Synthesis and stereoselective DNA binding abilities of new optically active open-chain polyamines

Article abstract of DOI:10.1021/jo0619837

The efficient synthesis of new open-chain enantiopure polyamines bearing (R,R)- and/or (S,S)-transcyclohexane-1,2-diamine moieties is described. The key step for the synthetic procedure is the selective monoalkylation of the cyclohexanebis(sulfonamide) co

Full text of DOI:10.1021/jo0619837

Synthesis and Stereoselective DNA Binding Abilities
of New Optically Active Open-Chain Polyamines
Carmen Pen˜a,† Ignacio Alfonso,*,‡ Blake Tooth,§ Nicolas H. Voelcker,§
and Vicente Gotor*,†
aDepartamento de Qu´ımica Orga´nica e Inorga´nica, Facultad de Qu´ımica, UniVersidad de OViedo, Julia´n
ClaVer´ıa, 8, E-33006 OViedo, Spain, Departamento de Qu´ımica Inorga´nica y Orga´nica, ESTCE,
UniVersidad Jaume I, Campus del Riu Sec, AVenida Sos Baynat, s/n, E-12071 Castello´n, Spain,
and School of Chemistry, Physics and Earth Sciences, Flinders UniVersity of South Australia,
Bedford Park, South Australia 5042, Australia
ialfonso@qio.uji.es; Vgs@fq.unioVi.es
ReceiVed September 26, 2006
The efficient synthesis of new open-chain enantiopure polyamines bearing (R,R)- and/or (S,S)-trans-
cyclohexane-1,2-diamine moieties is described. The key step for the synthetic procedure is the selective
monoalkylation of the cyclohexanebis(sulfonamide) core, which allows the subsequent functionalization
of this moiety. Compounds bearing different combinations of absolute configurations, length of the aliphatic
spacers and terminal groups have been prepared. As a demonstration of the potential utility of the obtained
compounds, the preliminary DNA binding abilities of some of them have been studied by UV-
measurements of melting temperatures (Tm). The effects of the absolute configuration of the corresponding
chiral centers and the length of the spacer separating the cyclohexanediamine moieties on the strength of
the interaction with DNA are also discussed.
Introduction
Thus, these compounds have a significant therapeutic potential
for the treatment of neurological diseases,3 in the development
of new antidiarrheals in AIDS-related cases,4 and as anti-cancer
agents.5 Most of the biological functions of polyamines relate
to their abilities to interact with polyanions such as nucleic acids
(DNA and RNA).6 Spermine, for example, is known to stabilize
DNA duplex and triplex7 and to promote conformational
changes of duplex DNA.8 There is also a proposed structure-
function relationship because different molecular architectures
display different spatial dispositions of the cationic ammonium
In general, interest in polyamine compounds is often based
on their polycationic nature at physiological pH. In that
environment, they play an essential role for many biological
processes,1 ranging from stabilization of membrane and mito-
chondria functions to facilitation of DNA transfection by phage.2
† Universidad de Oviedo.
‡ Universidad Jaume I.
§ Flinders University of South Australia.
(1) (a) Campbell, D. R.; Morris, D. R.; Bartos, D.; Daves, G. D., Jr.;
Bartos, F. AdVances in Polyamine Research; Raven Press: New York, 1978.
(b) Goldenburg, S. H.; Algranati, J. D. The Biology and Chemistry of
Polyamines; JCSU Press: Oxford, 1990. For some recent revisions on
polyamine synthesis, see: (c) Bender, J. A.; Meanwell, N. A.; Wang, T.
Tetrahedron 2002, 58, 3111. (d) Kuksa, V.; Buchan, R.; Lin, P. K. T.
Synthesis 2000, 1189. (e) Karigiannis, G.; Papaioannou, D. Eur. J. Org.
Chem. 2000, 1841.
(2) (a) Tabor, C. W.; Tabor, H. Annu. ReV. Biochem. 1984, 53, 749. (b)
Pegg, A. E. Biochem. J. 1986, 234, 249. (c) Pegg, A. E. Cancer Res. 1988,
759. (d) Schuber, F. Biochem. J. 1989, 260, 1.
(3) (a) Bergeron, R. J.; Weimar, W. R.; Wu, Q.; Austin, J. K., Jr.;
McManis, J. S. J. Med. Chem. 1995, 38, 425.
(4) Bergeron, R. J.; Yao, G. W.; Yao, H.; Weimar, W. R.; Sninsky,
R. B.; Feng, Y.; Wu, Q.; Gao, F. J. Med. Chem. 1996, 39, 2461.
10.1021/jo0619837 CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/10/2007
1924
J. Org. Chem. 2007, 72, 1924-1930

DNA Binding Abilities of Open-Chain Polyamines
groups able to interact with the phosphate chain of the
oligonucleotides. For instance, both the separation between
nitrogen atoms and their grade of substitution dramatically affect
the basicity of the compounds, determining their positive charge
density under physiological conditions. Accordingly, most of
the structural changes studied to date9 rely on spatial separation
of nitrogen atoms,10 on alkylation of terminal amino groups, or
on the construction of dendritic structures.11 Conformational
constrained analogues have been also prepared and tested for
biological activities.12 Especially, derivatives bearing optically
active pyrrolidyl moieties stabilize DNA duplexes and triplexes,
to an extent that critically depends on the absolute configuration
of the chiral centers.13 In spite of all these studies, a definitive
picture of the requirements for the polyamine-DNA supramo-
lecular structures has not yet emerged. In particular, the question
of how chirality on the polycationic polyamine can affect the
stability of its corresponding nucleic acid complexes has not
been investigated thoroughly, probably due to the difficulties
experienced in the synthesis of optically active polyamines.14
On the other hand, we had previously reported preliminary
studies toward the synthesis and DNA-binding abilities of an
optically active polyamine, having three cyclohexane rings in
its structure.15 Following on from this work, here we report an
extended application of the synthetic studies toward the selective
and efficient preparation of a larger family of open chain chiral
polyamines. Besides, we have selected some of the newly
prepared compounds for preliminary assays in polyamine-
oligonucleotide binding experiments.
