A charge-deficient analogue of spermine with chelating properties

Article abstract of DOI:10.1007/s11171-005-0037-1

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu²⁺ ions. Possible applications of this compound for studying enzymes o

Full text of DOI:10.1007/s11171-005-0037-1

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 3, 2005, pp. 271–278. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 303–311.
Original Russian Text Copyright © 2005 by Khomutov, Grigorenko, Skuridin, Demin, Vepsalainen, Casero, Woster.
A Charge-Deficient Analogue of Spermine
with Chelating Properties
1
A. R. Khomutov*, N. A. Grigorenko*, S. G. Skuridin*, A. V. Demin**, J. Vepsalainen***,
R. A. Casero****, and P. M. Woster*****
* Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia;
** Institute of Gene Biology, Russian Academy of Sciences, ul. Vavilova 34/5, Moscow, 117984 Russia
*** Department of Chemistry, University of Kuopio, P.O. Box 1627, Kuopio, FIN-70211 Finland
**** The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD, USA
***** Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA
Received June 3, 2004; in final form, July 5, 2004
Abstract—1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is
synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of
this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are dis-
cussed.
Key words: DNA, polyamines, spermine
1
INTRODUCTION
aminomethylene groups of Spd and Spm. These com-
pounds turned out to be effectors of the enzymes of
polyamine metabolism. They are capable of penetration
into various cell types and possess a comparatively low
cytotoxicity and a controlled catabolic stability [8–10].
An oxa analogue of Spd and a dioxa analogues of Spm
containing HNO group(s) instead of the central HNCH2
group(s) were also synthesized [11]; however, their bio-
chemistry has insufficiently been investigated up to
now.
The biogenic polyamines spermine, spermidine, and
putrescine are present in significant amounts in almost
all cell types and may be considered as universal low-
2
molecular regulators of cellular metabolism [1]. Bio-
logical effects of polyamines and their analogues are
determined by the geometry of molecule that provides
for the proper spatial arrangement of amino groups, the
degree of their protonation, and their ability to form a
network of hydrogen bonds while interacting with spe-
cific cellular binding sites. The majority of efforts to
correlate the biological activity of polyamine analogues
with their structures were devoted to the study of struc-
tural analogues of Spm and Spd, including the deriva-
tives with various substituents at the terminal nitrogen
atoms and analogues with conformationally restricted
fragments in the polyamine backbone (see reviews
[2, 3]).
The present paper is devoted to the principles of
design and synthesis of a new isosteric Spm analogue
with a charge-deficient central fragment, which, unlike
Spm, is an excellent chelator for Cu2+ ions.
RESULTS AND DISCUSSION
Trien, whose backbone includes three –ëç2ëç2–
groups, may be considered as an isosteric analogue of
Spd. A reduced to two methylene units distance
between secondary amino groups results in a strong
decrease in the basicity of secondary amino groups,
whereas the K‡ values of terminal amino groups
Investigations of the contribution of protonation of
the Spm/Spd amino groups to the biological effects of
polyamines are more limited. The analogues with pyri-
dinium cycle incorporated into its backbone [4] and
2,2-, 6,6-, and 7,7-difluorospermidines were synthe-
sized [5] in order to decreased the basicity of their
amino groups. The isosteric charge-deficient analogues remain almost the same as in parent Spd [12].
of Spd [6] and Spm [7] were obtained by substitution of
aminooxy (H2NO) groups with K‡ ~5 for the terminal
NH2
9.9
Spd
NH
H2N
1
Corresponding author; phone: +7 (095) 135-6065; e-mail:
pKa 10.9
8.4
alexkhom@genome.eimb.relarn.ru
2
Abbreviations: Ms, methanesulfonyl; Spd, spermidine (1,8-
NH
6.7
NH2
9.2
Trien
diamino-4-azaoctane; Spm, spermine (1,12-diamino-4,9-diaza-
dodecane); SpmTrien, 1,12-diamino-3,6,9-triazadodecane; and
Trien, triethylenetetramine.
NH
H2N
pKa
9.9
3.3
1068-1620/05/3103-0271 © 2005 Pleiades Publishing, Inc.

272
KHOMUTOV et al.
