Engineering pH-gated transitions for selective and efficient double-strand DNA photocleavage in hypoxic tumors

Article abstract of DOI:10.1021/jm2010282

We describe a family of hybrid compounds for the most efficient light-activated double-strand (ds) DNA cleavage known to date. This family represents the second generation of "switchable" molecular systems for pH-gated ds DNA-cleavage which combine a potent DNA-photocleaver and a pH-regulated part derived from a dipeptide. Design of the pH-switchable part utilizes amino groups of different basicity. Whereas the basic amino groups are protonated throughout the biologically relevant pH range, the pH-gating amines undergo protonation at the pH threshold which separates cancer and normal cells. Control over the reactivity and selectivity is achieved via transformation of the initial protonation state (a monocation or a dication) into a trication at the acidic pH. This change leads to an extraordinary increase in the efficiency of ds DNA cleavage leading to the ds:ss ratios comparable with the most efficient nonenzymatic ds DNA cleavers. Statistical analysis reveals that these high ds:ss ratios result from the combination of several factors: (a) true double-stranded cleavage, and (b) conversion of single-stranded (ss)-scission into ds cleavage. Considerable part of ds cleavage is also produced via the combination of ss cleavage events. (Figure presented)

Full text of DOI:10.1021/jm2010282

Article
pubs.acs.org/jmc
Engineering pH-Gated Transitions for Selective and Efficient Double-
Strand DNA Photocleavage in Hypoxic Tumors
Wang-Yong Yang, Saumya Roy, Boondaniwon Phrathep, Zach Rengert, Rachael Kenworthy,
Diego A. R. Zorio, and Igor V. Alabugin*
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, United States
S
* Supporting Information
ABSTRACT: We describe a family of hybrid compounds for
the most efficient light-activated double-strand (ds) DNA
cleavage known to date. This family represents the second
generation of “switchable” molecular systems for pH-gated ds
DNA-cleavage which combine a potent DNA-photocleaver
and a pH-regulated part derived from a dipeptide. Design of
the pH-switchable part utilizes amino groups of different
basicity. Whereas the basic amino groups are protonated
throughout the biologically relevant pH range, the pH-gating
amines undergo protonation at the pH threshold which
separates cancer and normal cells. Control over the reactivity and selectivity is achieved via transformation of the initial
protonation state (a monocation or a dication) into a trication at the acidic pH. This change leads to an extraordinary increase in
the efficiency of ds DNA cleavage leading to the ds:ss ratios comparable with the most efficient nonenzymatic ds DNA cleavers.
Statistical analysis reveals that these high ds:ss ratios result from the combination of several factors: (a) true double-stranded
cleavage, and (b) conversion of single-stranded (ss)-scission into ds cleavage. Considerable part of ds cleavage is also produced
via the combination of ss cleavage events.
single-strand (ss) DNA damage is easily repaired by enzymatic
processes,21 the repair of double-strand (ds) DNA cleavage is
more difficult and, thus, it can initiate self-programmed cell
death, or apoptosis. Therefore, ds DNA cleavage is a more
efficient tool for cancer therapy as long as it can be induced
selectively in cancer cells avoiding damage to healthy cells.
Light-activated DNA-cleavers provide spatial and temporal
control over DNA cleavage, allowing drug activation in the
right place and at the right time, when concentration of the
drug is highest in the cancer tissues.22 In our previous work, we
have added another level of selectivity via the development of
the first pH-controlled system for ds DNA cleavage (Figure 1).
This hybrid system combined an efficient DNA-cleaver capable
of operating within the physiological pH range when attached
to a pH-sensitive functionality.23,24
The new family of pH-dependent DNA photocleavers
displayed a number of unique properties. In particular, these
lysine conjugates showed efficient ds DNA cleavage (ss:ds
=2:1) rivaling the efficiency of calicheamicin,24 selective
cleavage at G-sites flanking AT-tracks,25 and ability to convert
ss DNA damage into ds DNA damage.23,26 We have also shown
that these compounds cleave intracellular DNA,27 display light-
induced cytotoxicity to several cancer cell lines,24 and are
susceptible to two-photon absorption (TPA) activation.28
INTRODUCTION
■
Molecular systems, where structure, reactivity, and biological
activity are “switchable” via an externally controlled factor,
create new opportunities for the design of drug delivery
systems,1 optical sensors,2,3 molecular switches/logic gate
mimics,4,5 and a variety of new materials.6 Out of the many
external stimuli, pH-gated “switching” is especially useful for
addressing biochemical and environmental processes which
depend on the acidity of the medium.7
In particular, the relatively acidic extracellular environment of
solid tumors8,9 lends itself for the design of tumor-specific pH-
activated chemical agents.10 Hyperglycemia and/or such drugs
as amiloride, nigericin, and hydralyzine, are able to lower the
intracellular pH of cancer cells as well. At dosages that do not
affect the normal cells, amiloride and nigericin has been
reported to drop the intracellular pH in a number of tumor cell
types from 7.2 to 6.2−6.6.11−14 When combined with
hyperglycemia and/or hypoxia, further acidification to pH as
low as 5.5 is possible.15,16
One way to take advantage of these differences involves the
development of pH-gated DNA cleaving agents.17 The promise
of DNA as a target for cancer therapy18 is illustrated by the
astounding biological activity of natural enediyne antibiotics.
These compounds, hailed as “the most potent family of
anticancer agents”,19 can induce ds-DNA cleavage via
abstraction of two hydrogen atoms, one from each strand of
DNA duplex, with the most efficient ds DNA-cleaver from this
family, calicheamicin, forming ∼25−33% of ds breaks.20 While
Received: August 2, 2011
Published: November 3, 2011
© 2011 American Chemical Society
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amino groups of the lysine residue free for being included into
pH-gating and facilitation of DNA-damage.
Potentially, the lysine group can interact with different forms
of DNA in a variety of ways. For example, a large body of
research documents the possibility of cross-link formation via a
reaction with nucleobases.30 Several mechanistic scenarios
operate for different types of oxidative DNA-damage (e.g., 8-
oxo-guanine (OG), guanine radical cation or oxidized 8-oxo-
guanine (OGox) Scheme 1).31 Although the selectivity of
nucleophilic attack of the lysine amino group at the oxidized
DNA depends on the type of DNA damage, both C5 and C8
attacks can lead, after a rearrangement, to the formation of
cross-linked spiro-adducts. Alternatively, formation of lysine-
DNA adducts can also occur through initial oxidation of lysine
and reaction of an N-centered radical with DNA.32 This DNA−
protein cross-linking can block DNA replication and, if not
repaired, cause cell death.
A very interesting model for the possible role of lysine in
converting OG sites into strand scission is suggested by the
mechanism of action of DNA glycosylase/β-lyase OGG1 in
which lysine acts both to displace the oxoguanine base (with
the formation of an abasic site) and to promote subsequent
elimination via an transient formation of a Schiff base.33
The oxidation of DNA and the formation of cross-linked
products in the above examples are caused by two independent
chemical agents. It is conceivable that hybrid agents which
combine photooxidant and lysine would accomplish such cross-
links in a more efficient manner.