FIGURE 1. Retrosynthetic analysis of the target chiral polyamines.
recently reported the efficient monoalkylation of chiral cyclo-
hexanebis(sulfonamide) in very high yields and selectivities.15
A thorough study showed that the polarity of the solvent for
the reaction is critical for the formation of the monoalkylated
derivative, due to the presence of folded conformations in polar
solvents, such as acetonitrile.16 Here, we have used this
monoalkylation pathway for the preparation of a new family of
chiral polyamines.
The structural variables for the generation of our small library
of compounds have been the absolute configurations of the chiral
centers of the cyclohexane moiety, the length of the aliphatic
spacer (n in Figure 1), and to a lesser extent, the terminal
alkylating group (R). Regarding the spacer (n), preliminary
molecular modeling suggested that a propylene spacer (n ) 1)
would separate the consecutive cyclohexanediamine moieties
by about 5.0 Å, which is close to the distance between phosphate
linkages of the same strand in a DNA molecule (Figure 2). On
the other hand, a hexamethylene spacer (n ) 4) would increase
the flexibility of the compound, separating the cylohexanedi-
amine cores up to ca. 9.0-10.0 Å, allowing the binding of the
protonated nitrogen atoms from the same polyamine to phos-
phate anions of complementary strands of the DNA-double
helix, within the minor groove. Consequently, these two aliphatic
spacers were selected. Also supported by molecular models and
previous experimental data,17 we anticipated that the absolute
configuration of the diamine would induce a preferred confor-
mation of the polyamine in a helix of different handness. By
preparing both enantiomers of the polyamines, we could also
test if the compounds are able to exhibit any steroselectivity in
DNA binding, as it is a chiral entity. Apart from the chiral
centers of the sugar backbone; the nucleic acid chirality is also
expressed through the helicity of the DNA hybridized mol-
ecule.18 Thus, it is well-known that most of the naturally
occurring forms of DNA are right-handed (B-DNA) while other
secondary structures are left-handed (Z-DNA).19 The biological
role of this change of helicity is still unknown and the
Results and Discussion
Molecular Design. For the efficient synthesis of the intended
polyamine compounds shown in Figure 1, bearing nonsym-
metrically substituted cyclohexanediamine moieties, a selective
monofunctionalization of this synthon was necessary. We have
(5) (a) Casero, R. A.; Woster, P. M. J. Med. Chem. 2001, 44, 1. (b)
Zou, Y.; Wu, Z.; Sirisoma, N.; Woster, P. M.; Casero, R. A., Jr.; Weiss,
L. M.; Rattendi, D.; Lane, S.; Bacchi, C. J. Bioorg. Med. Chem. Lett. 2001,
11, 1613. (c) Gerner, E. W.; Meyskens, F. L. Nature ReV. Cancer 2004, 4,
781.
(6) See, for instance: Lomadze, N.; Schneider, H.-J.; Albelda, M. T.;
Garc´ıa-Espan˜a, E.; Verdejo, B. Org. Biomol. Chem. 2006, 4, 1755 and
references therein.
(7) (a) Feurstein, B. G.; Patabhiraman, N.; Marton, L. J. Nucleic Acids
Res. 1990, 18, 1271. (b) Delcros, J. G.; Strukenboom, C. J. M.; Basu,
H. S.; Shafer, R. H.; Szollosi, J.; Feuerstein, B. G.; Marton, L. J. Biochem.
J. 1993, 291, 269.
(8) (a) Bancroft, D.; Williams, L. D.; Rich, A.; Egli, M. Biochemistry
1994, 33, 1073. (b) Korolev, N.; Lyubartsev, A. P.; Nordenskio¨ld, L.;
Laaksonen, A. J. Mol. Biol. 2001, 308, 907.
(9) (a) Ganem, B. Acc. Chem. Res. 1982, 15, 290. (b) Bergeron, R. J.
Acc. Chem. Res. 1986, 19, 105.
(10) Sukhanova, A.; Deˆvy, J.; Pluot, M.; Bradley, J.-C.; Vigneron,
J.-P.; Jardillier, J.-C.; Lehn, J.-M.; Nabiev, I. Bioorg. Med. Chem. 2001, 9,
1255.
(11) Kra¨mer, M.; Stumbe´, J.-F.; Grimm, G.; Kaufmann, B.; Kru¨ger, U.;
Weber, M.; Haag, R. ChemBioChem 2004, 5, 1081.
(12) (a) Rajeev, K. G.; Sanjayan, G. J.; Ganesh, K. N. J. Org. Chem.
1997, 62, 5169. (b) Reddy, V. K.; Valasinas, A.; Sarkar, A.; Basu, H. S.;
Marton, L. J.; Frydman, B. J. Med. Chem. 1998, 41, 4723. (c) Valasinas,
A.; Sarcar, A.; Reddy, V. K.; Marton, L. J.; Basu, H. S.; Frydman, B.
J. Med. Chem. 2001, 44, 390.
(13) Nagamani, D.; Ganesh, K. Org. Lett. 2001, 3, 103.
(14) (a) Tsubaki, K.; Kusumoto, T.; Hayashi, N.; Tanima, D.; Fuji, K.;
Kawabata, T. Tetrahedron: Asymmetry 2005, 16, 739. (b) Nefzi, A.;
Ostresh, J. M.; Yu, J.; Houghten, R. A. J. Org. Chem. 2004, 69, 3603. (c)
Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G. J. Org. Chem.
2001, 66, 874.
