Syntheses of [12]aneN3-oligopeptide conjugates as effective DNA condensation agents

Article abstract of DOI:10.1016/j.bmc.2012.03.022

With the aim to develop effective and low toxicity DNA condensation agents, a series of oligopeptide derived macrocyclic polyamine [12]aneN₃ conjugates 7a-h·3HCl have been designed and synthesized through multi-step amidation reactions. Structu

Full text of DOI:10.1016/j.bmc.2012.03.022

Bioorganic & Medicinal Chemistry 20 (2012) 2897–2904
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses of [12]aneN3–oligopeptide conjugates as effective DNA
condensation agents
Zhi-Fen Li a, Zhi-Fo Guo a, Hao Yan a, Zhong-Lin Lu a, , Da-Yong Wu b,
⇑
⇑
a College of Chemistry, Beijing Normal University, Beijing 100875, China
b Key Laboratory of Photochemical Conversion and Optoelectronic Materials, TIPC, CAS, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to develop effective and low toxicity DNA condensation agents, a series of oligopeptide
derived macrocyclic polyamine [12]aneN3 conjugates 7a–hꢀ3HCl have been designed and synthesized
through multi-step amidation reactions. Structure–property study through gel electrophoresis proved
that the conjugates containing high hydrophobic ending amino acids exhibited effective condensation
Received 20 January 2012
Revised 7 March 2012
Accepted 8 March 2012
Available online 24 March 2012
ability at concentration of 150–250 lM, which was further confirmed by dynamic light scattering and
atomic force microscopy experiments. EB displacement assay, ionic salt effect, and structure–property
relationship in gel electrophoresis indicated that DNA condensation resulted from both the electrostatic
interaction of [12]aneN3 unit and hydrogen-bonding/hydrophobic multi-interaction of oligopeptide moi-
ety in the conjugates with DNA. The reversible condensation process and their low cytotoxicity suggest
that the new condensing agents are potential for the development of non-viral vectors.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
[12]aneN3
Oligopeptide
DNA condensation
Gel-electrophoresis
Atomic force microscopy
1. Introduction
vectors more recently.21–23 In spite of the various non-viral vectors
that have been investigated, small organic molecules capable of
Gene therapy has attracted intensive attention as a promising
method for treating both congenital and acquired diseases such as
cancer, AIDS, Parkinson’s and Alzheimer’s diseases, arthritis, and
heart diseases. The challenge of successful gene therapy relies heav-
ily on the development of effective and safe carrier vectors capable
of compacting and delivering DNA. Viral vectors are the preferred
system in clinical trials due to their high efficiency, but their cyto-
toxicity, mutagenesis, carcinogenesis, and immune response limit
their wide use. To this end, rapid advances have recently been made
in the design and syntheses of non-viral gene vectors due to their
advantages including adjustable structure, non-immunogenicity,
and potential for large-scale production over viral vectors.1–4 Non-
viral gene vectors reported so far include multivalent cations de-
rived from small organic molecules such as spermine and
spermidine,5 as well as organic polymers such as polyamines,6,7
polysaccharides,8 cationic lipids,9–11 polypeptides,12–14 dendri-
mers15 and metal complexes.16–20 Macrocylic polyamines such as
[9]aneN3, [12]aneN3, and [12]aneN4, which has multiple nitrogen
atoms and unfolded conformations and can serve as suitable cat-
ionic units to interact with negative charged phosphate in the
DNA backbone and induce desired condensation, they have been
incorporated in cationic lipids and organic polymers and proved
their applicability as DNA condensation agents and non-viral gene
effectively condensing DNA remain largely undeveloped, especially
when considering the effective concentration and toxicity.
In this paper, we report herein on the preparation of a series of
oligopeptide derived macrocyclic polyamine conjugates (see
Scheme 1) and their capabilities of inducing DNA condensation.
By varying the structure of ending amino acid in the oligopeptide
moieties, structure–activity relationship of these compounds has
been examined by using gel electrophoresis, dynamic light scatter-
ing, and atomic force microscopy. The obtained data clearly dem-
onstrates that the conjugates containing more hydrophobic side
chains are highly efficient in condensing plasmid DNA into partic-
ulate structures and with lower toxicity.
2. Results and discussion
2.1. Preparation of [12]aneN3–oligopeptides
Studies on mechanism of DNA condensation indicate that major
factors responsible for effective condensation include electrostatic
force, groove binding, and/or intercalation. The structure of the de-
signed conjugates is similar to cationic liposome. It is anticipated
that the macrocyclic polyamine [12]aneN3 moiety will act as cat-
ionic unit responsible for electrostatic interaction, while the
oligopeptide unit will act as hydrophobic moiety in cationic lipo-
some to make multiple interaction with DNA and enhance the
condensation process. At the same time, the incorporation of oligo-
⇑
Corresponding authors. Tel./fax: +86 10 58801804.