The pH-gated behavior in this design is based on the
different properties of these two amino groups.34 The auxiliary
amino group is protonated at a wider range of physiological
conditions. This group enhances solubility of conjugates in
water, increases their affinity to the negatively charged
Figure 1. Comparison of C-lysine conjugates and N-lysine conjugates.
In this study, we describe the design and properties of light-
activated systems with the ratio of ds:ss cleavage exceeding that of
calicheamicin. Importantly, the ds-cleavage in such systems is
suitable for selective targeting of cancer cells because this
process is pH-gated: its efficiency increases dramatically at a
relatively narrow and predefined pH point close to the
threshold between cancerous and healthy cells.
The practical implementation of this strategy has been based
on attachment of the DNA-cleaving moiety to the carboxyl
group of lysine. This mode of attachment is different from a
more common formation of a classic peptide bond via the α-
amino lysine group.29 Importantly, this choice leaves both
a
Scheme 1. Multiple Roles of Lysine Residues in Processing DNA Damage
a
(top left) Formation of deoxyguanosine-lysine adducts via a two-step mechanism which involves chemical or photochemical DNA oxidation
followed by reaction of 8-oxoguanine with lysine.31 (top right) Conversion of ss damage into ds damage.26 (bottom left) Acceleration of strand
scission via Schiff base formation with lysine.33
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Figure 2. Structures of bis-lysine conjugates studied in this work.
backbone of DNA, and modulates the basicity of the less basic
(pKa∼7) α-amino group. This “pH-gating” amino group
undergoes protonation only under a smaller subset of
conditions which enables a switch in DNA binding and
photophysical properties of the conjugate at the desired pH
threshold. At this threshold, the DNA-cleaver is transformed
from a monocation to a dication, a change which leads to a
dramatic increase in the DNA-cleaving ability.
To increase the selectivity, the difference in reactivity of the
two forms should be as large as possible. A priori, one could
expect that the transformation from a monocation to a trication
would increase the contrast in reactivity even further. To test
the possibility of designing systems activated via conversion
into a multiprotonated state, we designed two types of bis-
lysine peptides: one connected through a usual peptide bond
via the α-amino group (5, 6 in Figure 2) and the other is
connected via the ε-amino group (3, 4 in Figure 2).
target compounds 3−6, respectively. The final products were
fully characterized by spectroscopic methods and used in the
DNA-photocleavage studies as TFA salts.
Acidity of pH-Gating Groups. Acid−base equilibrium of
α-amino groups is the key to pH-gated reactivity of these
compounds. To obtain accurate information about this
equilibrium, we have measured the pKa values of the three
ammonium groups using fluorescence and NMR spectroscopy.
The two methods differ in the type of information and amount
of detail that they provide.
Fluorescence Titrations. Fluorescence directly reflects
the protonation state of the α-amino group closest to the
chromophore because the donor lone pair of the chromophore
can quench the chromophore excited state via electron transfer
(Figure 4). Once the donor amine is protonated, this
quenching mechanism is eliminated and a large increase in
the fluorescence quantum yield is observed.
This design also allowed us to keep the same total number of
amino groups in the conjugate but vary their nature and relative
basicity (two α-/one ε-group in 3 and 4, two ε-/one α-group in
5 and 6). In each of the cases, the final protonation state
corresponds to a tricationic dipeptide moiety expected to
display the strongest binding to DNA (Figure 3). However, the
initial protonation state is expected to be different: mono-
cationic for conjugates 3 and 4 but dicationic for conjugates 5
and 6. Because the α-amino groups are responsible for the
observed pH-dependence in the mono lysine conjugates, we
expected that the above structural variations will have a large
impact on the pH-dependence of DNA cleavage by the new
bis-lysine conjugates.
All pH titration experiments were carried out with only
monoacetylene conjugates due to aggregation observed for the
more hydrophobic enediyne conjugates at the higher pH. The
pH titration with bis-lysine conjugate 3 showed only single
titration curve corresponding to pKa of 7.0. The similarity of
this value to the pKa of the α-amino group of monolysine
conjugate 1 (7.0) and the high efficiency of fluorescence
modulation suggest that this pH titration curve corresponds to
deprotonation of α-ammonium group.
In contrast, the pH titration with bis-lysine conjugate 5 has
two titration curves with the pKa values of 7.1 and 10.1.
Although conjugates 3 and 5 have a different number of ε-
ammonium groups (one for 3 and two for 5), the similarity in
the first pKa values indicates that the α-amino group basicity
does not depend strongly on the number of the ε-amino
groups. In the pH titration of compound 5, the increase in the
efficiency of fluorescence quenching due the first deprotonation
is less pronounced (20% vs 50% for 3), likely due to the greater
distance between the chromophore and the α-amino group.
The relatively inefficient quenching of the excitation by the α-
amino group should lead to the less pronounced efficiency of
pH-modulation for the di-ε conjugate 5. The unexpected
RESULTS AND DISCUSSION
■
Synthesis. The target peptide conjugates we synthesized by
coupling Boc-protected lysine dipeptides (9 and 10) with
anilines 7 and 8 using dicyclohexylcarbodiimide (DCC) and 1-
hydroxybenzotriazole (HOBt) as coupling reagents (Scheme
2). The synthesis of anilines 7 and 8 were described previously
by our group.23,24 Removal of Boc-groups of compounds 11−
14 with trifluoroacetic acid (TFA) in CH2Cl2 produced the
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Figure 3. Design and structural variations for the second generation of lysine conjugates.
response of fluorescence of compound 5 to the protonation
state of at least one of the two ε-amino groups may indicate
presence of a folded conformation in the solution where one of
the remote ammonium groups participates in a cation−π
interaction with the chromophore or H-bonding with the
amide group directly attached to the chromophore.
amines, suggesting a lack of direct intramolecular interactions,
e.g. H-bonding between the remote amino groups.
Interestingly, the very close pKa values of the two α-amino
groups in 3 indicate that this molecule is converted from a
monocation to a trication at a relatively narrow pH region
between pH 7 and 8 and should respond stronger to the proton
flux than the isomeric conjugate 5 (vide infra) (Table 1).