Trien can effectively penetrate into cells, and Spm pool may be considered as a criterion of its integral bio-
inhibits its uptake [13]. Unlike Spd, Trien is an effective chemical similarity or dissimilarity to Spm.
chelator of bivalent cations of transition metals and,
A rational strategy of the SpmTrien synthesis con-
due to an excellent stability of its complex with Cu2+
sists in a successive elongation of the polyamine ana-
(Kdis 20.4 at pH 14 and 14.0 at pH 7 [14]), Trien is
used for the treatment of Wilson’s disease at daily doses
up to 1.2–1.6 g [15], which indicates its low toxicity.
logue backbone by alkylation of the corresponding free
amines with appropriate active esters of alcohols. Such
an approach was used to prepare several polyamine
analogues (see [16,17] and references therein), and we
successfully used it previously to synthesize the ami-
nooxy analogue of Spm [7] and also α-methyl-Spd
[18].
The replacement of the Spd fragment in the Spm
molecule by a triethylenetetramine moiety gives rise to
earlier unknown SpmTrien.
NH
NH
H2N
H2N
The first approach to the preparation of SpmTrien
(Scheme 1) envisages the build up of its polyamine
backbone starting from a three carbon fragment, N-ben-
zyloxycarbonyl-3-aminopropanol.
SpmTrien
Spm
NH
NH2
NH2
NH
NH
It was converted into the corresponding mesylate,
which was treated without purification with an excess
N-(2-aminoethyl)-aminoethanol in THF. The unreacted
N-(2-aminoethyl)-aminoethanol was rapidly removed
in a vacuum of oil pump, the residue was dissolved in
1 M NaOH, extracted with dichloromethane, and, after
a flash chromatography on silica gel, crude (I) was
obtained. This appeared to contain the alkylation prod-
uct of the secondary amino group of N-(2-aminoethyl)-
aminoethanol as a minor hardly removable impurity.
The crude mono benzyloxycarbonylamino (I) were
SpmTrien can be considered as an isoster of Spm; how-
ever, unlike Spm, its molecule is asymmetric.
SpmTrien is charge-deficient in respect of its central
fragment, since its K‡ (N3, N6) < 7 and only three of its
amino groups are protonated at physiological pH value.
The presence of triethylenetetramine fragment pro-
vides SpmTrien with good chelating properties toward
the cations of transition metals. Note that neither Spm
nor its analogues possess such a combination of prop-
erties.
Thus, SpmTrien could be used in biochemical stud- acylated with benzyl chloroformate, which led, after
ies of polyamines: in the investigation of the peculiari- flash chromatography on silica gel, to pure trisbenzyl-
ties of Spm active transport into cells; as a new chemi- oxycarbonyl derivative (II). (II) was converted into
cal regulator of the enzymes of polyamine catabolism; mesylate, which was treated without purification with
and for studying the polyamine interaction with nucleic excess ethylenediamine in THF. The ethylenediamine
acids. The combination of properties of SpmTrien excess was distilled off in a vacuum, and the subse-
imply that its Spm-like effects may be at least pH- quent flash chromatography of the residue on silica gel
dependent at the interaction with the specific sites of gave trisbenzyloxycarbonyl-SpmTrien (III). The pro-
Spm binding. The ability or inability of SpmTrien to tective groups were removed from (III) by catalytic
support the cells growth with the depleted polyamine hydrogenation over Pd-black in a methanol–acetic acid
i, ii
CbzHN
OH
NH
CbzHN
OH
NH
(I)
Cbz
N
Cbz
N
i, iv
iii
NH
CbzHN
H2N
OH
CbzHN
· 5HCl
N
N
NH2
(II)
(III)
Cbz
Cbz
NH
NH
v, vi
NH
SpmTrien (IV)
NH2
Reagents: i, MsCl/Et3N/CH2Cl2; ii, H2NCH2CH2NHCH2CH2OH/THF;
iii, CbzCl/NaHCO3/THF/H2O; iv, H2NCH2CH2NH2/THF;
v, H2/Pd/AcOH/MeOH; vi, HCl/MeOH.