E-mail addresses: luzl@bnu.edu.cn (Z.-L. Lu), dayongwu@mail.ipc.ac.cn (D.-Y. Wu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.022

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Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
O
O
each conjugates clearly indicates that the electrostatic effects from
H
N
H2N
HCl
O
macrocyclic polyamines is one aspect in the DNA condensation
process, the interaction between the oligopeptides and DNA also
contributes to the process. From the above results, it clearly dem-
onstrates that the hydrophobicity of the terminal amino acids in
hexapeptides side chain plays an important role for the DNA con-
densation process.24 It can be seen that the efficient DNA conden-
sations were achieved with the terminal amino acids with higher
hydrophobic index (HI), such as leusine (HI = 1.1) in 7e, phenylal-
anine (HI = 1.2) in 7f, and tryptophan (HI = 0.81) in 7h; while poor
or no DNA condensation were found for the terminal amino acids
with lower hydrophobic index, such as tyrosine (HI = 0.26) in 7g
Boc
OH
N
H
N
H
N
N
N
O
2
O
O
N
Boc
i)
1
O
O
O
O
H
N
H
N
O
Boc
Boc
N
N
N
H
H
ii)
O
O
O
O
O
N
Boc
3
R
HClH2N
OH
O
N
H
H
N
H
N
l
M.25 Thus conjugates
7e, 7f, and 7h were studied in more detail in the following work.
O
and serine (HI = ꢁ0.18) in 7c even at 500
N
N
N
H
N
H
5a-h
O
O
O
N
iii)
Boc
4
2.3. Dynamic light scattering
O
O
O
H
R
H
N
H
N
Dynamic light scattering (DLS) is a powerful tool for measuring
particle size distributions (PSDs) in solution.26 Here, DLS technique
was used to investigate the DNA condensation in solutions in the
presence of different concentrations of 7e, 7f, and 7h, respectively.
As shown in Figure 2, the measurements were performed at DNA
N
O
Boc
N
N
N
H
N
H
N
H
iv)
O
O
O
N
Boc
6a-h
O
O
O
H
R
concentration of 9 lg/mL in Tris–HCl buffer (50 mM, pH 7.4) at
H
N
H
N
N
O
25 °C. The obtained results revealed that the hydrodynamic diam-
eters of DNA particles condensed by 7e, 7f, and 7h were ranging
from 174 to 318 nm, 208 to 365 nm, 196 to 563 nm, respectively.
The results are consistent with examples reported in literatures
that the sizes of the DNA nano-particles increased with the in-
crease of the concentrations of condensing agents.16 To be noticed,
there should be a saturation in the particle size at higher concen-
tration of the conjugates.18
NH
N
N
N
N
H
H
H
H
O
O
O
O
N
3HCl
a, R=H;
7a-h
b, R=CH3;
c, R=CH2OH;
d, R=CH(CH3)2
e, R=CH2CH(CH3)2; f, R=C6H5CH2; g, R=CH2C6H5OH; h, R=CH2C8H5NH
Scheme 1. Synthesis of [12]aneN3–CH2CO–hexapeptide conjugates 7a–h.
Reagents: (i) HATU, DIEA, CH2Cl2; (ii) LiOHꢀH2O, MeOH, H2O; (iii) HATU, DIEA,
DMF; (iv) MeOH/HCl.
2.4. Atomic force microscopy
Additional evidence supporting DNA condensation was pro-
vided by atomic force microscopy (AFM).16 Figure 3 shows the
typical AFM images of pUC18 in the absence and presence of 7e,
7f, and 7h at the concentration to induce complete condensation.
AFM image in the absence of the conjugates showed that naked
DNA formed closed loops displaying twists of its strands
peptides is expected to reduce the toxicity of the conjugates due to
their biocompatibility. Furthermore, the hydrophobic properties of
the oligopeptide moieties were varied by using different terminal
amino acid to study the structure–activity relationship.
All the peptides used in the study were synthesized by liquid
phase procedure. The synthetic route to the conjugates 7a–h is
shown in Scheme 1. Compound 1 was first prepared, which was
coupled with tetrapeptide 2 to give product 3. Later hydrolysis of
compound 3 by LiOH resulted in acid 4. The acid 4 was coupled
with a series of H2N-dipeptides 5a–h in the presence of HATU to
afford compounds 6a–h. The target conjugates 7a–h were obtained
by N-Boc deprotection (4 M HCl in methanol) of 6a–h. The yields of
the last two steps were about 40–60%. All new compounds were
characterized by 1H NMR, 13C NMR, IR, ESI-MS, or HR-MS (see Sup-
plementary data).