(16) Pen˜a, C.; Alfonso, I.; Gotor, V. Eur. J. Org. Chem. 2006, 3887.
(17) (a) Bru, M.; Alfonso, I.; Burguete, M. I.; Luis, S. V. Tetrahedron
Lett. 2005, 46, 7781. (b) Gonza´lez-AÄ lvarez, A.; Alfonso, I.; Lo´pez-Ortiz,
F.; Aguirre, A.; Garc´ıa-Granda, S.; Gotor V. Eur. J. Org. Chem. 2004, 1117.
(c) Fitzsimmons, P. M.; Jackels, S. C. Inorg. Chim. Acta 1996, 246, 301.
(18) Saenger, W. Principles of Nucleic Acids. Springer-Verlag: New
York, 1984.
(19) (a) Ha, S. C.; Lowenhaupt, K.; Rich, A.; Kim, Y.-G.; Nim, K. K.
Nature 2005, 437, 1183. (b) Lewis, F. D.; Zhang, L.; Liu, X.; Zuo, X.;
Tiede, D. M.; Long, H.; Schatz, G. C. J. Am. Chem. Soc. 2005, 127, 14445.
(c) Mao, C.; Sun, W.; Shen, Z.; Seeman, N. C. Nature 1999, 397, 144. (d)
Erlanson, D. A.; Glover, J. N. M.; Verdine, G. L. J. Am. Chem. Soc. 1997,
119, 6927. (e) Saenger, W.; Hunter, W. N.; Kennard, O. Nature 1986, 324,
385.
(15) Pen˜a, C.; Alfonso, I.; Voelcker, N. H.; Gotor, V. Tetrahedron Lett.
2005, 46, 2783.
J. Org. Chem, Vol. 72, No. 6, 2007 1925

Pen˜a et al.
FIGURE 2. Schematic representation for the proposed polyamine-DNA binding modes, where triprotonated polyamines in the minor groove bind
to phosphate groups from one strand (left) or both strands (right).
SCHEME 1. Synthesis of Polyamines 7a,ba
the presence of an excess of cesium carbonate as a base.
Conventional deprotection of trityl group in 3a,b followed by
mesylation of the obtained alcohol yielded the corresponding
electrophiles 5a,b in excellent overall yields (88-90%). Cou-
pling of 2 equiv of these cyclohexane-derived electrophiles with
1 equiv of bis(sulfonamide) 1 led to the pertosylated polyamines
(6a,b) in very good (65-70%) isolated yields after chromato-
graphic purification. Finally, acidic hydrolysis of all of the
sulfonamide groups gave the desired polyamines (7a,b), which
were transformed into the corresponding hydrochloric salt for
an easier manipulation and storage. All of these compounds were
characterized by their full spectroscopic and analytical data,
showing the expected C2 symmetries in their corresponding 1H
and 13C NMR spectra and confirming that no epimerization of
any chiral center occurred during our synthetic procedure.
Besides, no significant differences in yields or reaction times
were observed for n ) 1 and 4. The obtained overall yields for
the final polyamines 7a,b starting from bis(sulfonamide) 1 was
ca. 28%, in a six-step synthetic procedure.
To explore the stereochemical space (only all-R isomer
represented in Scheme 1), we have used either (R,R)-1 or (S,S)-1
as starting materials, finally leading to both enantiomers of
homochiral7a,bcompoundsforeverycase[namely(R,R,R,R,R,R)-
7a, (S,S,S,S,S,S)-7a, (R,R,R,R,R,R)-7b, and (S,S,S,S,S,S)-7b] in
comparable yields. Conversely, coupling of 2 equiv of (S,S)-5a
with one of (R,R)-1 led, after final Ts deprotection, to (S,S-
,R,R,S,S)-7a, in very similar yield to its diastereomeric coun-
terpart. Thus, it is noteworthy that our modular methodology
allows the introduction of different stereochemistries (and even
combinations of them) within the polyamino backbone.
a Reactants and conditions: (a) see refs 15 and 16; (b) MeI, Cs2CO3,
PhCH3 (quantitative); (c) TFA, MeOH, rt (88-92%); (d) MsCl, NEt3, DCM
(quantitative); (e) 0.5 equiv. of 1, Cs2CO3, PhCH3 (65-70%); (f) aq HBr,
PhOH (60-66%).
development of drugs able to stabilize any of the forms or to
selectively bind to only one of them is an exciting field of
research.20
Synthesis of Optically Active Polyamines. The proposed
compounds were prepared following the synthetic pathway
outlined in Scheme 1. The unsymmetrical derivatives 2a,b were
obtained by monoalkylation of 1 as previously described.15,16
Methylation of 2a,b was quantitatively performed with MeI in
In order to expand the synthetic application of our methodol-
ogy, we have finally tried to introduce functionality in the
terminal alkylating groups (R in Figure 1). With this aim in
mind, we prepared the polyamine (R,R,R,R,R,R)-13 featuring
terminal primary amino groups (Scheme 2). Alkylation of 2b
with N-(3-bromopropyl)phthalimide using cesium carbonate as
a base and in dry toluene led to compound 8, with an
unsymmetrically substituted cyclohexane-1,2-diamine moiety.
Conventional transformation of 8 into the electrophile 10 was
carried out in very good overall isolated yield (90%). Although
the subsequent coupling between 10 and 1 (2:1 molar ratio)
occurred in moderate yield, the starting materials could be easily
recovered after flash chromatography. An explanation for this
low reactivity could be the intermediate polarity of phthalimide
(20) (a) Wang, G.; Christensen, L. A.; Vasquez, K. M. Proc. Nat. Acad.
Sci. U.S.A. 2006, 103, 2677-. (b) Waring, M. Proc. Nat. Acad. Sci. U.S.A.