Double-Strand Cleavage of Supercoiled DNA. Encour-
aged by these results, we proceeded to investigate the pH-
dependency of DNA-cleavage. Not only did both bis-lysine
conjugates clearly show enhancement of DNA cleavage as a
function of pH, but also the amount of ds-DNA cleavage
produced by 15 μM of mono acetylene conjugates at pH 6
(Form III/Form II ratios of 2:1 and 1:1 for di-α 3 and di-ε 5,
respectively, Figure 6) represents the highest ds:ss ratios
reported in the literature, by far exceeding the analogous ratios
for calicheamicin (1:2/1:3),20 bleomycin (1:6−1:10)39−44 and
monolysine conjugates described by us earlier (1:2 at pH 6).24
The new conjugates reported in this work show significant
increase in the efficiency of ds-DNA cleavage in comparison to
the analogous monolysine conjugates. The correlation between
the efficiency of DNA cleavage and protonation state of the
1
NMR Titration. H NMR pH titrations provided further
insight into the acid−base equilibrium of these peptide systems
at the atomic resolution. This method allows independent
monitoring of each of the three protonation events through
observations of chemical shifts for hydrogens spatially close to
each of the respective amino groups35 during the pH (pD)
titration (Figure 5).36,37
The titration curves for 3 clearly showed that two α-amino
groups are less basic (pKa = 7.2, 7.3) than the ε-amino group
(pKa = 11.3). The NMR pKa values for 5 were 7.2 for α-amino
group and 10.2 for both ε-amino groups (Figure 5). These pKa
values are consistent with the values from the fluorometric
titrations. The close pKa values of the two α-amino groups (7.2
and 7.3) in 3 and of the two ε-amino groups (10.2) in 5
indicate again that the amino group basicity in these conjugates
is not strongly affected by the protonation state of other
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a
Scheme 2. Synthetic Schemes for the Preparation of Enediyne and Acetylene Conjugates
a
Reagents and conditions: (a) Lys(N-α-Boc)-OMe, EDCl, HOBt, DIPEA, CH2Cl2; (b) Lys(N-ε-Boc)-OMe, EDCl, HOBt, DIPEA, CH2Cl2; (c)
LiOH, THF:MeOH:H2O; (d) DCC, HOBt, CH2Cl2, then 7/8; (e) TFA/CH2Cl2 (1:1).
Figure 4. Schematic representation of electron transfer as means of quenching of fluorescence of an excited state chromophore and quantified
changes in fluorescence intensity as a function of pH and their fit to the Henderson−Hasselbalch equation for acid−base equilibrium for bis-lysine
di-α and di-ε conjugates 3 (top) and 5 (bottom).
dipeptide moieties illustrated by Figure 7 suggests that the
tricationic form plays a particularly significant role in the ds-
cleavage. Interestingly, although the correlation is observed for
both the di-α and di-ε peptides, it is stronger for the less
efficient di-ε dipeptide 5. At the lower pH, the di-α conjugate 3
is so efficient that further cleavage of DNA into even smaller
fragments is observed rendering the measurements less accurate
and suggesting even more efficient DNA cleavage that implied
by the ds:ss ratio alone. In addition, the plot of ds DNA
cleavage by 5 as a function of pH fits well to the Henderson−
Hasselbalch equation with 6.9 of pKa value (R2 = 0.961) (see
the Supporting Information).
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Figure 5. Chemical shift-pD titration plots for α- and ε-hydrogens in conjugates 3 and 5 (3 mM) in D2O.
Table 1. The pKa Values of Amino Groups in Conjugates 3 and 5 (The First pKa for Lysine Conjugate 1 and Lysine Is Given for
Comparison)
3
5
1
a
a
method
emission
7.0
1H NMR
emission
1H NMR
emission
7.0
lysine
b
pKa1
pKa2
pKa3
7.2
7.3
7.1
7.2
10.2
10.2
8.95
b
10.1
10.53
11.3
a
b
Data include a correction factor from ref 36. Data from ref 38.
Figure 6. (top) Quantified cleavage data for plasmid relaxation assay for DNA photocleavage with 15 μM of acetylenic conjugates 3 (left) and 5
(right) and 38 μM (bp) of pBR322 plasmid DNA at pH range of 6−9 after 10 min of UV (>300 nm) irradiation. Reported values represent the
average of three experiments. Code: form I, blue diamond; form II, red square; form III, green triangle.
Although the chemical mechanism for the DNA cleavage and
the possibility of DNA alkylation and DNA-lysine cross-linking
are under investigation at this point, the new conjugates
reported in this work unambiguously show significant increase
in the efficiency of DNA cleavage promoted by a change from a
monolysine to a bis-lysine moiety. Surprisingly, significant
DNA cleavage including some ds DNA cleavage is observed
even at pH 8, the conditions where the monolysine conjugates
do not cause this damage in appreciable amounts. This
observation suggests that bis-lysine acetylene conjugates are
overall more efficient DNA cleavers than their monolysine
conjugates.
By using the statistical test of Povirk,45 one can gain more
insight into the nature of the cleavage as long as there is a set
condition where all of the three DNA forms are present.46 This
test assumes a Poisson distribution of strand cuts and calculates
the average number of ss-(n1) and ds-breaks (n2) per DNA
molecule. As we observed complete disappearance of super-
coiled DNA after 10 min of irradiation in both the cases for
compound 3 as well as compound 5, we performed the DNA-
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Figure 7. Relative amounts of mono- (black), di- (green), and tricationic (red) forms as function of pH (top) and correlation of DNA ds-cleavage
with the trication mole fraction (bottom) for conjugates 3 (left) and 5 (right).
Figure 8. Photochemical cleavage of pBR322 plasmid DNA (38 μM/bp) by (a) 3 (15 μM) and (b) 5 (15 μM) in phosphate buffer (20 mM). Lane
1, DNA+hv for 10 min; lanes 2−9, DNA + 3/5 + hv for 0, 0.5, 1, 2, 4, 6, 8, and 10 min of irradiation (>300 nm) respectively at pH 6.0. Lane 10,
DNA+hv for 10 min; lanes 11−18, DNA + 3/5 + hv for 0, 0.5, 1, 2, 4, 6, 8, and 10 min of irradiation respectively at pH 7.0. Lane 19, DNA+hv for 10
min; lanes 20−27, DNA + 3/5 + hv for 0, 0.5, 1, 2, 4, 6, 8, and 10 min of irradiation respectively at pH 8.0.
photocleavage experiment as a function of irradiation time at
the pH conditions 6, 7, and 8 (Figure 8).
efficient ds-cleavage at pH 6 but the full statistical analysis is
impossible at the longer times due to disappearance of
supercoiled DNA. Compound 5 also displayed the high ds-
cleavage at pH 6.0, although it is less selective than compound
3 (see Supporting Information for quantitative analysis).
Because of the high reactivity of the conjugates, no
supercoiled DNA remained after 30 s of irradiation for
compound 5 in pH 6 at 15 μM concentration (Figure 10).
Lowering the concentration to 10 μM produced less DNA
cleavage, but due to absence of all the three DNA form in a
single lane it was impossible to calculate the n1/n2 ratio.
It is not possible to calculate the n1/n2 ratio for those times
where there is no supercoiled DNA present or no ds-cleavage
was found. For the conjugate 3 at pH 6, less than 17%
supercoiled DNA remains after 2 min irradiation and n1/n2
values could not be calculated for irradiation time longer than 2
min (Figure 9). The n1/n2 ratios at pH 6 slightly decrease with
the irradiation time, but there is not enough data to be certain
that this trend persists. Data showed low n1/n2 ratio at pH 7
which decreases further with time (from 4.4 at 2 min to 2.2
after 10 min) (Table 2). These data suggests the likeliness of
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Figure 9. Quantified data of plasmid relaxation assays with 15 μM of mono acetylene conjugate 3.