(Fig. 3a).27 Upon the addition of 150
lM of 7e, the DNA molecules
were induced to form nanoparticles with a diameter of 30–294 nm
(Fig. 3b), The diameters of the DNA nanoparticles condensed by 7f
and 7h at concentrations of 250 lM were 20–69 and 36–214 nm,
respectively (Fig. 3c and d). The AFM images clearly demonstrate
the effective DNA condensation ability of 7e, 7f, and 7h. The parti-
cle sizes revealed by AFM were smaller than DLS as literature re-
ported, which is can be attributed to the different sampling
techniques used in these two experiments.28
2.5. EB displacement assay
2.2. Retardation of gel electrophoresis
Ethidium bromide (EB) displacement assays were conveniently
used to assess the binding ability of compound 7e, 7f, and 7h with
DNA. EB has weak fluorescence, its fluorescence is greatly enhanced
when intercalating with DNA. It is known that this enhanced fluo-
rescence can be reduced or quenched by the addition of other com-
pounds having the ability to bind to DNA, which can be used to
evaluate the binding ability of other molecules. As shown in Figure
4, the fluorescence intensity of EB-bound CT-DNA was reduced
upon the addition of conjugates 7e, 7f, and 7h. The apparent binding
The ability of each conjugates to condense a plasmid DNA was
evaluated by gel electrophoresis assay. As shown in Figure 1, as
the concentration of the conjugates 7a–h increased, the amount
of supercoiled DNA gradually diminished. For conjugates 7a–c,
7d and 7g, complete retardation of electrophoresis was not ob-
served even when the concentration reached to 500
gates 7e, 7f, and 7h, the migration of DNA are completely retarded
at 200, 250, and 250 M, respectively.
lM. For conju-
l
At pH 7.4, all the plasmid DNA phosphate functions are nega-
tively charged, and the [12]aneN3 unit in the conjugates 7a–h are
positively charged. The decrease of the mobility of plasmid DNA
as the increase of the conjugate concentrations is mainly due to
the charge neutralization. However, the different performance of
constants (Kapp
)
were calculated to be (1.9 0.2) ꢂ 105 Mꢁ1
,
(1.4 0.1) ꢂ 105 Mꢁ1, and (1.2 0.1) ꢂ 105 Mꢁ1 for 7e, 7f, and 7h,
respectively. These binding constants are close to those of the
bifunctional compounds containing two [12]aneN3 moieties, which

Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
2899
(a) DNA 0.1 0.2
0.3 0.4 0.5 (b) DNA 0.1
0.2 0.3 0.4 0.5 (c) DNA 0.1 0.2
0.3
0.4 0.5
(d) DNA 0.1 0.2
0.3
0.4 0.5 (e) DNA 0.05 0.1 0.15 0.2 0.25 (f) DNA0.05 0.08 0.1 0.15 0.2 0.25 0.3
(g) DNA 0.05 0.1 0.2 0.3 0.4 0.5 (h) DNA 0.05 0.1 0.15 0.2 0.25 0.3 0.4
Figure 1. Agarose gel electrophoresis assay to investigate the pUC18 DNA condensation in the presence of [12]aneN3–CH2CO–hexapeptide conjugates 7a (a), 7b (b), 7c (c), 7d
(d), 7e (e), 7f (f), 7g (g), and 7h (h) at different concentrations in Tris–HCl buffer (50 mM, pH 7.4, and 37 °C). The values shown on the figures are concentrations (mM) of the
conjugates. The most left lane in each figure indicates the mobility of DNA alone in the gel.
therapeutic gene into the target cell nucleus. Several experiments
7e
7h
7f
have been applied to modulate the DNA dissociation from the con-
densates, such as pH jump,31 linkage break,32 and temperature.33
In this work, high concentration of NaCl solution was used to eval-
uate the reversibility of DNA condensation. As shown in Figure 6, it
is obvious that the condensed DNA could be partially released after
being treated with 200 mM of NaCl, this clearly indicates that the
DNA condensation induced by 7e, 7f, and 7h was reversible. On
the other hand, there are still large amount of the condensates kept
in the loading well, which showed their stability of the conden-
sates, especially at the lower concentration of NaCl.