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

A CHARGE-DEFICIENT ANALOGUE OF SPERMINE
273
i
ii, iii
H2N
OH
CbzNH
OH
NH
CbzNH
NH
N
N
OH
(V)
OH
NH
(VI)
Cbz
Cbz
Cbz
Cbz
N
i
ii, iv
CbzNH
N
CbzNH
NH
N
N
NH2
(VII)
(VIII)
Cbz
Cbz
NH
H2N
v, vi
NH
NH2 · 5HCl
SpmTrien (IV)
Reagents:
i, CbzCl/NaHCO3/THF/H2O; ii, MsCl/Et3N/CH2Cl2; iii, H2NCH2CH2OH/THF;
iv, H2NCH2CH2CH2NH2/THF; v, H2/Pd/AcOH/MeOH; vi, HCl/MeOH.
Scheme 2.
mixture, and the resulting SpmTrien pentaacetate was
converted into pentahydrocloride.
A greater complexation capacity of SpmTrien for
Cu2+ ions than that of Spm was confirmed in another
model. SpmTrien demonstrated the ability to destroy
complex three-dimensional molecular constructs
formed on the basis of linear double-stranded DNA
molecules fixed within the spatial structure of choles-
teric liquid-crystalline dispersion and crosslinked by
planar polymer bridges. These consisted of alternating
daunomycin molecules and Cu2+ ions [21], which is
schematically presented in Fig. 2.
According to the results of work [21], the formation
of such molecular constructs is accompanied by the
appearance of an extra intense band in CD spectrum in
the region of daunomycin absorption (Fig. 3a, curve 2).
Obviously, the elimination of Cu2+ ions from the poly-
meric chelate bridge as a result of introduction into the
system of such an effective chelator of Cu2+ ions as
SpmTrien (Fig. 4) would lead to the destruction of the
polymer bridge and, consequently, to the disappearance
of the anomal band in the CD spectrum of the DNA
The second approach to the synthesis of SpmTrien
envisages the build up of the analogue backbone start-
ing from a two-carbon fragment (Scheme 2). This
approach allowed the avoidance of the ambiguously
proceeding stage of the primary amino group alkylation
in the presence of unprotected secondary amino group.
Methodologically, this scheme is similar to the first
approach and also is a five-stage process. However, the
isolation and purification procedures for the intermedi-
ates are less laborious. The overall yields of SpmTrien
(IV) were ~20% in both cases.
It is known that Spm and terminally asymmetric bis-
alkylated polyamine analogues can protect DNA from
the free-radical damage under the conditions of Fenton
reaction [19]. This property of Spm was proved to be
due to its direct reaction with free radicals [20].
SpmTrien is an isosteric analogue of Spm and, in the
case of free radicals generated in the system ç2é2/Cu2+,
SpmTrien protects DNA far better than Spm (Fig.1).
One can see from Fig. 1 that, at 25 µM concentration
and the molar ratio SpmTrien–Cu2+ of 1.25 : 1,
SpmTrien effectively prevents the degradation of
pUC19 DNA (lane 5) and a twofold increase in the
SpmTrien concentration hardly improves the protection
of DNA (lane 6). The content of DNA circular form at
25 µM concentration of SpmTrien is much lower then
that in the presence of 1 mM Spm (cf. lanes 5 and 4 in
Fig. 1). The observed differences in the protective prop-
erties of Spm and SpmTrien seem not to be due to the
differences in their DNA-binding properties or to the
reactivity as free radical scavengers. A more likely
explanation of the inhibition of DNA damage is the for-
mation of stable complex between SpmTrien and Cu2+,
which affects the production of free radicals in the
Cu2+/H2O2 system.
CF
LF
SCF
1
2
3
4
5
6
Fig. 1. Electrophoresis in 0.7% agarose gel of the degrada-
tion products of supercoiled DNA pUC19 effected by the
H O /Cu treatment. Lanes: 1, pUC19; 2, pUC19 + PBS,
60 min, 37°C; 3, pUC19 + PBS + 20 µM CuCl + 30 µM
H O , 60 min, 37°C; 4, the same as 3 + 1 mM Spm; 5, the
same as 3 + 25 µM SpmTrien; 6, the same as 3 + 50 µM
SpmTrien. CF, circular; LF, linear; and SCF, supercoiled
form of DNA, respectively.
2+
2
2
2
2
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

274
KHOMUTOV et al.