2000, 97, 11685. (c) Otokiti, E. O.; Sheardy, R. D. Biochemistry 1997, 36,
11419. (d) Misra, V. K.; Honig, B. Biochemistry 1996, 35, 1115. (e) Sheardy,
R. D.; Levine, N.; Marotta, S.; Suh, D.; Chaires, J. B. Biochemistry 1994,
33, 1385. (f) Hardin, C. C.; Walker, G. T.; Tinoco, I., Jr. Biochemistry
1988, 27, 4178. (g) Walker, G. T.; Stone, M. P.; Krugh, T. R. Biochemistry
1985, 24, 7471.
1926 J. Org. Chem., Vol. 72, No. 6, 2007

DNA Binding Abilities of Open-Chain Polyamines
SCHEME 2. Syntheses of Polyamine 13a
in the heating and the annealing melting processes were
observed, both temperatures are given for all of the examples
in Table 1.
Almost all of the tested polyamines stabilized the DNA
double helix, as shown by the increasing melting temperature
effects (∆Tm > 0, Table 1). This stabilization most likely arises
from the electrostatic interaction between phosphate backbone
of the DNA and partially protonated polyamine. While all of
the studied polyamines are expected to be triprotonated at neutral
pH, the observed differences in the DNA binding must come
from other structural factors, different to the charge density.21
In general, polyamines 7a,b bound more strongly to AT-rich
than to CG-rich sequences, as the increase in the melting
temperatures is higher (∆Tm ≈ 10-11 °C for AT-rich sequence
but ∆Tm ≈ 0-6 °C for CG-rich sequence). Regarding the
chirality of the polyamine, interesting differences were found,
which are highlighted in Figure 3. For the AT-rich sequence,
when comparing entries 2 and 3, a subtle difference was
obtained for the binding with both enantiomers of 7a. Although
the difference in melting of diastereomeric pairs is small (∆∆Tm
≈ 1.5 °C), it is above the experimental error and suggests that
the all-R enantiomer of 7a binds slightly more strongly to the
oligonucleotide than the all-S isomer. Besides, when the aliphatic
spacer is longer (entries 4 and 5) this stereoselectivity is
suppressed, probably due to larger flexibility of 7b or to a
different mode of binding, as proposed in Figure 2. Interestingly,
for the CG-rich sequence, the observed stereoselectivity is much
higher and reversed. Thus, in this case, when comparing results
with both enantiomers of the short polyamine 7a, we found that
the all-R isomer has little effect on the CG-rich DNA melting,
while the all-S isomer produced a ∆∆Tm ≈ 5 °C, which is
remarkable for an energetic difference of diastereomeric
polyamine-DNA noncovalent complexes.22 These results sup-
port that, in this case, (S,S,S,S,S,S)-7a binds much more strongly
to a CG-rich sequence than (R,R,R,R,R,R)-7a. Once again, this
difference is reduced (almost abolished) for the longer deriva-
tives 7b. Thus, the larger aliphatic spacer does not seem to
improve the binding but rather reduced the stereoselectivity
observed for the shorter derivatives.
a Reactants and conditions: (a) N-(3-bromopropyl)phthalimide, Cs2CO3,
cat. TBABr, PhCH3 (75%); (b) TFA, MeOH, rt (90%); (c) MsCl, NEt3,
DCM (quantitative); (d) 0.5 equiv of 1, Cs2CO3, PhCH3 (30%); (e)
hydrazine, MeOH (75%); (f) aq HBr, PhOH (77%).
group which could facilitate folded conformers both in polar
and nonpolar environments, as previously observed for the same
residue.16 The protected polyamine 11 is a very valuable
compound as it contains orthogonally protected secondary and
primary nitrogen atoms. Thus, hydrazinolysis of 11 selectively
deprotected the terminal amino groups, leading to the diami-
nopolysulfonamide 12. This intermediate could be easily used
for further transformations, such as elongation, polymerization
of dendrimer synthesis. Instead, we finally deprotected the
secondary amino groups to yield the desired polyamino com-
pound 13. Once again, the spectroscopic data confirms the lack
of epimerization during the synthetic route.
In order to check the effect of the electrostatic interactions
between the phosphate backbone and the polyamonium moieties,
measurements at high salt concentration (5 mM Tris, 100 mM
NaCl, 1 mM MgCl2, pH ) 7.11) were performed in the case of
the system with the best enantiodiscrimination, the CG-rich
sequence and both enantiomers of polyamine 7a. In the absence
of polyamine, but in high salt concentration, a stabilization of
the double helix was observed [Tm ) 72.0 °C (heating) and
Tm ) 71.8 °C (annealing)] as expected. In the presence of 40
µM (R,R,R,R,R,R)-7a, there is no appreciable effect [Tm)
DNA Binding Studies. In order to study potential biological
and therapeutic applications of this family of compounds, we
have investigated their DNA binding abilities. As the polyamine-
oligonucleotide interaction increases the thermal stability of the
double helix, we have performed preliminary UV melting
experiments of DNA strands in the absence and in the presence
of some of the synthesized polyamines. A melting temperature
shift in the presence of the different polyamines is indicative
of the strength of their interaction with DNA. For a clearer
interpretation, we have selected the polyamines having different
length of the aliphatic spacers (n ) 1, 4) and different absolute
configurations of the chiral centers, but all of them having a
terminal methyl group and being homochiral. Thus, we could
directly compare the effects of two variables (spacer length and
chirality) in the DNA binding abilities. Besides, two different
oligonucleotides were checked: one AT-rich and another CG-
rich sequences, which are expected to favor B and Z-DNA
forms, respectively.18,19 As, in some cases, smooth differences
(21) (a) Gonza´lez-AÄ lvarez, A.; Alfonso, I.; D´ıaz, P.; Garc´ıa-Espan˜a, E.;
Gotor, V. Chem. Commun. 2006, 1227. (b) Alfonso, I.; Dietrich, B.;
Rebolledo, F.; Gotor, V.; Lehn, J. M. HelV. Chim. Acta 2001, 280. (c)
Alfonso, I.; Rebolledo, F.; Gotor, V. Chem. Eur. J. 2000, 6, 3331.