Table 2. Statistical Analysis of the Single-Strand and Double-
Strand Break Formation by 3 as a Function of Irradiation
a
Time at pH 6.0, 7.0, and 8.0
no. of ss-breaks (n1) and ds-
relative amounts (%) at pH 6.0 breaks (n2) per molecule
time/min
form I
form II
form III n1
n2
0.16
n1/n2
0.5
1
26.5
30.2
17.3
0.0
60.0
52.7
54.2
65.1
48.1
35.0
21.3
13.5
17.1
28.4
34.9
51.9
65.0
78.7
1.17
0.99
1.36
7.5
4.8
3.4
0.21
0.40
0.54
1.08
1.85
3.69
2
4
6
0.0
Figure 10. Photochemical cleavage of pBR322 supercoiled DNA (38
μM/bp) by 5 (10 μM) in phosphate buffer (20 mM). Lane 1, DNA
+hv for 10 min; lanes 2−9, DNA + 5 + hv for 0, 0.5, 1, 2, 4, 6, 8, and
10 min of irradiation (>300 nm), respectively, at pH 6.0.
8
0.0
10
0.0
no. of ss-breaks (n1) and ds-
breaks (n2) per molecule
relative amounts (%) at pH 7.0
time/min
form I
form II
form III
n1
n2
n1/n2
percentage of DNA cleavage decreases from 155% (200%
cleavage corresponds to full conversion of supercoiled DNA to
linear DNA = full ds DNA cleavage) to 136% to 32% for
conjugate 5 and from 159% to 75% to 50% for conjugate 3
(upon 15 →10 → 5 μM changes, data for the 10 μM
concentrations are shown in Figure 11-1). Although the
concentration effects are significant for both mono acetylene
conjugates, the acetylene conjugate 3 with two α- and one ε-
amino groups is affected by the concentration change much
more than the isomeric bis-lysine conjugate 5, which has two ε-
amino groups available (Figure 11a). These results suggests
that, although α-amino groups play the key role in pH-
regulation, the ε-amino groups which are analogous to amino
groups in the histone proteins may be important for further
“processing” of the oxidative DNA damage, possibly with the
involvement of processes outlined in Scheme 1. In addition, the
greater fluorescence enhancement upon the binding of
conjugate 5 to DNA relative to that for compound 3 (both
at pH 6) suggests that the chromophore moiety of 5 is placed
deeper inside of the hydrophobic environment of DNA at the
lower pH. The tighter binding of compound 3 in its tricationic
state can also be partially responsible for the relative
insensitivity of its DNA cleavage to the secondary factors.
At 10 μM concentration, both the full cleavage of supercoiled
DNA and the appearance of ds cleavage (average ss/ds ratio of
1.7:1, Figure 11b) by the “1α,2ε” conjugate 5 were observed
only at pH 6. At pH 7, ∼40% of supercoiled DNA remained
intact and no ds-damage was detected (∼50% of supercoiled
DNA was intact at pH 8). The particularly large increase in the
0.5
1
64.6
52.7
45.8
22.6
24.7
20.9
11.7
35.4
47.3
41.5
52.4
47.2
51.0
47.8
0.0
0.0
0.44
0.64
0.64
1.15
1.01
1.17
1.47
0.00
0.00
0.15
0.33
0.39
0.39
0.68
2
12.7
25.0
28.1
28.1
40.5
4.4
3.5
2.6
3.0
2.2
4
6
8
10
no. of ss-breaks (n1) and ds-
breaks (n2) per molecule
relative amounts (%) at pH 8.0
time/min
form I
form II
form III
n1
n2
n1/n2
0.5
1
86.4
83.9
77.8
58.6
40.8
42.3
26.3
13.6
16.1
22.2
41.4
59.2
45.6
52.3
0.0
0.0
0.15
0.18
0.25
0.00
0.90
0.72
1.06
0
0
2
0.0
0
4
0.0
0
6
0.0
0
8
12.1
21.3
0.14
0.27
5.3
3.9
10
a
Amount of form II DNA was calculated by deducting the amount of
form II DNA present in the control experiment at irradiation time = 0
(22%). See the Supporting Information for the calculated n1/n2 ratios
without this correction.
These data suggests that the high Form III/Form II ratios
result, at least partially, from the combination of very efficient
multiple ss-scission. The decreasing n1/n2 ratio also suggests
that conversion of ss-damage into ds-damage may also occur as
described in Scheme 1.
The additional concentration effects on the efficiency of
DNA cleavage are summarized in Figure 11. At pH 6, the total
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Figure 11. Quantified data of plasmid relaxation assays with 10 μM of mono acetylene conjugate 3 and 5 (a and b) and 10 μM of enediyne
conjugates 4 and 6 (c and d). Reported values represent the average of four experiments.
Figure 12. Effect of hydroxyl radical/singlet oxygen scavengers (20 mM) upon the efficiency of DNA cleavage at pH 6, 7, and 8 by 15 μM of 3 (left)
and 15 μM of 5 (right) after 10 min irradiation. Color coding: blue, form I; red, form II; green, form III.
Figure 13. Quantified data of plasmid relaxation assays with 5 μM of enediyne conjugates 4 (left) and 6 (right).
total percentage of DNA cleavage at the threshold separating
acidified and normal tissues (136% (pH 6) ≫ 61% (pH 7)) is
promising for selective targeting of acidic cancers.
The differences between conjugates 3 and 5 in regards to the
chemical mechanism of the DNA cleavage were further
illustrated by the effects of common scavengers for hydroxyl
radical (glycerol, DMSO)47 and singlet oxygen (NaN3)48 in the
plasmid relaxation assays (Figure 12). While the protecting
effect by glycerol and DMSO was small, the protection of DNA
cleavage by NaN3 was noticeable with both conjugates, except
for the “2ε,1α” conjugate 5 at pH 6. Failure of scavengers to
prevent DNA cleavage at these conditions is, again, consistent
with the relatively tight binding and a direct mechanism for
DNA modification by conjugate 5.
Enediyne conjugates showed a different DNA cleavage
pattern (Figure 13c,d) in comparison with the mono acetylene
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Figure 14. DIC (top) and fluorescence (bottom) cell images of A375 cells without compound (a) and with 30 μM of 3 (b), 4 (c), 5 (d), and 6 (e) at
pH 7.4 (compound numbers are also shown in the top right corners of the images).
conjugates. First, efficient photoreactivity is observed at the
lower concentrations. At pH 6, 17% and 13% of ds DNA
cleavage was observed, respectively, for the enediyne conjugates
4 and 6 at concentrations as low as 5 μM (Figure 13). At this
concentration, 92% and 77% of total DNA damage by 4 and 6
compare favorably with the total damage by 10 μM of mono
acetylene conjugate 5 in Figure 13. However, in spite of the
efficient ds DNA cleavage of enediyne conjugates at the lower
concentration, there was no big increase in DNA damage at 10
μM. We attribute this unexpected result to the aggregation of
enediyne conjugates which have more hydrophobic core.49
Interestingly, with 10 μM of enediynes, ds DNA cleavage was
observed at all pH conditions including pH 8. This observation
indicates that enediyne conjugates may have another
mechanism of DNA damage which is not concentration-
dependent (at least, for ds DNA cleavage) and less pH-
dependent overall. More detailed studies of the chemical
mechanism responsible for the highly efficient DNA cleavage
are needed, but photo C1−C5 cyclization of TFP-substituted
enediynes to the corresponding indenes involves the net
transfer of four hydrogen atoms from the environment,50 which
may be responsible for the different photoreactivity of enediyne
conjugates.
