α-carboxyl group to
the amino group to produce compound 16 and a radical of the
amino acid residue. Subsequently the radial may react with
the surrounding molecules and/or further decompose to form
various products.
Naphthalimides represent an important class of DNA
binders that have been extensively explored as antitumor
agents, and a few of them have been shown to exhibit the
activity of photoinduced DNA cleavage under UV light
irradiation.11,12 It is well known that UV-light can cause DNA
damage. The above results have shown the photocleavage
property of 4-α-amino acid substituted naphthalimides, this
property may endow them with the ability to photocleave DNA.
Fig. 1 Photocleavage of 4-amino substituted naphthalimides. Top:
structures of 4-amino substituted naphthalimides; 1–7: substituted by
α-amino acids; 8–15: substituted by other amino compounds. Bottom:
TLC analysis of 4-amino substituted naphthalimides (1–15) after
exposure to daylight for 2 h; lane a: without irradiation; lane b: irradiated
in 1% TFA; lane c: irradiated in ethanol; lane d: irradiated in 1% ammonia.
product (Fig. S2†) only showed 1H signals higher than 6.5 ppm To prove this hypothesis, we synthesized a lysine substituted
and 13C signals higher than 105 ppm, suggesting that no naphthalimide, DNLys (N-dimethylaminopropyl-4-(1-carboxyl-
saturated alkyl was linked on the product. The EI-MS showed 5-amino-amylamino)-1,8-naphthalimide), and its photo-
the molecular ion peak at m/z 212, which corresponded to cleaved product, DNNH (N-dimethylaminopropyl-4-amino-1,8-
compound 16 (Fig. 1).
naphthalimide) (Fig. 3 and S7†). The introduction of the
The other part of the photocleaved products of 4-α-amino dimethylaminopropyl group and lysine is to increase the
acid substituted naphthalimides was investigated with com- affinity of naphthalimide to DNA, because the amino groups
pound 6, because the nitrobenzoyl moiety of 6 can be moni- bear positive charges under physiological conditions9d and
tored using a UV detector (270 nm). HPLC analysis showed can enhance the DNA cleavage efficiency of the light-activated
that a peak (15 min) of the photocleaved product only with the DNA cleaver.13 The absorption and emission spectra of DNLys
UV absorption at 270 nm (without absorption at 430 nm) was and DNNH showed that the maximum absorption was in the
observed (Fig. S3†). However, this product was unstable, and range of 435–445 nm (Fig. S8†), and the maximum emission
changed to many other compounds during purification was in the range of 545–550 nm (Fig. S9†). The addition of
(Fig. S4 and S5†). In the ESI-MS analysis of the reaction DNA slightly decreased their absorption and slightly increased
mixture of photocleaved 6, we found one group of molecules their emission.
with molecular weights of 296, 280, 264, 250, and 232, and the
The photocleavage experiment of DNLys was performed
MS/MS analysis of these molecules (296, 280, 264 and 250) under a LED array (3 W; 465–470 nm). 82% of DNLys was
showed fragment ions of 233 and 150, which suggests a series observed to be cleaved to DNNH after exposure to blue light in
of molecules containing the nitrobenzoyl moiety (Fig. S6†).
methanol for 1 h (Fig. S10†). However, the photocleavage of
Based on the structure of 4-α-amino acid substituted DNLys in phosphate buffered saline (PBS, pH 7.4) was much
naphthalimides, the formation of a five-membered-ring intra- slower than that in methanol (Fig. 3), only 23% of DNLys was
molecular hydrogen-bond between the α-carboxyl group and cleaved in 4 h. It is interesting that the addition of DNA could
the amino group may be critical for the photocleavage of these accelerate the photocleavage of DNLys in PBS, and 19%, 32%
4-α-amino acid substituted naphthalimides, which can explain and 63% of it were cleaved in 1, 2 and 4 h (Fig. 3). These
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Fig. 3 The structure of DNLys/DNNH and HPLC analysis of the photo-
cleaved DNLys (200 µM). (a) Photocleaved in pH 7.4 PBS; (b) photo-
cleaved in pH 7.4 PBS in the presence of CT-DNA (100 µM in base pairs).
Samples were irradiated under 465–470 nm LED light for 1, 2, and 4 h at
room temperature, and then applied for the RP-HPLC assay, mobile
phase: methanol–H2O (0.1% TFA), 4 : 6; detected at 245 nm.
Fig. 4 Cleavage of closed supercoiled pBR322 DNA (0.5 µg/20 µL) irra-
diated under blue LED light at room temperature. a. (left) Agarose gel
assay of DNA cleaved at different concentrations (µM) of DNLys or
DNNH after 0.5 h irradiation; (right) quantified plots of DNA as a function
of DNLys concentration. b. (left) Gel assay of DNA cleaved by DNLys (5
µM) after irradiation for different times (min); (right) quantified plots of
DNA as a function of irradiation time. c. Gel (left) and quantified (right)
assay of effects of additives on DNA cleavage by DNLys (5 µM) after
0.5 h irradiation, Ctr 1: DNA only without irradiation; Ctr 2: DNA only;
Ctr 3: DNA + DNLys; additives: DTT (50 mM); NaN3 (100 mM); SA
(salicylic acid, 20 mM); His (histidine, 5 mM); EtOH (ethanol, 2 M).
results suggest that the interaction of DNA and DNLys could
promote the photocleavage of DNLys and implied the feasi-
bility for DNA cleavage. These results further confirm that
4-α-amino acid substituted naphthalimides can be photo-
cleaved by visible light and produce stable fluorescent 4-amino
naphthalimides.