(22) For some recent examples, see: (a) Gaudry, E.; Aubard, J.; Amouri,
H.; Levi, G.; Cordier, C. Biopolymers 2006, 82, 399. (b) Xu, Y.; Zhang,
Y. X.; Sugiyama, H.; Umano, T.; Osuga, H.; Tanaka, K. J. Am. Chem.
Soc. 2004, 126, 6566. (c) Smith, J. A.; Collins, J. G.; Patterson, B. T.;
Keene, F. R. Dalton Trans. 2004, 1277. (d) Zinchenko, A. A., Sergeyev,
V. G.; Kabanov, V. A.; Murata, S.; Yoshikawa, K. Angew. Chem., Int. Ed.
2004, 43, 2378. (e) Munk, V. P.; Diakos, C. I.; Messerle, B. A.; Fenton, R.
R.; Hambley, T. W. Chem. Eur. J. 2002, 8, 5486. (f) Honzawa, S.; Okubo,
H.; Anzai, S.; Yamaguchi, M.; Tsumono, K.; Kumagai, I. Bioorg. Med.
Chem. 2002, 10, 3213. (g) Ji, L.-N.; Zou, X.-H.; Liu, J.-G. Coord. Chem.
ReV. 2001, 216, 513. (h) Becker, H.-C.; Norden, B. J. Am. Chem. Soc. 2000,
122, 8344.
J. Org. Chem, Vol. 72, No. 6, 2007 1927

Pen˜a et al.
TABLE 1. UV (260 nm) Melting Temperatures (Tm ( 0.5 °C) of DNA (2 µM) Sequences in the Absence and in the Presence of 40 µM of
Different Polyamines
5′-GCC AAG AAA GAA AAA AGA CGC
3′-CGG TTC TTT CTT TTT TCT GCG
5′-CCT TCG CCT CGC ACA TAG CC
3′-GGA AGC GGA GCG TGT ATC GG
entry
1
polyamine
T
m (°C)
∆Tma (°C)
∆∆Tmb (°C)
Tm (°C)
∆Tma (°C)
∆∆Tmb (°C)
heating
annealing
heating
annealing
heating
annealing
heating
annealing
heating
annealing
44.8
44.5
56.6
56.0
55.1
54.5
56.5
55.9
56.0
55.5
0
0
54.2
53.0
54.3
54.2
59.3
58.8
59.3
58.0
60.4
58.2
0
0
2
3
4
5
(R,R,R,R,R,R)-7a
(S,S,S,S,S,S)-7a
(R,R,R,R,R,R)-7b
(S,S,S,S,S,S)-7b
11.8
11.5
10.3
10.0
11.7
11.4
11.2
11.0
1.5
1.5
0.1
1.2
5.1
5.8
5.1
5.0
6.2
5.2
5.0
4.6
0.5
0.4
1.1
0.2
a Observed increase of Tm in the presence of the corresponding polyamine. b Difference of ∆Tm produced by enantiomers of the each corresponding
polyamine (given at the isomer inducing the highest Tm shift).
FIGURE 3. Selected UV-melting traces for (A) AT-rich and (B) CG-rich model sequences in the absence (green) and in the presence of polyamines
(R,R,R,R,R,R)-7a (red) and (S,S,S,S,S,S)-7a (blue).
72.1 °C (heating) and Tm ) 71.8 °C (annealing)]. In addition,
the melting experiment with its enantiomer gave very similar
results [Tm ) 73.1 °C (heating) and Tm ) 71.6 °C (annealing)].
These results support the importance of the electrostatic
interaction in the polyamine binding to the DNA strands. On
the other hand, the stereoselectivity previously observed was
almost abolished after elimination of this charge-charge
interaction, by competition with an excess of both sodium and
magnesium cations.
Although we are aware that the data here presented does not
allow extracting definitive conclusions, the current work reports
a remarkable stereoselective DNA molecular recognition event
produced by a chiral polyamine compound. Our preliminary
data seems to indicate that the DNA with an AT-rich sequence
binds preferentially to polyamines containing (R,R)-cyclohexane-
1,2-diamine moieties while the CG-rich sequence prefers to bind
to the (S,S) enantiomer. However, more synthetic and analytical
work has to be done to optimize the strength and selectivity of
binding to DNA and to verify the proposed model for the
interaction.
an interesting stereoselective interaction between some of the
new polyamino derivatives and model olygonucleotides. The
easy synthesis and high modularity of our synthetic approach
open up possibilities for expanding the methodology to longer
and more elaborated systems which could improve the selectivity
and stability of the interaction with DNA strands and, thus,
present interesting biological functions. The potential of these
new molecules ranges from chiral anion recognition21 to
separation and transport.23 Biotechnological applications in
nonviral gene transfection technology24 as well as in medicinal
chemistry for new anti-cancer drugs25 are also foreshadowed
for this family of compounds. Further studies regarding these
topics are underway in our laboratories.
Experimental Part
Synthesis of Different Stereoisomers of Polyamines 7a,b.
Tosylated Polyamine (R,R,R,R,R,R)-6a. To a solution of 0.5 mmol
of (R,R)-1 in 10 mL of dry toluene was added 10 mmol (3.1 g) of
Cs2CO3, and the suspension thus obtained was stirred at 75 °C for
(23) Bianchi, A.; Bowman-James, K.; Garc´ıa-Espan˜a, E. Supramolecular
Chemistry of Anions; VCH: Weinheim, 1997.