To test the DNA damaging ability of the conjugates in cells,
we performed single-cell gel electrophoresis (SCGE) assay. The
control SCGE assay results for undamaged cells and
commercially obtained cells with DNA damage are given for
comparison (Figure 15a,b). SCGE assay with healthy cells
Absence of ds damage at pH 7 at the 5 μM concentration of
enediynes 4 and 6 is particularly encouraging from a practical
perspective. The differences in the efficiency and pH-depend-
ence of DNA cleavage by enediynes have two probable origins.
Our earlier work suggested that enediyne and acetylene
conjugates often bind DNA in a different way (e.g.,
intercalation vs groove binding)24 and also may cause DNA
damage via different mechanisms, e.g., C1−C5 cyclization vs
alkylation.51
Figure 15. Images of SCGE assays. (a) Undamaged control cells; (b)
control cells with damaged DNA; (c) no compound + UV; (d) A375
cells + UV + compound 3.
Cellular Uptake of Bis-lysine Conjugates, Intracellular
DNA Damage, and Cell Proliferation Studies. Exper-
imental data (both the differential interference contrast (DIC)
and fluorescence cell images) displaying uptake bis-lysine
conjugates by melanoma cells are given in Figure 14-4. Whereas
no significant fluorescence was detected from the cells without
conjugates (Figure 14a), the cells became fluorescent after 30
min incubation with the conjugates, (Figure 14b,c,d,e),
indicating that all four conjugates can penetrate the cellular
membrane. The fluorescence is relatively well-distributed
throughout the whole cell suggesting that the bis-lysine
conjugates are capable of entering the nuclei as well.
showed no tails indicative of DNA damage (Figure 15a),
whereas the damaged cells produced the characteristic tails, the
size of which correlates with the amount of DNA damage in
these cells (Figure 15b).
Experiments evaluating light-induced DNA damage in
melanoma cells are summarized in Figure 15c,d. In the absence
of the photoactivated conjugates, irradiation for 10 min does
not produce efficient DNA damage (Figure 15c). In contrast,
photochemical activation of compound 5 produced very
efficient intracellular DNA damage in the A375 cells (Figure
15d). These results confirm that compound 5 can penetrate
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into the cancer cell nucleus and damage highly compacted
DNA upon photoactivation.
To further elucidate the cellular effects of these compounds,
we tested their ability to cause DNA damage in A375
melanoma cells. Figure 16 (left) showed that the conjugate 5
cells. This protonation leads to the extraordinary increase in
reactivity under the conditions suitable for selective targeting of
cancer cells.
The two alternative approaches to the construction of the
dipeptide moiety allowed us to change the relative number of
the α-amino vs ε-amino groups and achieve better control over
the reactivity and selectivity of the ds DNA cleavage.
Depending on the structure, the conjugates exist as either
monocations or dications at the neutral pH but are transformed
into trications at the acidic pH. The efficiency of ds-cleavage is
increased dramatically at the slightly acidic pH (<7), where it
exceeds the ds:ss ratio of calicheamicin. We have also
demonstrated that these conjugates are also capable of the
efficient penetration through cellular membranes and induction
of intracellular DNA damage. The high phototoxicity displayed
toward melanoma cells with CC50 in the 10−7 M range suggests
that this class of hybrid molecules has potential value for the
development of future light-activated anticancer agents.
Figure 16. (left) UV activated compound KK (1a,2e)-Ac (5) causes
DNA damage in A375 cells. A375 cells were treated as described in the
Experimental Section. After UV irradiation cells were harvested at 6,
24, and 48 h, DNA was extracted and analyzed by gel electrophoresis.
Smaller − and + symbols indicates absence or presence of conjugate 5.
Time points are indicated by numbers in hours; M stands for DNA
marker. Bigger − and + indicates whether cells were UV irradiated (20
min). (right) Cell proliferation assay using human melanoma A375 cell
line with compound 3 (green squares), 4 (red triangles pointing up), 5
(blue triangles pointing down), and 6 (black diamonds) after 10 min
of irradiation at 360 nm. The cell counts were normalized to the
experiment with UV irradiation.
EXPERIMENTAL SECTION
■
General Information. 1H, 13C NMR spectra were collected on a
Bruker 400 and 600 MHz NMR spectrometer. Mass spectrometry data
was collected on a Jeol JMS-600H. UV spectra were recorded on a
Shimadzu UV-2100. Fluorescence spectra were obtained with SPEX
FluoMax spectrofluorimeter using right-angle geometry. pH was
adjusted with AB 15 plus pH meter (Accument) after standardization
at 25 °C. All buffers were prepared and pH-adjusted at room
temperature (25 °C). Purity of biologically important compounds is
≥95%. Reagent kit for single cell gel electrophoresis assay kit,
CometAssay, and control cells containing different levels of DNA
damage, CometAssay Control Cell, were purchased from Trevigen,
Inc. The CC0 sample corresponds to cells with undamaged DNA.54
Miligel FisherBiotech Horizontal Electrophoresis System was used for
electrophoresis. An Olympus BX61 microscope attached with the
DP71 color digital camera was used to take fluorescence images of
SCGE assay.
had the ability to target and cause DNA damage in a UV
dependent manner. DNA degradation was observed at all-time
points, and it also progressively increased with time. The
continuous increase in DNA degradation is considered as one
of the signs of apoptosis due to the release of endogenous
endonucleases,52 whereas the appearance of fragmented DNA
on the gel was similar to that reported earlier by Wyllie and co-
workers for the fraction of apoptotic cell lysates which has been
cross-linked to the nucleus with the involvement of histone
proteins.53
Finally, we investigated phototoxicity of the four conjugates
against the melanoma cells. Gratifyingly, all four conjugates (3,
4, 5, and 6) are inactive in the dark and do not inhibit cell
proliferation at the concentration <10 μM (see the Supporting
Information). On the other hand, all the conjugates displayed
photoinduced cytotoxicity against this cancer cell line at the
submicromolar levels. Conjugate 5 displayed a particularly
strong phototoxicity toward the human melanoma A375 cell
line at the nanomolar range (CC50 = 2.7 × 10−7 M after 10 min
of irradiation using 360 nm light) (Figure 16).
(S)-Methyl 6-((S)-2,6-Bis((tert-butoxycarbonyl)amino)-
hexanamido)-2-((tert-butoxycarbonyl)amino)hexanoate (9).
The acid, Boc-L-lysine-OH (550 mg, 1.52 mmol) was dissolved in
CH2Cl2 (10 mL) and cooled to 0 °C. Then it was sequentially treated
with HOBt (308 mg, 2.28 mmol) and EDCI (436 mg, 2.28 mmol).