(a)
(b)
DNA
CH
O
3
OCH
O
O
OH
3
NH
CH
2
HO
HO
3
Neighboring layers
from the DNA
molecules
O
O
O
O
Cu
O
O
CH
3
OH
O
OH
NH
2
Polymer chelate
bridge
CH O
3
O
O
O
O
CH
3
Cu
CH
O
3
OCH
O
O
3
NH
2
HO
HO
DNA molecules
CH
3
O
O
O
O
O
Cu
O
CH
3
OH
O
OH
NH
2
CH O OH
O
O
CH
3
3
DNA
Fig. 2. (a) Schematic representation of polymeric crosslink between two neighboring DNA molecules (the view along the long axis
of DNA molecule) and (b) a hypothetical structure of molecular construct (in accordance with [21]) prepared from DNA cholesteric
liquid-crystalline dispersion.
1
5
0
–500
0
–400
(‡)
1
(b)
4
–800
–1000
–1500
3
2–7
–1200
2
400 450 500 550 600 650 700
400 450 500 550 600 650 700
λ, nm
Fig. 3. CD spectra of DNA molecular construct (curve 2) before and (a, curves 3–5) after treatment with SpmTrien and Spm (b,
–5
curves 3–7). 1, DNA (5.6 µg/ml, daunomicyn (2.7 × 10 M), PEG
(170 mg/ml) in 0.01 M sodium phosphate buffer (pH 7)
4000
containing 0.3 M NaCl (the CD spectrum of cholesteric liquid-crystalline dispersion of DNA–daunomycin complex); 2, the same
as 1 but in the presence of 1 × 10 å CuCl ; (a) the same as 2, but in the presence of 3, 2.5 × 10 M; 4, 4.9 × 10 M; and 5, 7.3 ×
10 M SpmTrien; (b) the same as 2, but in the presence of 3, 2.5 × 10 M; 4, 4.9 × 10 M; 5, 1.5 × 10 M, 6, 2.4 × 10 M; and
–5
–6
–6
2
–6
–6
–6
–5
–5
−5
7, 4.9 × 10 M Spm.
molecular construct. Therefore, the amplitude of this
band can be used as an analytical criterion that allows a
comparison of Cu2+-chelating properties of SpmTrien
and Spm.
Treatment of the DNA molecular construct with
SpmTrien at concentration of 7.3 × 10–6 M at a
SpmTrien–Cu2+ molar ratio of 0.7 : 1 resulted in disap-
pearance of the anomal band in the CD spectrum
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

A CHARGE-DEFICIENT ANALOGUE OF SPERMINE
275
(Fig. 3a, curve 5). The interaction of SpmTrien with
DNA could not be the reason of the CD spectral
changes, since the Spm 2.5 × 10–6–4.9 × 10–5 M con-
centrations exert no effect (Fig. 3b, curves 3–7). There-
fore, the observed changes in CD spectrum were due to
a strong complexation of SpmTrien with Cu2+.
Thus, the presence of triethylenetetramine fragment
in the SpmTrien molecule results in appearance in this
isosteric analogue of an ability to effectively chelate
Cu2+ ions. Such a property is new for Spm analogues. A
unique combination of central fragment charge defi-
ciency of central fragment with significant chelating
properties makes SpmTrien and its derivatives a useful
tool for the investigation of polyamine cellular func-
tions and enzymes of their metabolism.
H2N
NH
NH
Cu2+
NH
H2N
2+
Fig. 4. The structure of Cu –SpmTrien complex.
500 DRX (Germany) at the working frequency of
500.1 MHz for protons and 125.8 MHz for 13C nuclei in
CDCl3 (if other is not indicated) using TMS (CDCl3)
and sodium 3-trimethylsilylpropanesulfonate (D2O) as
internal standards. Chemical shifts are given in ppm,
and constants of spin–spin interaction in Hz.
EXPERIMENTAL
9-(Benzyloxycarbonylamino)-3,6-diazanonan-1-
ol (I). A solution of MsCl (0.87 ml, 0.011 mol) in dry
dichloromethane (10 ml) was added for 15 min to a
stirred and cooled (0°C) solution of N-Cbz-3-amino-
propanol (2.09 g, 0.01 mol) and Et3N (2.08 ml,
0.015 mol) in dry dichloromethane (50 ml). Stirring
was continued for 1 h at 0°C and for 1 h at 20°C. The
reaction mixture was poured into 1 M NaHCO3 (30 ml);
the organic layer was separated, successively washed
with water (10 ml), 0.5 M H2SO4 (3 × 30 ml), water
(20 ml), 1 M NaHCO3 (30 ml), and water (2 × 10 ml);
dried with MgSO4; and evaporated in a vacuum to dry-
ness. The residue was dissolved in THF (25 ml), cooled
to 0°C, and freshly distilled N-(2-aminoethyl)-aminoet-
hanol (20.0 ml, 0.2 mol) was added in one portion. The
reaction mixture was kept for 24 h at 20°C, THF and
unreacted amine were then distilled off in a vacuum.