600
500
400
300
200
100
50
100
150
200
250
2.8. Cytotoxicity assay
concentration/µM
Figure 2. Hydrodynamic diameter distributions of pUC18 DNA particles condensed
As potential gene vectors, the condensing agents are desired to
have low toxic effect on cells. Hence the cytotoxicity profiles for 7e,
7f, and 7h were evaluated by the MTT assay against T98G and
HepG2 cell lines.26 As shown in Figure 7, three compounds did
not exhibit obvious cytotoxicity towards T98G and HepG2. At the
by 7e, 7f, and 7h at concentrations of 50, 100, 150, 200, 250
Scattering angle of 90° at 25 °C. The DNA concentration is
expressed as mean S.D. n = 10.
l
M, respectively.
9 lg /mL. Values
concentration of 250 lM for the conjugates 7e, 7f, and 7h, the
clearly indicates that oligopeptide moieties in 7e, 7f, and 7h facili-
viabilities of T98G were 96%, 78% and 80%, and those of HepG2
were 97%, 83% and 85%, respectively. It can be seen that the toxic-
ity of oligopeptide derived macrocyclic polyamines are less than
those of the bifunctional condensing agents we developed in the
previous work.27 The incorporation of oligopeptide unit into mac-
rocyclic ployamines really benefits their biocompatibility. Lower
toxicity of the three conjugates provides the possibility for the
development of non-viral gene vector.
tate the interaction between the conjugates and the plasmid DNA.
2.6. Ionic strength effect
To evaluate the contribution of the electrostatic effect in the
DNA condensation induced by 7e, 7f, and 7h, the effect of ionic
strength in buffer solutions was examined. As shown in Figure 5,
the amount of uncondensed supercoiled DNA increased along the
increase of NaCl concentrations from 0 to 400 mM, which obvi-
ously implies that the condensing agents bind largely with the an-
ionic phosphate in DNA via electrostatic interactions. Addition of
excess salt partially neutralizes the phosphate backbone, which
in turn decrease the binding of the conjugates with DNA.29,30
3. Conclusion
In this study, a series of [12]aneN3–CH2CO–hexapeptide conju-
gates were designed, synthesized, and fully characterized. Their
capabilities in condensing DNA were studied with gel electrophore-
sis, fluorescence spectra, dynamic light scattering, and atomic force
microscopy techniques. It proved that the high hydrophobic prop-
erty of the oligopeptides unit in the conjugates favors the DNA con-
densation process. The condensation of plasmid DNA can be
2.7. Release of the compact DNA
The release process of DNA condensates plays a crucial role in
determining the effectiveness of the condensates to deliver the

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Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
Figure 3. AFM Images of pUC18 DNA (9
l
g/mL) and its condensation induced by the conjugates: (a) DNA (b) DNA + 150 lM 7e; (c) DNA + 250 lM 7f. (d) DNA + 250 lM 7h.
(a)
(b)
600
600
500
400
300
200
100
0
500
400
300
200
100
0
550 600 650 700 750 800
wavelength(nm)
550 600 650 700 750 800
wavelength(nm)
7e
7f
1.5
(c)600
(d)
7h
500
400
300
200
100
0
1.0
0.5
0.0
0.0
0.2
0.4
0.6
0.8
1.0
550 600 650 700 750 800
wavelength(nm)
concentration/mM
Figure 4. Fluorescence quenching curves of EB bound ct-DNA by 7e, 7f, and 7h in 5 mM Tris–HCl/50 mM NaCl solution, pH 7.4, kex = 537 nm, [EB] = 4
[conjugate] = 0–0.9 mM, 25 °C. The arrow shows the intensity changes on increasing concentration of 7e (a), 7f (b), and 7h (c).
lM, DNA = 40 lM,
occurred at 150
l
M of the conjugate, 37 °C and pH 7.4, and the pro-
(200 mM) NaCl. The effective DNA condensation ability of the con-
jugates 7e, 7f, and 7h can be attributed to the electrostatic effect
cess is partially reversible in the presence of higher concentration

Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
2901
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
(b)
(a)
(b)
Figure 5. Agarose gel electrophoresis assay to investigate the effect of ionic strength of DNA condensation by 150
lM 7e (a), 150
lM 7f (b) and 220
l
M 7h (c) in Tris–HCl
buffer (50 mM, pH 7.4), [DNA] = 9 lg/mL. Lane 1, DNA control; Lane 2–7, [NaCl] = 0, 50, 100, 200, 300, 400 mM.