After 15 min, amine Lys(N-α-Boc)-OMe (475 mg, 1.82 mmol) was
added to the reaction mixture followed by DIPEA (0.8 mL, 4.56
mmol). After stirring for 12 h at room temperature, the reaction
mixture was extracted with ethyl acetate, washed with saturated NH4Cl
solution, water, brine, dried (Na2SO4), filtered, and concentrated in
vacuo. Chromatographic purification (SiO2, 30% ethyl acetate in
hexane eluant) of the residue provided dipeptide 9 (700 mg, 81%) as
1
white solid. H NMR (400 MHz, CDCl3): δ 6.23 (bs, 1H), 5.28 (bs,
1H), 4.63 (bs, 1H), 4.24 (m, 1H), 4.03−3.95 (m, 1H), 3.73 (s, 3H),
3.33−3.16 (m, 2H), 3.10 (dd, J = 13.0, 6.7 Hz, 2H), 1.89−1.73 (m,
2H), 1.73−1.57 (m, 4H), 1.56−1.29 (m, 33H). 13C NMR (100 MHz,
CDCl3): δ 173.3, 172.4, 156.1, 155.8, 155.5, 79.7, 78.9, 54.2, 53.4, 53.2,
52.1, 39.9, 38.7, 32.2, 31.8, 29.5, 28.9, 28.4, 28.3, 22.6. MS (ESI): m/z
(%) 611 (100) [M + Na]+, 589 (20) [M + H]+. HRMS (ESI): calcd
for C28H52N4O9Na [M + Na]+ 611.36320, found 611.36505.
(S)-Methyl 2-((S)-2,6-Bis((tert-butoxycarbonyl)amino)-
hexanamido)-6-((tert-butoxycarbonyl)amino)hexanoate (10).
The dipeptide 10 was prepared by using the same procedure as
synthesis of compound 9 in 90% yield. 1H NMR (400 MHz, CDCl3):
δ 6.65 (d, J = 7.7 Hz, 1H), 5.21 (bs, 1H), 4.69 (bs, 1H), 4.62−4.53 (m,
1H), 4.12−4.03 (m, 1H), 3.73 (s, 3H), 3.17−3.03 (m, 4H), 1.91−1.78
(m, 2H), 1.75−1.57 (m, 3H), 1.55−1.24 (m, 34H). 13C NMR (100
MHz, CDCl3): δ 172.6, 172.2, 156.2, 156.1, 155.8, 79.9, 79.0, 54.2,
52.3, 51.8, 40.1, 39.8, 31.9, 31.7, 29.5, 29.3, 28.4, 28.3, 22.4. MS (ESI):
m/z (%) 611 (100) [M + Na]+. HRMS (ESI): calcd for
C28H52N4O9Na [M + Na]+ 611.36320, found 611.36283.
CONCLUSION
■
In conclusion, this work describes the second generation of pH-
gated hybrid molecules which produces the most efficient light-
activated double-strand DNA cleavage known to date. These
hybrid compounds consist of two functional parts: an efficient
DNA-photocleaving agent and a pH-regulated part derived
from a basic dipeptide (bis-lysine). In the design of the pH-
switchable part, we took advantage of the different basicity of α-
and ε-amino groups of lysine. The more basic ε-amino groups
are protonated throughout all biologically relevant pH range,
whereas the α-amino groups undergo selective protonation at
the desired pH threshold which separates cancer and normal
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t e r t - B u t y l N - [ ( 1 S ) - 5 - [ [ ( 2 S ) - 2 , 6 - B i s ( t e r t -
butoxycarbonylamino)hexanoyl]amino]-1-[[4-[2-(2,3,5,6-tetra-
fluoro-4-pyridyl)ethynyl]phenyl]carbamoyl]pentyl]carbamate
(11). To a solution of dipeptide 9 (300 mg, 0.522 mmol) in
THF:MeOH:H2O (3:1:1, 5 mL) at 0 °C, LiOH·H2O (65 mg, 2.40
mmol) was added and stirred at the room temperature for 3 h. The
reaction mixture was then acidified to pH 2 with 1N HCl. It was
extracted with ethyl acetate, washed with brine, dried (Na2SO4),
filtered, and concentrated in vacuo to get the crude acid.
40.4, 40.3, 32.3, 30.6, 28.2, 28.1, 26.4, 22.6. MS (ESI): m/z (%) 696
(100) [M + H]+. HRMS (ESI): calcd for C32H30F8N7O2 [M +H]+
696.23332, found 696.23180.
t e r t - B u t y l N - [ ( 5 S ) - 5 - [ [ ( 2 S ) - 2 , 6 - B i s ( t e r t -
butoxycarbonylamino)hexanoyl]amino]-6-oxo-6-[4-[2-(2,3,5,6-
tetrafluoro-4-pyridyl)ethynyl]anilino]hexyl]carbamate (13).
The peptide conjugate 13 was prepared by using the same procedure
as synthesis of compound 11 in 68% yield. 1H NMR (400 MHz,
CDCl3): δ 9.13 (bs, 1H), 7.81−7.73 (m, 2H), 7.54 (d, J = 8.7 Hz, 1H),
6.72 (bs, 1H), 5.80 (bs, 1H), 4.84−4.67 (m, 2H), 4.62−4.52 (m, 1H),
4.12−3.99 (m, 1H), 3.19−3.02 (m, 4H), 2.12−1.64 (m, 5H), 1.63−
1.33 (m, 34H). 13C NMR (100 MHz, CDCl3): δ 173.1, 170.4, 156.7,
156.6, 156.2, 144.6, 144.4 (dm, J = 245 Hz), 141.6 (dm, J = 270 Hz),
140.5, 133.2, 119.7, 117.6, 115.5, 107.1, 80.6, 79.3, 73.2, 55.5, 53.9,
40.0, 39.2, 31.4, 30.9, 29.5, 28.5, 28.4, 28.3, 22.8, 22.3. MS (ESI): m/z
(%) 845 (100) [M + Na]+. HRMS (ESI TOF): calcd for
C40H54F4N6O8Na [M + Na]+ 845.38369, found 845.38472.