The residue was poured into 2 M NaOH (20 ml);
extracted with dichloromethane (2 × 20 ml); organic
layer was separated, washed with water (2 × 3 ml);
dried with ä2ëé3 and evaporated in a vacuum. The res-
idue was dissolved in åÂéç–Et3N (8 : 2, 10 ml),
applied onto a silica gel column (125 g) and eluted with
8 : 2 åÂéç–Et3N. The fractions containing (I) were
evaporated in a vacuum and dried in a vacuum over
P2O5. This resulted in (I); a viscous oil; yield 1.8 g
3-Aminopropanol, ethanolamine, ethylenediamine,
1,3-diaminopropane, N-(2-aminoethyl)-aminoethanol,
benzyloxycarbonyl chloride, and mesyl chloride were
purchased from Fluka (Switzerland); polyethylenegly-
col 4000 Da from Ferak (Germany); and Spm, dauno-
mycin, Tris-base, ethidium bromide, Na2-EDTA, and
agarose from Sigma (United States). Other reagents
were manufactured in Russia. N-Benzyloxycarbonyl-3-
aminopropanol was prepared as described in [22].
Supercoiled DNA pUC19 was isolated from
Escherichia coli strain DH5α, which was transformed
with pUC19 DNA, and purified by a CsCl-gradient
centrifugation as described in [23]. Chicken erythro-
cyte DNA with molecular mass of ~(0.3–0.7) × 106 Da
was from Reanal (Hungary); it was additionally puri-
fied from trace proteins and low-molecular polysac-
charides by the procedure [24]. Concentrations of
DNA and daunomycin were spectrophotometrically
determined in aqueous salt solutions, using the molar
absorption coefficients ε260 6600 å–1 cm–1 [23] and
ε
475 12000 å–1 cm–1 [25], respectively. The DNA-con-
taining molecular construct was prepared as previ-
ously described in [26, 27].
TLC was carried out on precoated Kieselgel 60 F254
plates (Merck, Germany) using the following elution
systems: (A) 9 : 1 chloroform–methanol, (B) 9 : 1 diox-
ane–25% ammonia, (C) 95 : 5 chloroform –methanol,
(D) 95 : 5 dioxane–25% ammonia, (E) 97 : 3 dioxane–
25% ammonia, and (F) 4 : 2 : 1 : 2 n-butanol–acetic
acid–pyridine–water. Kieselgel (40–63 µm, Merck,
Germany) was used for column chromatography; elu-
tion systems are indicated in the text. TLC spots of
compounds were visualized in UV-light and also using
color reaction with ninhydrin.
(60%); Rf 0.18 (B); 1H NMR: 7.35–7.24 (5 H, m, C6H5),
5.70 (1 I, bs, NHCbz), 5.07 (2 H, Ò, CH2Ph), 3.62–3.54
(2 H, m, CH2OH), 3.30–3.20 (2 H, m, CH2NHCbz),
2.75–2.62 (6 H, m, CH2NH), 2.57–2.47 (2 H, m,
CH2NH), 2.20 (3 H, bs, NH + OH), 1.68–1.58 (2 H, m,
CH2CH2CH2).
N3,N6,N9-Tris(benzyloxycarbonyl)-9-amino-3,6-
diazanonan-1-ol (II). Benzyl chloroformate (1.5 ml,
12 mmol) was added in three portions at 15-min inter-
vals to a cooled (0°C) and vigorously stirred mixture of
(I) (1.5 g, 5 mmol), THF (10 ml), water (3 ml),
Absorption spectra were registered using Specord
M40 (Germany), and CD-spectra using a portable
dichrometer SKD-2 (Institute of Spectroscopy, Russian
Academy of Sciences, Troitzk, Moscow oblast). 1H and NaHCO3 (0.84 g, 10 mmol), and 10 M NaOH (0.3 ml).