Conclusions
(24) (a) Kirby, A. J.; Camillery, P.; Engberts, J. B. F. N.; Feiters, M. C.;
Nolte, R. J. M.; So¨derman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.;
Garc´ıa-Rodr´ıguez, C.; Gue´dat, P.; Kremer, A.; McGregor, C.; Perrin, C.;
Ronsin, G.; van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 42, 1448. (b)
Luo, D.; Saltzman, W. M. Nature Biotech. 2000, 18, 33.
(25) For some recent revisions, see: (a) Huang, Y.; Pledgie, A.; Casero,
R. A., Jr.; Davidson, N. E. Anti-Cancer Drug 2005, 16, 229. (b) Moinard,
C.; Cynober, L.; Bandt, J.-P. Clin. Nutr. 2005, 24, 184. (c) Seiler, N. Curr.
Drug Targets 2003, 4, 565.
We describe in this paper the efficient synthesis of a new
family of open-chain optically active polyamines, bearing three
fragments of trans-cyclohexane-1,2-diamine in their stuctures.
Different combinations of the absolute configurations of the
chiral centers, aliphatic spacers between cyclohexanediamine
moieties, and terminal groups are implemented within the
polyamino backbone. Preliminary DNA binding studies showed
1928 J. Org. Chem., Vol. 72, No. 6, 2007

DNA Binding Abilities of Open-Chain Polyamines
0.5 h. After that time, 1 mmol of (R,R)-5a was added, and the
mixture was stirred at the same temperature for 5 days. Then, the
reaction was extracted with CH2Cl2 and 1 N HCl, and the organic
layers were dried and evaporated. The title compound was purified
by flash chromatography with 5.8% AcOEt in CH2Cl2 as eluent:
yield ) 70%; white foamy solid; mp 138-142 °C; [R]20D ) +7.1
(c ) 0.59, CHCl3); Rf (5.8% AcOEt/CH2Cl2) 0.1; 1H NMR (CDCl3,
300 MHz) δ (ppm) 0.71-1.81 (bm, 23H), 1.96-2.23 (m, 5H), 2.43
(m, 18H), 2.54 (s, 6H), 2.87-3.40 (bm, 8H), 3.60-4.24 (m, 6H),
6.97-7.55 (m, 12H), 7.55-8.19 (m, 12H); 13C NMR (CDCl3, 75
MHz) δ (ppm) 20.9 (CH3), 24.0 (CH2), 24.6 (CH2), 24.9 (CH2),
27.9 (CH2), 27.9 (CH3), 28.2 (CH2), 30.2 (CH2), 31.0 (CH2), 41.3-
(CH2), 56.0 (CH2), 57.5 (CH), 58.0 (CH), 126.7 (CH), 126.8 (CH),
126.9 (CH), 129.0 (CH), 129.0 (C), 136.2 (C), 137.9 (C), 138.1
(C), 142.3 (C), 142.5 (C); ESI-MS (m/z) 1397.2 [(M + Na)+, 100].
Anal. Calcd for C68H90N6O12S6 C, 59.36; H, 6.59; N, 6.11. Found:
C, 59.03; H, 6.75; N, 6.01
(CH3), 42.5 (CH2), 42.6 (CH2), 57.0 (CH), 57.5 (CH), 57.6 (CH);
HRMS/EI calcd for C26H54N6 450.4410, found 450.4414.
Polyamine (S,S,S,S,S,S)-7a. The synthesis of the title compound
was performed as for (R,R,R,R,R,R)-7a, but starting from (S,S,S,S,S,S)-
6a, showing the expected spectroscopic and analytical data: yield
61%; [R]25 ) +48.5 (c ) 0.32, MeOH).
Hg
Polyamine (R,R,R,R,R,R)-7b. The synthesis of the title com-
pound was performed as for (R,R,R,R,R,R)-7a, but starting from
(R,R,R,R,R,R)-6b: yield 66%; [R]20Hg ) -36.7 (c ) 0.49, MeOH);
1H NMR (D2O, 300 MHz) δ (ppm) 1.10-1.76 (m, 34H), 1.95-
2.23 (m, 6H), 2.66 (s, 6H), 2.80-3.00 (m, 4H), 3.01-3.24 (m,
4H), 3.24-3.49 (m, 4H); 13C NMR (D2O, 75 MHz) δ (ppm) 20.8
(CH2), 20.9 (CH2), 21.1 (CH2), 24.6 (CH2), 24.6 (CH2), 24.8 (CH2),
24.8 (CH2), 25.0 (CH2), 30.0 (CH3), 43.0 (CH2), 45.0 (CH2), 57.1
(CH), 56.7 (CH), 57.2 (CH); ESI-MS (m/z) 767.5 [(M + Na)+,
50]. Anal. Calcd for C32H82N6Cl6: C, 50.32; H, 10.82; N, 11.00.
Found: C, 50.15; H, 10.98; N, 10.80.
Tosylated Polyamine (S,S,S,S,S,S)-6a. The synthesis of the title
compound was performed as for (R,R,R,R,R,R)-6a, but coupling
(S,S)-1 with (S,S)-5a, showing the expected spectroscopic and
Polyamine (S,S,S,S,S,S)-7b. The synthesis of the title compound
was performed as for (R,R,R,R,R,R)-7a, but starting from (S,S,S,S,S,S)-
6b, showing the expected spectroscopic and analytical data: yield
analytical data: yield 70%; [R]20 ) -7.2 (c ) 0.48, CHCl3).
60%; [R]20 ) +36.5 (c ) 0.89, MeOH).