1
2
3
4
1
2
3
4
1
2
3
4
(a)
(b)
(b)
Figure 6. Agarose gel electrophoresis assay to investigate the reversibility of DNA condensation in Tris–HCl buffer (50 mM, pH 7.4): (a) DNA + 150
DNA + 250 M 7f + NaCl; (c) DNA + 250 M 7h + NaCl. Lane 1, DNA control, lane 2–4, [NaCl] = 0, 100, 200 mM.
lM 7e + NaCl; (b)
l
l
resulted from the positive charged [12]aneN3 moiety and the hydro-
gen bonding and hydrophobic multi-interactions from oligopeptide
unit in the conjugates. For 7h, aromatic–aromatic interaction
should also be a potential contributor. The lower cytotoxicity of
the three [12]aneN3–oligopeptide condensing agents make them
desirable for the development of non-viral gene vectors. Further
investigations are still undergoing in this lab.
4.2. Synthesis
Details for the preparation and characterization of compounds
1, 2, 3, 4, 5a–h, and 6a–h can be found in the Supplementary data.
A typical procedure for the preparation of compounds 7a–hꢀ3HCl
was described as follows.
Acetyl chloride (14.0 g, 178 mmol) dissolved in methanol
(30 mL) at ice bath, then the Boc-protected compound 6a–h was
added. The mixture was stirred at room temperature for 12 h.
The solvent was removed at reduced pressure and the remaining
solid was washed with chloroform and dried in vacuum to give
7a–hꢀ3HCl.
4. Experimental section
4.1. Materials and methods
All chemicals and reagents were obtained commercially and used
without further purification. All amino acid, HATU and DIEA were
obtained commercially from Beijing Ou He Company (China). Calf
thymus DNA (CT-DNA) and plasmid DNA (pUC18) were purchased
from Solarbio Company (China). The concentrations of CT-DNA were
determined by UV spectroscopy at 260 nm, taking 6600 Mꢁ1 cmꢁ1
as the molar absorption coefficient. All solvents and reagents were
of analytical grade and were used as received. Ultrapure milli-Q
4.2.1. [12]aneN3–CH2COLeuAlaLeuAlaLeuGlyOCH3ꢀ3HCl, 7aꢀ3HCl
Yield, 81%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 4.35–4.22
(m, 5H), 3.88–3.72 (m, 2H), 3.60 (s, 3H), 3.52 (s, 2H), 3.15–3.04
(m, 12H), 2.02 (s, br, 2H), 1.86 (s, br,4H), 1.58 (s, br, 4H), 1.50–1.39
(m, 5H), 1.19 (t, J = 6.9 Hz, 6H), 0.89–0.81 (m, 18H). 13C NMR
(101 MHz, DMSO) d (ppm) 172.3, 171.8, 171.6, 171.5, 171.1, 170.0,
54.7, 51.6, 51.3, 50.8, 50.7, 48.0, 41.0, 40.8, 40.6, 40.4, 24.1, 24.0,
23.9, 23.0, 22.9, 21.5, 21.4, 17.8, 17.7. IR (KBr, cmꢁ1): 3435, 3274,
2957, 1736, 1633, 1548, 1443, 1383, 1365, 1212, 1161. HRMS-ESI:
m/z Calcd for C38H72N9O8 [M+H]+ 782.5504, Found: 782.5516.
water (18.25 MX) was used in all DNA condensation assays.
1H NMR and 13C NMR spectra were obtained on a Bruker Avance
III 400 MHz spectrometer at 25 °C. Chemical shifts were referenced
on residual solvents peaks. ESI-MS and HRMS were acquired on a
Waters Quattro Mocro spectrometer and Bruker Daltonics Bio
TOF mass spectrometer, respectively. IR spectra were taken on a
Nicolet 380 spectrometer. Fluorescence spectra were recorded on
a Varian Cary Eclipse Spectrometer. All experiments were per-
formed in Tris buffer (5 mM Tris–HCl/50 mM NaCl, pH 7.4) at room
temperature. Hydrodynamic diameters (DLS) were determined
using a DynaPro Nanostar dynamic laser light scattering apparatus.
Atomic force microscopy (AFM) images were obtained with a
Veeco NanoScope IIIa atomic force microscope. UV/Vis spectra
were measured on a Varian Cary 300 UV/Vis spectrophotometer
using solutions in 1.0 cm quartz cuvettes.
4.2.2. [12]aneN3–CH2COLeuAlaLeuAlaLeuAlaOCH3ꢀ3HCl 7bꢀ3HCl
Yield, 88%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 4.35–
4.21(m, 6H), 3.59 (s, 3H), 3.53 (s, 2H), 3.13–3.03 (m, 12H), 2.02
(s, br, 2H), 1.83 (s, br,4H), 1.61–1.55 (m, 3H), 1.50–1.41 (m, 6H),
1.33–1.16 (m, 9H), 0.92–0.80 (m, 18H).13C NMR (101 MHz, DMSO)
d (ppm) 172.9, 172.0, 171.9, 171.7, 171.3, 54.7, 51.9, 51.4, 51.0,
50.7, 48.2, 47.5, 40.9, 40.7, 40.6, 24.2, 24.1, 24.0, 23.1, 21.6, 21.4,
17.9, 17.7, 16.8. IR (KBr, cmꢁ1): 3436, 3270, 3078, 2957, 1736,
1632, 1548, 1452, 1383, 1368, 1218, 1158.HRMS-ESI: m/z Calcd
for C39H74N9O8 [M+H]+ 796.5660, found: 796.5666.