13C NMR spectra were measured on a Bruker Avance Stirring was continued for 1 h at 0°C and then for 3 h at
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

276
KHOMUTOV et al.
20°C, organic layer was separated, the aqueous phase (V) was evaporated and dried over P2O5 in a vacuum;
was extracted with dichloromethane (2 × 5 ml), and the
combined organic layers were evaporated to dryness in
a vacuum. The residue was dissolved in dichlo-
romethane (20 ml); the solution was successively
washed with 0.5 M HCl (3 × 10 ml), water (2 × 10 ml),
and 0.5 M NaHCO3 (2 × 10 ml); dried with MgSO4; and
evaporated in a vacuum. The residue was dissolved in
system (C) (7 ml) and applied onto a silica gel column
(125 g), which was eluted with system (C) to give 2.4 g
of crude (II). This was again purified on the same col-
umn eluted with 96 : 4 chloroform–methanol mixture to
give (II); a viscous oil; yield 1.9 g (68%); Rf 0.45 (C);
yield 13 g (70%); a viscous oil, which solidified on stor-
age; Rf 0.30 (A); 1H NMR: 7.40–7.20 (10 H, m, C6H5),
5.53 (0.5 H, bs, NHCbz), 5.31 (0.5 H, bs, NHCbz), 5.06
(2 H, Ò, CH2Ph), 5.04 (2 H, Ò, CH2Ph), 3.71 (2 H, m,
CH2OH), 3.50–3.25 (6 H, m, CH2N), 2.86 (1 H, bs,
OH).
N6,N8-Bis(benzyloxycarbonyl)-8-amino-3,6-diaza-
octan-1-ol (VI). A solution of MsCl (0.61 ml, 7.8 mmol)
in dry dichloromethane (5 ml) was added for 10 min to
a stirred and cooled (0°C) solution of (V) (2.65 g,
7.1 mmol) and Et3N (1.15 ml, 8.3 mmol) in dry dichlo-
romethane (20 ml). Stirring was continued for 1 h at
0°C and for 1 h at 20°C. The reaction mixture was
treated with 1 M NaHCO3 (20 ml); the organic layer
was separated, washed with 0.5 M H2SO4 (3 × 15 ml),
water (10 ml), 1 M NaHCO3 (20 ml), and water (10 ml);
dried with MgSO4; and evaporated to dryness in a vac-
uum. The residue was dissolved in dry THF (7 ml),
cooled to 0°C, and ethanolamine (4.2 ml, 68 mmol) was
added in one portion. The reaction mixture was kept for
1H NMR: 7.40–7.20 (15 H, m, C6H5), 5.48 (1 H, bs,
NHCbz), 5.17–4.98 (6 H, m, CH2Ph), 3.75–3.53 (2 H,
m, CH2OH), 3.52–2.95 (10 H, m, CH2NCbz), 2.36
(1 H, bs, OH), 1.61–1.45 (2 H, m, CH2CH2CH2).
N6,N9,N12-Tris(benzyloxycarbonyl)-1,12-diamino-
3,6,9-triazadodecane (III). A solution of MsCl
(0.25 ml, 3.2 mmol) in dry dichloromethane (5 ml) was
added for 20 min to a stirred and cooled (0°C) solution
of (II) (1.7 g, 3 mmol) and Et3N (0.7 ml, 5 mmol) in dry
dichloromethane (20 ml). Stirring was continued for 6 h at 20°C and 48 h at 20°C, and THF and unreacted
1 h at 0°C and for 1 h at 20°C. The reaction mixture was amine were distilled off in a vacuum. The residue was
poured into 1 M NaHCO3 (10 ml); the organic layer
was separated, washed with water (8 ml), 0.5 M H2SO4
(3 × 7 ml), water (8 ml), 1 M NaHCO3 (5 ml), and water
(2 × 10 ml); dried with MgSO4; and evaporated in a vac-
uum. The residue was dissolved in dry THF (10 ml),
cooled to 0°C, and ethylenediamine (4 ml, 60 mmol)
was added in one portion. The reaction mixture was
kept for 14 h at 20°C, and THF and unreacted diamine
were then distilled off in a vacuum. The residue was
dissolved in 8 : 2 methanol–Et3N (10 ml) and the result-
ing solution was separated into two parts, each part was
purified on a silica gel column (125 g) eluted with the
same solvent mixture to get (III); a viscous oil after
drying in a vacuum over P2O5; yield 1.07 g (59%); Rf
dissolved in 2 M NaOH (10 ml) and extracted with
chloroform (2 × 7 ml); organic layer was separated,
washed with water (2 × 2 ml); dried with ä2ëé3 and
evaporated in a vacuum. The residue was dissolved in
100 : 0.8 dioxane–25% ammonia (10 ml); applied onto
a silica gel column (130 g) and eluted with the same
solvent mixture. The resulting (VI) was dried in a vac-
1
uum over P2O5; yield 2.05 g (70%); Rf 0.39 (E); H
NMR: 7.34–7.26 (10 H, m, C6H5), 5.70 (1 I, bs,
NHCbz), 5.10 (2 H, s, CH2Ph), 5.05 (2 H, s, CH2Ph),
3.58–3.52 (2 H, m, CH2OH), 3.43–3.34 (6 H, m,
CH2NCbz), 2.83–2.64 (4 H, m, CH2NH), 2.25 (2 H, bs,
NH + OH).
