was collected, which was purified in a dialysis membrane (Sigma
benzoylated tubing) for 48 h, treated with Dowex Type I resin and
lyophilized (64%). H NMR (500 MHz, D2O, 300 K) d 5.20 (d,
J = 3.3 Hz, 7H, H1), 4.00 (t, J = 9.7 Hz, 7H, H5), 3.92 (t, J =
9.5 Hz, 7H, H3), 3.65 (dd, J = 3.5 Hz, J = 9.5 Hz, 7H, H2), 3.61
(d, J = 14.9 Hz, 7H, H6), 3.43–3.41 (m, 14H, H4, H6ꢀ) ppm; 1H
NMR (500 MHz, DMSO-d6, 300 K) d 7.87 (br, s, 7H, C6-NH),
Atomic force microscopy
Samples were examined using a Digital Instruments Multimide
AFM with Nanoscope III controller, operating in Tapping mode.
The Si AFM tips, having a typical radius of curvature of
7 nm, and mounted on cantilevers with a resonant frequency of
approximately 300 kHz, were supplied by NANOSENSORS. The
images had a 512 × 512 pixel resolution with the scan range
varying between 1 lm × 1 lm and 6 lm × 6 lm. The tip was
operated at a scan rate of 1–3 Hz (lines s−1). AFM images of
the plasmid pcDNA3 (5446 bases) alone and in the presence of 4b
were collected. 1 ll of pcDNA3 solution (4.4 lg ll−1) was diluted to
100 ll with HEPES–NaOH buffer (pH 7.4) resulting in a 44 ng ll−1
solution. The buffer solution was prepared with ultrapure water
(18.2 MX) and filtered through a 0.22 lm Millipore filter prior to
use. For the DNA condensation studies, 10 ll of pcDNA3 solution
were mixed with 10 ll solution (6 × 10−3 M) of 4b and left for
incubation for 1 h at room temperature. Then 5 ll of the above
mixture were placed on a mica surface and left to dry under a
stream of air for 30 min. For the control experiment, 5 ll of
pcDNA3 solution were used in the same way.
1
=
7.24 [br, s, 21H, C( NH)-NH2], 5.97 (br, s, 7H, OH2), 5.85 (br, s,
7H, OH3) 4.96 (br s, 7H, H1), 3.83 (br, m, 7H, H5), 3.64 (br,
m, 7H, H3), 3.53 (m, 7H, H6), 3.44–3.35 (br, m, 21H, H6ꢀ, H2,
H4) ppm; 13C NMR (125 MHz, D2O, 300 K) d 158.2 (C ), 102.2
=
(C1), 82.9 (C4), 72.8 (C5), 72.1 (C2), 71.2 (C3), 42.6 (C6) ppm;
15N NMR (50.66 MHz, 1.5 M in D2O, 300 K, D1 = 60 s) d 78.5
=
( NH), 72.3 (-NH2) ppm. ESI-MS (positive ion mode), calcd:
M+ = 1422.4. Found: m/q 204.3 ([M + 7H+]/7, 100%), 1423
(MH+, 5%). Exact mass (MALDI) for [C49H91O28N21·HCl·Na]+
calcd 1480.6006. Found 1480.7453 (Found: C, 31.0; H, 5.8;
N, 15.4. C49H91O28N21·8HCl·9H2O requires C, 31.4; H, 6.3;
N, 15.7%).
Octakis(6-guanidino-6-deoxy)-c-cyclodextrin (4c)
Acknowledgements
The title compound was obtained using octakis(6-amino-deoxy)-
c-cyclodextrin (3c) (0.561 g, 0.44 mmol) using the same equivalents
of reagents as described above for 4a. A white solid was recovered,
which was purified in a dialysis membrane (Sigma benzoylated
tubing) for 48 h, treated with Dowex Type I resin and either
The financial support of GSRT programs “Aristeia” (Excellence
in the Research Institutes in the frame of articles 4 & 6 of
N.2860/00 and EU regulations 1260/99 and 438/01) to K. E.,
and PRAXE for partial support to N. Mourtzis is appreciated.
The NCSR “Demokritos” scholarships to N. M. and C. A. are
acknowledged. The authors also thank Ms M. Archimandriti
and Dr J. Nolan for help with the AFM measurements, Mr
E. Ghika for spectrophotometric chloride determinations, and
Dr M. Paravatou and P. Klimentzou for kindly providing the
plasmid DNA.
1
lyophilized or water was evaporated to dryness (48%). H NMR
(500 MHz, D2O, 300 K) d 5.14 (br, 8H, H1), 4.05 (m, 8H,
H5), 3.90 (t, J = 9.3 Hz, 8H, H3), 3.63 (dd, J = 3.6 Hz, J =
9.8 Hz, 8H, H2), 3.58 (d, J = 14.5 Hz, 8H, H6), 3.54–3.45
(m, 16H, H4, H6ꢀ) ppm; 13C NMR (125 MHz, D2O, 300 K),
=
158.0 (C ), 101.0 (C1), 81.0 (C4), 73.0 (C5), 72.9 (C2), 72.8
(C3), 41.0 (C6) ppm; ESI-MS (positive ion mode) calcd: M+
=
1625.6. Found m/q 204.3 ([M + 8H+]/8, 100%), 408 ([M +
4H+]/4, 12%), 543 ([M + 3H+]/3, 17%), 813 ([M + 2H+]/2,
7%); Exact mass (MALDI) calcd for [C56H104O32N24·4HCl·Na]+ =
1791.6213. Found: 1791.6920 (Found: C, 33.9; H, 6.4; N, 16.2.
C56H104O32N24·8HCl·5H2O requires C, 33.5; H, 6.1; N, 16.7%).
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Agarose gel electrophoresis
Ultrapure calf thymus DNA (∼8.6 MDa, >13 kb, Sigma) (1 ll of
a 12.5 lg per 50 ll solution in doubly distilled, autoclaved water)
was mixed with increasing volumes (2, 5, 10 ll) of solution (0.1045
mM) of 4a, 4b or 4c in order to achieve a DNA : compound mass :
charge ratio of 5 : 1, 2 : 1 and 1 : 1, respectively. Each mixture
was diluted with water to a total volume of 20 ll and incubated
for approximately 45 min at room temperature. Control samples
with guanidine hydrochloride (10 ll of 0.1706 mM solution,
DNA : compound mass : charge ratio 1 : 1) and aCD, bCD,
cCD (10 ll of 0.1045 mM solutions) were prepared in the same
way. After incubation, 2 ll of a dye (0.4% Bromophenol Blue,
67% sucrose) were added to each sample that was then loaded in
a 1% agarose gel containing 10 lg ethidium bromide [10 mg ml−1
per 100 ml TAE buffer (40 mM Tris-acetate, 1 mM EDTA)] and
electrophoresed, using kHindIII as the molecular weight marker
(lane 1). For exact volumes of solutions used, see supporting infor-
mation.†
130 | Org. Biomol. Chem., 2007, 5, 125–131
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