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Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
172.7, 172.0, 171.9, 171.7, 171.6, 171.2, 54.9, 51.8, 50.6, 50.1,
7e
7f
7h
T98G
48.0, 40.9, 40.8, 40.6, 24.2, 24.1, 24.0, 23.1, 22.8, 21.6, 21.5, 21.4,
21.3, 17.9. IR (KBr, cmꢁ1): 3418, 2959, 2872, 1732, 1635, 1548,
1455, 1386, 1365, 1271, 1119. HRMS-ESI: m/z Calcd for
100
80
60
40
20
0
(a)
C
42H80N9O8 [M+H]+ 838.6130, found: 838.6133.
4.2.6. [12]aneN3–CH2COLeuAlaLeuAlaLeuPheOCH3ꢀ3HCl 7f ꢀ3HCl
Yield, 88%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 7.27–7.17
(m, 5H), 4.45–4.41 (m, 1H), 4.35–4.19 (m, 5H), 3.62 (s, 3H), 3.55
(s, 2H), 3.15–2.88 (m, 12H), 2.03 (s, br, 2H), 1.88 (s, br, 4H), 1.60–
1.34 (m, 9H), 1.20 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H), 0.88–
0.78 (m, 18H). 13C NMR (101 MHz, DMSO) d (ppm) 171.9, 171.7,
171.5, 171.2, 137.1, 129.0, 128.1, 126.4, 54.8, 53.4, 51.8, 51.3,
48.1, 48.0, 40.9, 40.6, 36.5, 24.2, 24.0, 23.0, 22.9, 21.6, 21.5, 21.4,
17.8, 17.7. IR (KBr, cmꢁ1): 3422, 3066, 2958, 1735, 1643, 1541,
1456, 1386, 1369, 1222, 1167, 702. HRMS-ESI: m/z Calcd for
0
50
100
150
200
250
Concentration/(µM)
C
45H78N9O8 [M+H]+ 872.5973, found: 872.5969.
HepG2
7e
7f
7h
100
80
60
40
20
0
(b)
4.2.7. [12]aneN3–CH2COLeuAlaLeuAlaLeuTyrOCH3ꢀ3HCl 7gꢀ3HCl
Yield, 83%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 6.96 (d,
J = 8.3 Hz, 2H), 6.64 (d, J = 8.3 Hz, 2H), 4.364–22(m, 6H), 3.54 (s,
5H), 3.15–3.03 (m, 12H), 2.90–2.78 (m, 2H), 2.03 (s, br, 2H), 1.84
(s, br, 4H), 1.59–1.35 (m, 9H), 1.20 (d, J = 7.0 Hz, 3H), 1.15 (d,
J = 7.0 Hz, 3H), 0.88–0.79 (m, 18H). 13C NMR (101 MHz, DMSO) d
(ppm) 171.9, 171.9, 171.8, 171.6, 171.5, 171.2, 156.0, 129.9,
126.9, 115.0, 54.9, 53.8, 53.8, 51.7, 51.7 51.3, 50.9, 50.7, 48.1,
47.9, 40.9, 40.8, 40.6, 35.8, 24.2, 24.0, 23.1, 23.0, 21.6, 21.6, 21.4,
17.9, 17.8. IR (KBr, cmꢁ1): 3278, 2957, 2871, 1742, 1650, 1538,
1512, 1451, 1387, 1369, 1217, 1170, 1053, 848, 753. HRMS-ESI:
m/z Calcd for C45H78N9O9 [M+H]+ 888.5923, found: 888.5940.