1
N3,N6,N8-Tris(benzyloxycarbonyl)-8-amino-3,6-
diazaoctan-1-ol (VII). Benzyl chloroformate (0.8 ml,
5.4 mmol) was added in two portions at a 20-min inter-
val to a cooled (0°C) and vigorously stirred mixture of
(VI) (1.95 g, 4.7 mmol), THF (6 ml), water (2 ml),
NaHCO3 (0.7 g, 8.3 mmol), and 10 M NaOH (0.3 ml).
Stirring was continued for 1 h at 0°C and then for 3 h at
20°C. Organic layer was separated, the aqueous phase
was extracted with dichloromethane (5 ml), and the
combined organic layers were evaporated to dryness in
a vacuum. The residue was dissolved in chloroform,
dried with MgSO4, and evaporated in a vacuum. The
residue was dissolved in 99 : 1 chloroform–methanol
(15 ml); applied onto a silica gel column (130 g) and
eluted with the same solvent mixture. This resulted in
(VII); yield 2.19 g (85%); Rf 0.6 (C); 1H NMR: 7.34–
7.23 (15 H, m, C6H5), 5.10–4.98 (6 H, m, CH2Ph), 3.75–
3.63 (2 H, m, CH2OH), 3.54–3.05 (10 H, m, CH2NCbz),
0.20 (D); H NMR: 7.42–7.22 (15 H, m, C6H5), 5.53
(1 H, bs, NHCbz), 5.17–4.97 (6 H, m, CH2Ph), 3.47–
2.93 (10 H, m, CH2NCbz), 2.85–2.43 (6 H, m, CH2NH),
1.60–1.19 (5 H, m, CH2CH2CH2 + NH + NH2).
N3,N5-Bis(benzyloxycarbonyl)-5-amino-3-azapen-
tan-1-ol (V). Benzyl chloroformate (15 ml, 0.11 mol)
was added in five portions with 20-min intervals to a
cooled (0°C) and vigorously stirred mixture of N-(2-
aminoethyl)-aminoethanol (5.2 g, 0.05 mol), THF
(100 ml), water (17 ml), NaHCO3 (8.4 g, 0.1 mol), and
10 M NaOH (2.5 ml). Stirring was continued for 1 h at
0°C and for 3 h at 20°C. Organic layer was separated
and evaporated to dryness in a vacuum. The residue was
dissolved in EtOAc (100 ml); the solution was succes-
sively washed with 1.0 M HCl (3 × 40 ml), water (2 ×
40 ml); dried with MgSO4; and evaporated in a vacuum.
The residue was dissolved in chloroform (25 ml) and
applied onto a silica gel column (300 g) eluted first with
chloroform and then with a system (A). The fraction of 2.36 (1 H, bs, OH).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

A CHARGE-DEFICIENT ANALOGUE OF SPERMINE
277
N1,N3,N6-Tris(benzyloxycarbonyl)-1,12-diamino-
3,6,9-triazadodecane (VIII). A solution of MsCl
and the combined filtrates were evaporated in a vac-
uum. The residue was dissolved in hot methanol (9 ml),
(0.3 ml, 3.8 mmol) in dry dichloromethane (5 ml) was and 5 M HCl (1.4 ml, 7 mmol) and hot EtOH (20 ml)
added for 10 min to a stirred and cooled (0°C) solution
of (VII) (1.95 g, 3.5 mmol) and Et3N (0.55 ml, 4 mmol)
in dry dichloromethane (20 ml). Stirring was continued
for 1 h at 0°C and for 1 h at 20°C. Reaction mixture was
treated with 1 M NaHCO3 (5 ml); the organic layer was
separated, successively washed with 0.5 M H2SO4 (3 ×
4 ml), water (4 ml), 1 M NaHCO3 (5 ml), and water
(3 ml); dried with MgSO4; and evaporated in a vacuum.