D
Hg
Tosylated Polyamine (R,R,R,R,R,R)-6b. The synthesis of the
title compound was performed as for (R,R,R,R,R,R)-6a, but coupling
(R,R)-1 with (R,R)-5b: yield 68%; white solid; mp ) 111-
Polyamine (S,S,R,R,S,S)-7a. The synthesis of the title compound
was performed as for (R,R,R,R,R,R)-7a, but starting from (S,S-
,R,R,S,S)-6a: yield 68%; decompose without melt; [R]20Hg ) +8.8
(c ) 0.25, MeOH); 1H NMR (D2O, 300 MHz) δ (ppm) 0.99-2.29
(m, 28H), 2.70 (s, 6H), 2.90-3.49 (m, 14H); 13C NMR (D2O, 75
MHz) δ (ppm) 21.3 (CH2), 21.4 (CH2), 21.6 (CH2), 23.3 (CH2),
24.8 (CH2), 25.4 (CH2), 25.6 (CH2), 30.4 (CH3), 42.5 (CH2), 54.1
(CH2), 57.0 (CH), 57.7 (CH), 57.8 (CH); HRMS-EI (m/z) calcd
for C26H54N6 450.4410, found 450.4414.
115 °C; [R]20 ) -9.4 (c ) 0.65, CHCl3); Rf (5.8% AcOEt/CH2-
D
Cl2) 0.1; 1H NMR (CDCl3, 300 MHz) δ (ppm) 0.98-1.90 (m, 44H),
2.39 (s, 18H), 2.59 (s, 6H), 2.85-3.30 (m, 8H), 3.65-4.00 (m,
6H), 7.25 (m, 12H,), 7.59-7.84 (m, 12H); 13C NMR (CDCl3, 75
MHz) δ (ppm) 21.2 (CH3), 24.6 (CH2), 25.2 (CH2), 26.6 (CH2),
26.8 (CH2), 28.6 (CH3), 29.6 (CH2), 30.2 (CH2), 32.0 (CH2), 32.3
(CH2), 44.0 (CH), 44.2 (CH), 56.8 (CH2), 57.4 (CH), 58.3 (CH2),
127.0 (CH), 127.1 (CH), 129.3 (CH), 129.4 (CH), 136.3 (C),138.6
(C), 138.8 (C), 142.6 (C),143.0 (C); ESI-MS (m/z) 1481.2 [(M +
Na)+, 80]. Anal. Calcd for C74H102N6O12S6: C, 60.88; H, 7.04; N,
5.76. Found: C, 60.56; H, 7.34; N, 5.65.
Synthesis of Polyamine (R,R,R,R,R,R)-13. Pertosylated Oc-
taamine (R,R,R,R,R,R)-11. In a flask under nitrogen atmosphere,
0.3 mmol of (R,R)-1, a large excess of Cs2CO3 (1.1 g, 3.3 mmol),
and catalytic TBABr (0.18 mg, 0.06 mmol) were suspended in 10
mL of dry toluene. The reaction mixture was stirred and heated to
reflux for 0.5 h. After that, 0.6 mmol of (R,R)-10 was added. After
5 days at reflux, the solvent was evaporated, and the crude product
extracted with 1 N HCl and CH2Cl2. The combined organic layers
were dried and concentrated in vacuum. The title compound was
finally isolated after flash chromatographic purification: yield 30%;
Tosylated Polyamine (S,S,S,S,S,S)-6b. The synthesis of the title
compound was performed as for (R,R,R,R,R,R)-6a, but coupling
(S,S)-1 with (S,S)-5b, showing the expected spectroscopic and
analytical data: yield 65%; [R]20 ) +9.8 (c ) 0.52, CHCl3).
D
Tosylated Polyamine (S,S,R,R,S,S)-6a. The synthesis of the title
compound was performed as for (R,R,R,R,R,R)-6a, but coupling
(R,R)-1 with (S,S)-5a, showing the expected spectroscopic and
white foamy solid; mp 117-119 °C; [R]20 ) -32.7 (c ) 0.40,
D
CHCl3); Rf (CH2Cl2/AcOEt 10:1) 0.28; IR (cm-1) (KBr) 1710; 1H
NMR (CDCl3, 300 MHz) δ (ppm) 1.06-2.20 (m, 44H), 2.35 (m,
18H), 2.90-3.85 (m, 22H), 7.20 (m, 12H), 7.66-7.80 (m, 16H);
13C NMR (CDCl3, 75 MHz) δ (ppm) 21.3 (CH3), 25.2 (CH2), 26.8
(CH2), 29.2 (CH2), 29.7 (CH2), 31.7 (CH2), 32.0 (CH2), 42.0 (CH2),
44.1 (CH2), 44.3 (CH2), 58.4 (CH), 58.6 (CH), 60.2 (CH2), 123.0
(CH), 127.0 (CH), 127.2 (CH), 127.3 (CH) ,129.4 (CH), 132.0 (C),
133.7 (C), 137.9 (C), 138.7 (C), 142.8 (C), 143.0 (C), 167.1 (C);
ESI-MS (m/z) 1825.5 [(M + Na)+, 80]. Anal. Calcd for
C94H116N8O16S6: C, 62.50; H, 6.47; N, 6.20. Found: C, 62.30; H,
6.80; N, 6.11.
analytical data: yield 68%; [R]20 ) +1.4 (c ) 0.4, CHCl3); Rf
D
1
(5.8% AcOEt/CH2Cl2) 0.1; H NMR (CDCl3, 300 MHz) δ (ppm)
1.00-1.35 (m, 10H), 1.35-1.81 (m, 12H), 1.96-2.26 (m, 6H), 2.42
(s, 18H), 2.56 (s, 6H), 3.22-3.64 (m, 7H), 3.69-4.00 (m, 7H),
7.31 (dd, J ) 8.1 Hz, J ) 2.2 Hz,12H), 7.68 (d, J ) 8.3 Hz ,6H),
7.75 (d, J ) 8.3 Hz ,6H); 13C NMR (CDCl3, 75 MHz) δ (ppm)
21.4 (CH3), 24.7 (CH2), 25.2 (CH2), 28.4 (CH2), 28.6 (CH3), 31.1
(CH2), 33.7 (CH2), 40.8 (CH2), 57.4 (CH), 58.3 (CH2), 60.2 (CH2),
65.8 (CH2), 77.2 (CH), 127.3 (CH), 127.3 (CH), 129.6 (CH), 129.7
(CH), 136.4 (C),138.0 (C), 143.2 (C), 143.3 (C); ESI-MS (m/z)
1397.2 [(M + Na)+, 20]. Anal. Calcd for C74H102N6O12S6: C, 60.88;
H, 7.04; N, 5.76. Found: C, 60.75; H, 7.40; N, 5.53.