0
50
10 0
15 0
20 0
250
4.2.8. [12]aneN3–CH2COLeuAlaLeuAlaLeuTrpOCH3ꢀ4HCl 7h ꢀ4HCl
Yield, 87%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 7.45 (d,
J = 7.9 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.05 (t,
J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.47 (t, J = 6.8 Hz, 1H),
4.36–4.23 (m, 5H), 3.52 (s, 3H), 3.47 (s, 2H), 3.16–3.04 (m, 14H),
2.02 (s, br, 2H), 1.86 (s, br, 4H), 1.61–1.51 (m, 4H), 1.47–1.40 (m,
5H), 1.20 (d, J = 7.1 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H), 0.88–0.80 (m,
18H).13C NMR (101 MHz, DMSO) d (ppm) 172.1, 172.0, 171.8,
171.7, 171.6, 171.3, 136.0, 127.1, 123.6, 121.0, 118.5, 118.0,
111.5, 109.2, 54.7, 53.4, 53.1, 51.9, 51.8, 51.3, 50.9, 48.0, 40.9,
40.8, 40.7, 26.9, 24.3, 24.1, 23.1, 21.7, 21.5, 17.8, 16.8. IR (KBr, cm
ꢁ1): 3276, 3060, 2956, 2928, 2871, 1729, 1694, 1629, 1541, 1454,
1387, 1369, 1215, 1165, 748, 708. HRMS-ESI: m/z Calcd for
Concentration/(µM)
Figure 7. Relative viabilities of T98G and HepG2 cells in the presence of different
concentrations of 7e, 7f, and 7h after incubation at 37 °C for 24 h. Each point
represents the means of three experiments.
4.2.3. [12]aneN3–CH2COLeuAlaLeuAlaLeuSerOCH3ꢀ3HCl 7cꢀ3HCl
Yield, 80%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 4.35–4.21
(m, 6H), 3.75–3.67 (m, 2H), 3.62–3.60 (s, 5H), 3.15–3.04 (m,
12H), 2.03 (s, br, 2H), 1.85 (s, br, 4H), 1.62–1.54 (m, 3H), 1.48–
1.39 (m, 6H), 1.19 (t, J = 6.5 Hz, 6H), 0.89–0.80 (m, 18H). 13C NMR
(101 MHz, DMSO) d (ppm) 172.0, 171.9, 171.6, 171.5, 171.2,
170.8, 61.1, 54.8, 54.7, 51.7, 51.3, 51.2, 50.9, 50.7, 48.5, 48.0, 40.9,
40.8, 40.6, 24.2, 24.0, 23.1, 21.5, 21.4, 17.9, 17.7. IR (KBr, cmꢁ1):
3430, 3270, 2956. 2871, 1733, 1693, 1630, 1545, 1454, 1380,
1365, 1233, 1155. HRMS-ESI: m/z Calcd for C39H74N9O9 [M+H]+
812.5610; found: 812.5609.
C
47H79N10O8 [M+H]+ 911.6082, found: 911.6072.
4.3. Agarose gel electrophoresis
Negatively supercoiled pUC18 DNA (0.4
treated with each conjugate 7a–h in Tris–HCl (2 l
l
L, 450
L, 50 mM, pH
7.4), followed by dilution with the water to a total volume of
20 L. The samples were then incubated at 37 °C for 1 h. Then
the loading buffer 2 L (10 mM Tris–HCl, pH 7.6, 0.03% bromophe-
lg/mL) was
4.2.4. [12]aneN3–CH2COLeuAlaLeuAlaLeuValOCH3ꢀ3HCl 7dꢀ3HCl
Yield, 88%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 4.37–4.23
(m, 5H), 4.16–4.12 (m, 1H), 3.61 (s, 3H), 3.44 (s, 2H), 3.15–3.03
(m, 12H), 2.05–2.00 (m, 3H), 1.84 (s, br,4H), 1.62–1.53 (m, 4H),
1.48–1.39 (m, 5H), 1.33–1.16 (m, 6H), 0.94–0.81 (m, 24H). 13C
NMR (101 MHz, DMSO) d (ppm) 172.2, 171.9, 171.7, 171.5, 171.3,
57.3, 55.0, 51.7, 51.2, 50.9, 50.0, 40.8, 40.7, 40.6, 29.9, 24.2, 24.0,
23.0, 21.6, 21.5, 21.4, 18.9, 18.2, 17.9, 17.7. IR (KBr, cmꢁ1): 3431,
3275, 3072, 2958, 2862, 1732, 1634, 1546, 1455, 1386, 1370,
l
l
nol blue, 0.03% xylene cyanol, 60% glycerol, 60 mM EDTA) was
added to the mixtures. The solutions were analyzed by electropho-
resis for 35 min at 70 V on a 1.0% agarose gel in 1 ꢂ TAE buffer. The
gel was stained with Goldview II (1.0
an UVP EC3 visible imaging system.
lg/mL) and photographed on
1216, 1159.HRMS-ESI: m/z Calcd for
C
41H78N9O8 [M+H]+
824.5973, found: 824.5980.