The above prepared crude acid was dissolved in CH2Cl2 (10 mL)
and cooled to 0 °C. It was sequentially treated with HOBt (105 mg,
0.783 mmol) and DCC (161 mg, 0.783 mmol). After 15 min, amine 7
(205 mg, 0.783 mmol) was added to the reaction mixture and stirred
for 12 h, raising the temperature to rt. The reaction mixture was
extracted with ethyl acetate, washed with saturated NH4Cl solution,
saturated NaHCO3, 1 N HCl, water, brine, dried (Na2SO4), filtered,
and concentrated in vacuo. Chromatographic purification (SiO2, 50%
ethyl acetate in hexane eluant) of the residue provided compound 11
(S)-2,6-Diamino-N-((S)-6-amino-1-oxo-1-((4-((perfluoropyri-
din-4-yl)ethynyl)phenyl)amino)hexan-2-yl)hexanamide Tritri-
fluoroacetic Acid Salt (5). The Boc-deprotected compound 5 was
prepared by using the same procedure as synthesis of compound 3 in
1
(258 mg, 60%) as yellow solid. H NMR (400 MHz, CDCl3): δ 9.12
(bs, 1H), 7.65 (d, d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 6.51
(bs, 1H), 5.62 (bs, 1H), 5.31 (bs, 1H), 4.66 (bs, 1H), 4.25−4.13 (m,
1H), 4.09−3.99 (m, 1H), 3.52−2.97 (m, 4H), 1.98−1.89 (m, 1H),
1.87 −1.78 (m, 1H), 1.77 −1.62 (m, 4H), 1.61−1.30 (m, 33H). 13C
NMR (100 MHz, CDCl3): δ 172.8, 171.3, 156.3, 143.5 (dm, J = 245
Hz), 141.7 (dm, J = 264 Hz), 133.3, 119.6, 117.6, 115.5, 107.0, 80.6,
80.2, 79.2, 73.2, 55.1, 54.5, 49.2, 39.9, 33.9, 29.7, 28.8, 28.5, 28.4, 25.6,
24.9, 22.7, 22.6. MS (ESI): m/z (%) 845 (100) [M + Na]+. HRMS
(ESI): calcd for C40H54F4N6O8Na [M + Na]+ 845.38369, found
845.38406.
1
95% yield. H NMR (600 MHz, CD3OD): 7.71 (d, J = 8.7 Hz, 2H),
7.59 (d, J = 8.7 Hz, 2H), 4.51 (dd, J = 8.6, 5.2 Hz, 1H), 3.99 (t, J = 6.4
Hz, 1H), 2.92 (m, 4H), 1.98−1.42 (m, 12H). 13C NMR (100 MHz,
CD3OD): δ 172.6, 170.4, 163.4, 163.2, 144.9 (dm, J = 251 Hz), 143.3
(dm, J = 279 Hz), 142.1, 134.3, 121.2, 118.5, 117.1, 107.5, 74.0, 55.9,
54.1, 40.6, 40.4, 32.7, 32.1, 28.4, 28.1, 24.1, 22.6. MS (ESI): m/z (%)
523 (100) [M + H]+. HRMS (ESI): calcd for C25H31F4N6O2 [M +
H]+ 523.24446, found 523.24465.
t e r t - B u t y l N - [ ( 5 S ) - 5 - [ [ ( 2 S ) - 2 , 6 - B i s ( t e r t -
butoxycarbonylamino)hexanoyl]amino]-6-[3,4-bis[2-(2,3,5,6-
tetrafluoro-4-pyridyl)ethynyl]anilino]-6-oxo-hexyl]carbamate
(14). The peptide conjugate 14 was prepared by using the same
(S)-2,6-Diamino-N-((S)-5-amino-6-oxo-6-((4-((perfluoropyri-
din-4-yl)ethynyl)phenyl)amino)hexyl) hexanamide Tritri-
fluoroacetic Acid Salt (3). Boc-protected compound 11 (100 mg,
0.08 mmol) was dissolved in CH2Cl2 (2 mL) and cooled to 0 °C,
followed by addition of trifluoroacetic acid (2 mL). Reaction mixture
was stirred for 4 h and was poured in a centrifuge tube containing
anhydrous diethyl ether (15 mL). Immediately solid came out, and it
was centrifuged. The solid material was washed with anhydrous diethyl
ether (2 × 15 mL) to get pure TFA-salt 3 (95 mg, 90%) as white solid.
1H NMR (400 MHz, CD3OD): 7.78 (d, J = 8.6 Hz, 2H), 7.64 (d, J =
8.6 Hz, 2H), 4.04 (t, J = 6.6 Hz, 1H), 3.82 (t, J = 6.6 Hz, 1H), 3.29−
3.17 (m, 2H), 2.92 (t, J = 7.8 Hz, 2H), 2.06−1.76 (m, 4H), 1.73−1.57
(m, 4H), 1.56−1.39 (m, 4H). 13C NMR (100 MHz, CD3OD): δ
170.2, 169.1, 163.4, 163.0, 144.8 (dm, J = 245 Hz), 143.3 (dm, J = 264
Hz), 141.6, 134.3, 121.1, 117.4, 118.4, 107.3, 74.0, 55.2, 54.3, 40.3,
40.2, 32.3, 32.1, 29.9, 28.1, 23.3, 23.0. MS (ESI): m/z (%) 523 (100)
[M + H]+. HRMS (ESI): calcd for C25H31F4N6O2 [M + H]+
523.24446, found 523.24481.
1
procedure as synthesis of compound 11 in 62% yield. H NMR (400
MHz, CDCl3): δ 9.29 (bs, 1H), 8.16 (bs, 1H), 8.09−7.97 (m, 1H),
7.64 (d, J = 8.7 Hz, 1H), 6.59 (bs, 1H), 5.95 (bs, 1H), 4.84−4.50 (m,
3H), 4.08−4.01 (m, 1H), 3.23−3.03 (m, 4H), 1.96−1.31 (m, 39H).
MS (ESI): m/z (%) 1018 (100) [M + Na]+. HRMS (ESI TOF): calcd
for C47H53F8N7O8Na [M + Na]+ 1018.37256, found 1018.37108.
( S ) - 2 , 6 - D i a m i n o - N - ( ( S ) - 6 - a m i n o - 1 − 1 ( ( 3 , 4 - b i s -
((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-1-oxohexan-2-
yl)hexanamide Tritrifluoroacetic Acid Salt (6). The Boc-depro-
tected compound 6 was prepared by using the same procedure as
synthesis of compound 3 in 86% yield. 1H NMR (400 MHz, CD3OD):
8.14 (d, J = 1.8 Hz, 1H), 7.79 (dd, J = 8.6, 1.8 Hz, 1H), 7.73 (d, J = 8.6
Hz, 1H), 4.54 (d, J = 8.7, 5.1 Hz, 1H), 3.99 (t, J = 6.3 Hz, 1H), 2.99−
2.91 (m, 4H), 2.03−1.79 (m, 4H), 1.79−1.60 (m, 4H), 1.66−1.45 (m,
4H). 13C NMR (150 MHz, CD3OD): δ 172.8, 169.1, 163.1, 145.7,
144.1, 142.6, 142.3, 135.2, 125.6, 124.4, 122.9, 119.8, 104.3, 103.8,
78.5, 78.2, 55.9, 54.1, 40.5, 40.4, 32.6, 32.4, 28.4, 28.2, 24.1, 22.8. MS
(ESI): m/z (%) 696 (100) [M + H]+. HRMS (ESI): calcd for
C32H30F8N7O2 [M + H]+ 696.23332, found 696.23180.
t e r t - B u t y l N - [ ( 1 S ) - 5 - [ [ ( 2 S ) - 2 , 6 - B i s ( t e r t -
butoxycarbonylamino)hexanoyl]amino]-1-[[3,4-bis[2-(2,3,5,6-
tetrafluoro-4-pyridyl)ethynyl]phenyl]carbamoyl]pentyl]-
carbamate (12). The peptide conjugate 12 was prepared by using
1
the same procedure as synthesis of compound 11 in 60% yield. H
Plasmid DNA Photocleavage. pBR322 plasmid DNA (4361 b/
p; from BioLabs Inc., 1 μg/μL solution in 10 mM Tris-HCl (pH 8.0),
and 1 mM EDTA buffer) was diluted to a concentration of 0.01 μg/
μL. The solution containing cleavage agent, DNA (38 μM/bp) in 20
mM sodium phosphate buffer was incubated for 1 h at 30 °C. Samples
were placed on ice at a distance of 20 cm from 200 W Hg−Xe lamp
(Spectra-Physics, Laser & Photonics Oriel Instruments with long pass
filter with 324 nm cut-on wavelength).