Hexatosylated Octaamine (R,R,R,R,R,R)-12. In a flask under
nitrogen atmosphere, 0.051 mmol of (R,R,R,R,R,R)-11 was dissolved
in 0.3 mL of toluene, and an excess of hydrazine (0.04 mL, 0.51
mmol) was added. The mixture was heated at 120 °C for 1 day.
Then, the crude reaction was filtered, and the final compound was
obtained after evaporation of the solvent to dryness. Also, for
suitable storage, the HCl salt was prepared by addition of
concentrated HCl to a methanolic solution: yield 75%, pale red
hygroscopic solid, decompose without melt; [R]20D ) -15.2 (c )
Polyamine (R,R,R,R,R,R)-7a. Compound (R,R,R,R,R,R)-6a (0.1
mmol) was dissolved in HBr (48% in water) and phenol (1.6 mmol).
The mixture was heated to reflux for 5 days, and then the cold
mixture was extracted with CH2Cl2. The aqueous layer was basified
with NaOH and thoroughly washed with CH2Cl2. The combined
organic fractions were evaporated to dryness giving rise to the title
compound. For longer storage and better characterization, it was
more convenient to transform the polyamine into the corresponding
HCl salt, which was prepared by the treatment of (R,R,R,R,R,R)-
7a in methanolic HCl solution: yield ) 60%; white hygroscopic
solid; decompose without melt; [R]25Hg ) -48.4 (c ) 0.50, MeOH);
1H NMR (D2O, 300 MHz) δ (ppm) 1.18-1.81 (bm, 18H), 1.98-
2.67 (m, 10H), 2.69 (s, 6H), 2.99-3.22 (m, 4H), 3.22-3.64 (bm,
10H); 13C NMR (D2O, 75 MHz) δ (ppm) 21.2 (CH2), 21.3 (CH2),
21.4 (CH2), 22.9 (CH2), 24.8 (CH2), 25.1 (CH2), 25.3 (CH2), 30.6
1
0.37, CHCl3); H NMR (CDCl3, 300 MHz) δ (ppm) 1.14-2.10
(m, 44H), 2.39-2.69 (m, 22H), 2.69-4.00 (m, 18H), 7.10-7.42
(m, 12H), 7.61-7.80 (m, 12H); 13C NMR (CDCl3, 75 MHz) δ
(ppm) (all of the aliphatic signals seemed to be broad) 21.3 (CH3),
25.2 (CH2), 26.8 (CH2), 29.2 (CH2), 29.7 (CH2), 31.7 (CH2), 32.0
(CH2), 42.0 (CH2), 44.1 (CH2), 44.3 (CH2), 58.4 (CH), 58.6 (CH),
60.2 (CH2), 123.0 (CH), 127.0 (CH), 127.2 (CH), 127.3 (CH), 129.4
(CH), 132.0 (C), 133.7 (CH), 137.9 (C), 138.7 (C), 142.8
J. Org. Chem, Vol. 72, No. 6, 2007 1929

Pen˜a et al.
(C), 143.0 (C), 167.1 (C); ESI-MS (m/z) 774.2 [(M + 2H)+2, 20].
Anal. Calcd for C78H114Cl2N8O14S6: C, 56.74; H, 6.96; N, 6.79.
Found: C, 56.35; H, 7.23; N, 6.58.
Acknowledgment. Financial support from the Spanish
Ministerio de Educacio´n y Ciencia (CTQ-2004-04185), the
Principado de Asturias (IB-05-109), and the Australian Research
Council is gratefully acknowledged. I.A. thanks MEC for
personal financial support (Ramo´n y Cajal Program).
Polyamine (R,R,R,R,R,R)-13. Tosyl group deprotection was
carried out as in (R,R,R,R,R,R)-6a. Also in this case, the title
compound was isolated and stored as the HCl salt: Yield: 77%;
1
white hygroscopic solid; [R]20 ) -39.9 (c ) 0.51, MeOH); H
Supporting Information Available: General experimental
methods, detailed procedures, and characterization for all of the
synthetic intermediates [(R,R)-3a,b, (S,S)-3a,b, (R,R)-4a,b, (S,S)-
4a,b, (R,R)-5a,b, (S,S)-5a,b, (R,R)-8, (R,R)-9, and (R,R)-10], as well
as copies of 1H, 13C, and DEPT NMR spectra for all of the described
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
D
NMR (D2O, 300 MHz) δ (ppm) 1.20-1.84 (m, 34H), 1.93-2.30
(m, 10H), 2.88-3.50 (m, 22H); 13C NMR (D2O, 75 MHz) δ (ppm)
21.0 (CH2), 23.6 (CH2), 25.0 (CH2), 25.2 (CH2), 36.3 (CH2), 42.5
(CH2), 45.4 (CH2), 57.0 (CH), 57.4 (CH); ESI-MS (m/z) 311 [(M
+ 2H)+2, 65], 208.0 [(M + 3H)+3, 100]. Anal. Calcd for C36H84-
Cl8N8: C, 47.37; H, 9.28; N, 12.28. Found: C, 47.12; H, 9.55; N,
12.03.
JO0619837
1930 J. Org. Chem., Vol. 72, No. 6, 2007