4.4. Dynamic light scattering
4.2.5. [12]aneN3–CH2COLeuAlaLeuAlaLeuLeuOCH3ꢀ3HCl 7eꢀ3HCl
Yield, 87%. 1H NMR (400 MHz, DMSO+D2O) d (ppm) 4.36–4.17
(m, 6H), 3.59 (s, 3H), 3.46 (s, 2H), 3.15–3.03 (m, 12H), 2.02 (s, br,
2H), 1.85 (s, br, 4H), 1.60–1.42 (m, 12H), 1.34–1.16 (m, 6H),
0.89–0.81 (m, 24H). 13C NMR (101 MHz, DMSO) d (ppm) 173.7,
Dynamic laser light scattering (DLS) equipment was used to
determine the average size of DNA nanoparticles condensed by dif-
ferent agents at 25 °C. The scattering angle was set to 90°. Typi-
cally, 10 runs were measured for each solution, with the average
of all the runs reported. DNA solutions (9 lg/mL) were prepared

Z.-F. Li et al. / Bioorg. Med. Chem. 20 (2012) 2897–2904
2903
in the presence of each conjugate 7e, 7f, and 7h (Tris buffer 50 mM,
pH 7.4) with deionised Milli-Q water (18.25 M ). The mixture was
allowed to stand for 5 min at room temperature and then 40 L of
the solution was transferred into the standard quartz cuvette for
measurement.
4.9. Cytotoxicity assay
X
l
The cytotoxicity of compound 7e, 7f, and 7h toward HepG2 and
T98G cell lines were tested by MTT assays (MTT = 3-(4, 5-dimeth-
ylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). HepG2 cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gib-
co) supplemented with fetal bovine serum (15%, v/v) and T98G
cells were cultured in minimum essential medium (MEM, Gibco)
supplemented with fetal bovine serum (10%, v/v) in a humid atmo-
sphere containing 5% CO2 at 37 °C. After 48 h of incubation in the
medium, the cells were seeded in 96-well plates at 5 ꢂ 105 cells
per well and cultured for another 24 h. Then the cells were treated
with different concentrations of 7e, 7f, and 7h for 24 h, after which
4.5. Atomic force microscopy
A stock solution of pUC18 DNA (450
lg/mL) was diluted to
9
lg/mL (Tris buffer 5 mM, pH 7.4, MgCl2 1 mM) in the presence
of each conjugate 7e, 7f, and 7h. MgCl2 were used optimally in or-
der to facilitate the adhesion of DNA onto mica for better viewing.
Then the corresponding solutions were left to equilibrate at 37 °C
for 1 h. Subsequently, samples were studied by AFM in air condi-
tion. Freshly cleaved mica was used as substrate for all AFM imag-
ing. Pretreatment of mica was necessary to promote electrostatic
time the medium was removed and 10
added to wells along with 90 L of culture medium. The cells were
incubated for 4 h and the MTT containing medium was replaced
with 110 L of DMSO. Finally the plates were oscillated for
lL of MTT (5 mg/mL) was
l
l
immobilization between the condensates and mica. Thus 15 lL of
10 min to fully dissolve the formazan crystal formed by living cells
in the wells. The absorbance of the purple formazan was recorded
at 490 nm using a Bio-Rad 680 plate reader. The relative viability of
the cells was calculated based on the data of four parallel tests by
comparing to the controls.
a 10 mM NiCl2 solution was deposited for 2 min onto the surface
of mica. The mica was then thoroughly rinsed with pure water to
prevent the formation of salt crystals on the surface. A total of
10 lL of DNA solution was spotted onto the pretreated mica and
incubated for 5 min. After which the mica was thoroughly rinsed
with water and dried under a gentle steam of argon. AFM images
were obtained in the air at room temperature with a Veeco atomic
force microscope. Scan were run at a rate of 1–3 Hz operating in
tapping mode using conical-shaped Si tips integrated to nano-crys-
talline Si cantilevers with an average resonance frequency of
280 kHz. The images were analyzed with the software accompany-
ing with the imaging module.
Acknowledgments
The authors gratefully acknowledge the financial assistances
from the Fundamental Research Funds for the Central Universi-
ties (2009SC-1); Beijing Municipal Commission of Education;
Program for New Century Excellent Talents at Universities
(NCET-08-0054), the Ministry of Education of China; and the
Nature Science Foundation of China (20972019).
4.6. EB displacement assay
Supplementary data
A Cary Eclipse Luminescence Spectrometer was used for the EB
displacement assay to confirm the DNA binding ability of the DNA
condensing agents in Tris buffer (50 mM, pH 7.4) at room temper-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.022.
ature. CT-DNA (100
lM) was first treated with EB (ethidium
bromide, 10 M), then the conjugate 7e, 7f, and 7h was added,
l
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