Electrophoretic Analysis. The gel electrophoresis was carried
out in 1× TBE buffer at 80 V using Miligel FisherBiotech horizontal
electrophoresis system. All gels were run on 1% agarose slab gels.
Before loading, the DNA samples were mixed with 0.33 volume of
tracking dye containing bromophenol blue (0.25%) and glycerol
(30%) in water. After staining in ethidium bromide solution (2 μg/
mL) for 1 h, the gel was washed with water and pictures were taken.
The relative quantities of the supercoiled and nicked, and linear DNA
were calculated by integrating the “area” of each spot by the image
analyzer software Total/Lab (Nonlinear Dynamics Ltd., UK). The
amount of supercoiled DNA was multiplied by a factor of 1.4 to
NMR (400 MHz, CDCl3): δ 9.54 (bs, 1H), 8.06 (d, J = 8.5 Hz, 1H),
7.73 (bs, 1H), 7.61 (d, J = 8.5 Hz, 1H), 6.69 (bs, 1H), 5.78−5.14 (bs,
2H), 4.69 (bs, 1H), 4.33−4.17 (m, 1H), 3.40−2.97 (m, 4H), 2.00−
1.19 (m, 39H). 13C NMR (100 MHz, CDCl3): δ 175.4, 174.3, 158.7,
158.2, 157.9, 146.2, 146.1, 143.7, 142.5, 135.2, 125.5, 124.5, 122.9,
119.5, 104.6, 103.9, 80.9, 80.7, 79.9, 57.0, 56.3, 41.1, 40.1, 33.2, 32.9,
30.6, 30.1, 28.9, 28.8, 27.1, 26.7, 24.3. MS (ESI): m/z (%) 1018 (100)
[M + Na]+. HRMS (ESI): calcd for C47H53F8N7O8Na [M + Na]+
1018.37256, found 1018.37340.
( S ) - 2 , 6 - D i a m i n o - N - ( ( S ) - 5 - a m i n o - 6 - ( ( 3 , 4 - b i s -
((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-6-oxohexyl)-
hexanamide Tritrifluoroacetic Acid Salt (4). The Boc-depro-
tected compound 4 was prepared by using the same procedure as
synthesis of compound 3 in 85% yield. 1H NMR (600 MHz, CD3OD):
8.21 (s, 1H), 7.86−7.76 (m, 2H), 4.10−4.03 (m, 1H), 3.78−3.68 (m,
1H), 3.25−3.15 (m, 1H), 2.98−2.89 (m, 2H), 2.09−1.77 (m, 4H),
1.76−1.41 (m, 8H). 13C NMR (100 MHz, CD3OD): δ 170.2, 169.5,
163.5, 163.3, 162,.9, 159.4, 145.7, 144.2, 142.5, 141.8, 135.3, 125.7,
124.6, 123.1, 120.2, 117.7, 104.3, 103.8, 78.6, 78.3, 55.3, 54.4, 52.7,
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account for reduced ethidium bromide intercalation into supercoiled
DNA.
ACKNOWLEDGMENTS
■
I.A. is grateful to the National Science Foundation (CHE-
0848686) and James & Esther King Biomedical Research
Program (09KC-03) for support of this research project.
Spectrometric Determinations of pKa. pH of 10 μM of
compound solution in H2O was adjusted with 10, 100 mM HCl
(aq) and NaOH (aq) solution under nitrogen purged condition.
Excitation wavelength was 330 nm and polymethacrylate fluorimeter
cuvettes were used.
ABBREVIATIONS USED
■
Alkaline Single-Cell Gel Electrophoresis Assay. LMAgarose was
melt in boiling water bath for 5 min and placed in 37 °C water bath for
at least 20 min to cool. Cells at 2 × 105/mL were combined with
molten LMAgarose at a ratio of 1: 10 (v/v) and 50 μL of the mixture
was transferred on CometSlides and lysed overnight at 4 °C. After
that, the slides were immersed in alkaline solution prepared freshly
with NaOH (0.6 g), 200 mM EDTA (250 μL), and dH2O (49.75 mL)
for 20 min at room temperature, in the dark. Then, the slides were
removed from alkaline solution and washed by immersing in 1× TBE
buffer for 5 min twice. After adding alkaline electrophoresis buffer 1
mm above slides, the voltage at 1 V per cm was applied and the
volume of buffer adjusted to reach 300 mA. Electrophoresis was run at
4 °C for 30 min. The samples were dried at ≤45 °C for 15 min, and
100 μL of diluted SYBR Green I was placed on the gels and the slides
were stored at refrigerator. After 5 min, excess SYBR solution was
removed by gentle tapping, and the slides were completely dried at
room temperature in the dark. The fluorescence images were taken by
epifluorescence microscopy.
Cell Culture. The human malignant melanoma cell line A375
(CRL-1619) was obtained from the American Type Culture
Collection (ATCC). The cell line were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum (FBS) and
penicillin/streptomycin (Life Technologies). Cells were propagated
according to ATCC guidelines and maintained in a 37 °C incubator
with 5% CO2 atmosphere.
MTT Assay. Half-maximal Inhibitory concentrations (CC50) for
the compounds tested were determined using the metabolic assay
MTT (dimethyl thiazolyl diphenyl tetrazolium salt) that measures
conversion of MTT into formazan by the mitochondrial reductase
producing a purple color. A375 cell lines were seeded at 2500 cells per
well in a 96-well plate. Seven 2-fold serial dilutions of the compounds
were added to the cells in triplicate. After 4 h of incubation with the
compounds, cells were UV-irradiated for 10 min at 365 nm (UV
transluminator, Spectronomics Corp. model TR-365R). After 72 h,
MTT (Sigma cat. no. M2128) was added to cells to a final
concentration of 1.25 mg/mL. After 60 min cells were centrifuged
at 900g for 5 min at room temperature. Media was replaced with
DMSO, and absorbance was measured in a multidetection microplate
reader Synergy HT, from BIOTEK at 600 nm. Graphical
representation was generated using the software Prism version 4
from GraphPad.
ss, single-stranded; ds, double-stranded; TPA, two-photon
absorption; OG, 8-oxo-guanine; TFA, trifluoroacetic acid; DIC,
differential interference contrast; SCGE, single-cell gel electro-
phoresis
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Fluorescence data, diagrams for plasmid relaxation assay, H
and 13C NMR spectroscopic data for new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-850-644-5795. E-mail: alabugin@chem.fsu.edu.
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