The DNA photocleavage experiments were performed using responsible for DNA cleavage by light-activated DNLys, stan-
a closed supercoiled pBR322 DNA under irradiation with a dard scavengers and inhibitors of reactive oxygen species were
blue LED at room temperature. The cleaved DNA was analyzed added respectively to the reaction system, i.e. singlet oxygen
on 1% agarose gel. After cleavage, supercoiled pBR322 DNA scavengers (sodium azide (NaN3) and histidine (His)), super-
(form I) would convert to relaxed circular DNA (form II). oxide radical scavenger (dithiothreitol (DTT)), and hydroxyl
As shown in Fig. 4a, DNLys did not cleave DNA without radical scavengers (salicylic acid (SA) and ethanol (EtOH)). As
irradiation. After irradiation for 0.5 h, DNA cleavage was shown in Fig. 4c, a certain inhibition effect of all the test
observed in the presence of different concentrations of DNLys, scavengers was observed. Among them, the singlet oxygen
the cleavage efficiency increased with the increase of DNLys scavenger, NaN3 and histidine showed the strongest and the
concentration. 2–5 µM DNLys could cause most of the DNA to second strongest inhibition effect on the DNA cleavage. DNA
be cleaved under irradiation. As expected, 30 µM DNNH photocleavage is usually caused by reactive oxygen species or
(photocleavage product of DNLys) did not cause notable DNA the direct reaction with the light-activated DNA cleaver.13b–d
cleavage under the same conditions. The kinetics of DNA This set of results suggests that the DNA photocleavage in the
photocleavage showed that the intact DNA greatly decreased presence of DNLys may involve the reactive oxygen species,
with the irradiation time, and most of the plasmid DNA was especially the singlet oxygen.15
consumed within 30 min of irradiation (Fig. 4b). The control
The preceding results have shown that the DNA cleavage
experiments showed that very little DNA damage was observed was only caused by the photocleavage of DNLys, and not
in the absence of DNLys even after irradiation for 60 min. caused by the irradiated DNNH, suggesting that the singlet
Since DNNH also undergo the photo-induced ICT process, oxygen was not generated from the photosensitized 4-amino-
these results suggest that the DNA cleavage was related to naphthalimide. The photocleavage of DNLys produced a
the photocleavage of DNLys, and not to the ICT excited state of radical of amino acid residues, thus the singlet oxygen may be
4-aminonaphthalimides.
generated by the reaction of dissolved oxygen with the excited
The photoinduced DNA damage by synthetic DNA cleavage radical states. Even so, we could not exclude the direct mech-
agents usually involves the reactive oxygen species, such as anism of DNA damage by the excited radical states because
hydroxyl radicals, singlet oxygen and superoxide anions.14 of the high activity of the radical. Further research will focus
In order to determine whether the reactive oxygen species are on the mechanistic details of photocleavage of 4-α-amino acid
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substituted naphthalimides, as well as of the DNA cleavage by
the irradiated DNLys.
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The above results showed that 4-α-amino acid substituted
naphthalimides could be photoactivated with blue light and
release a fluorescent product, 4-amino naphthalimide. The
4-α-amino acid substituted naphthalimides can be easily syn-
thesized by substitution reaction of 4-bromine-1,8-naphthal-
imides with different α-amino acids. Furthermore, through the
reaction with the amino acid residues, the 4-α-amino acid sub-
stituted naphthalimides (e.g. lysine and glutamic acid substi-
tuted) could be further linked to peptides, proteins and other
biologically interesting molecules. In addition, the substituent
at the N-atom at the 9-position of naphthalimides (imide
N-atom) can be easily changed to different molecules by reac-
tion of 4-bromine-1,8-naphthalanhydride with the corres-
ponding compounds containing the NH2 group. Therefore the
4-α-amino acid substituted naphthalimide fluorophore could
act as a multifunctional platform for the construction of light-
control systems by linking different functional molecules. The
visible light activation makes the constructed systems hold the
potential in biological applications, e.g. spatial and temporal
control of drug delivery. The fluorescence emission of
4-α-amino acid substituted naphthalimides and their photo-
cleaved products makes the constructed systems work in
visualization. The photoactivation of DNLys showed the photo-
cleavage ability towards DNA, suggesting its potential appli-
cation in phototherapy after further modifying the 4-α-amino
acid substituted naphthalimides to enhance the DNA binding
ability.
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Conclusions
In summary, we describe the photocleavage property of
4-α-amino acid substituted naphthalimides upon blue light
irradiation. The photocleavage of these molecules occurred at
the C–N bond between 4-amino and the amino acid residue
and released a fluorescent product, 4-aminonaphthalimide.
DNA was found to enhance the photocleavage of a lysine-sub-
stituted naphthalimide (DNLys), which caused the cleavage of
DNA. Because of the ease of the modification on the 4-position
and 9-position of the naphthalimide ring, this finding
provided a multifunctional platform for the construction of
light-control systems.
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We gratefully acknowledge the financial support from
the grant 973 Program (2011CB935800, 2011CB911000 and
2013CB933700) and NSF of China (21275149, 21375135,
21205124 and 21321003).
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