The residue was dissolved in dry THF (5 ml), cooled to
0°C, and 1,3-diaminopropane (2.5 ml, 30 mmol) was
added in one portion. The reaction mixture was kept for
6 h at 0°C, and then 24 h at 20°C; and THF and unre-
acted diamine were then distilled off in a vacuum. The
residue was dissolved in 1 M NaOH (10 ml) and
extracted with chloroform (20 ml). The organic layer
was separated, washed with water (2 × 3 ml), dried with
ä2ëé3 and evaporated in a vacuum. The residue was
dissolved in system (E) (10 ml); applied onto a silica
gel column (130 g) and eluted with the same system.
The resulting (VIII) was dried in a vacuum over ê2é5;
were added. After cooling, the solution was evaporated
in a vacuum to one half of initial volume, then boiling
EtOH (15 ml) was added, and the resulting mixture was
left at –20°C overnight. The precipitate was filtered,
washed with cooled EtOH, and dried in a vacuum over
P2O5/KOH. The yield of (IV) pentahydrochloride 0.19 g
(70%), the preparation was identical to the sample
obtained by method A.
Protection of supercoiled DNA from the free-
radical H2O2/Cu2+ damage by Spm and SpmTrien
(IV). A supercoiled DNA plasmid pUC19 (200 ng) was
incubated for 60 min at 37°C in PBS (pH 7.4), contain-
ing 30 µå ç2é2 and 20 µM CuCl2 in total volume
30 µl (mixture A). The incubation in PBS without addi-
tion of ç2é2 and CuCl2 was used as a control. The pro-
tective effects of Spm and SpmTrien were investigated
incubating mixture A containing either 1 mM Spm or
SpmTrien at a concentration of 25 or 50 µM for 60 min
at 37°C. Aliquots (5 µl) of the reaction mixtures were
analyzed by electrophoresis in 0.7% agarose gel, con-
taining 40 mM Tris-acetate buffer (pH 7.4) and 1 mM
ÖDíÄ-Na2. Gel was stained with ethidium bromide
(2 µg/ml) for 10 min, the stain was washed out with
water, and gel was photographed in UV-light. The
results are presented at Fig. 1.
1
yield 1.5 g (71%); Rf 0.15 (E); H NMR: 7.40–7.25
(15 H, m, C6H5), 5.10–4.98 (6 H, m, CH2Ph), 3.49–
3.05 (10 H, m, CH2NCbz), 2.85–2.46 (6 H, m, CH2NH),
1.72 (3 H, s, NH), 1.65–1.44 (2 H, m, CH2CH2CH2).
1,12-Diamino-3,6,9-triazadodecane pentahydro-
chloride (IV). MethodA.Approximately 1 ml of a sus-
pension of Pd-black in methanol was added to a solu-
tion of (III) (0.97 g, 1.7 mmol) in 1 : 1AcOH–methanol
mixture (20 ml) and hydrogenation at atmospheric
pressure was continued until the end of CO2 evolution
(~2.5 h). The catalyst was filtered off and washed with
methanol, and the combined filtrates were evaporated
in a vacuum. The residue was dissolved in hot methanol
(14 ml), and after the addition of 5 M HCl (3.5 ml) and
hot EtOH (15 ml) the mixture was left overnight at 0°C.
The precipitate was filtered off, washed with cold 1 : 1
methanol–ethanol mixture and dried in a vacuum over
P2O5/KOH to give (IV) pentahydrochloride; yield 0.52 g
(79%); mp.: 265–266°C (decomp.); Rf 0.24 (F); MS,
m/z: [M + H]+ 204.05 (C9H25N5; calculated [M + H]+
204.22); 1H NMR (D2é): 3.58–3.47 (10 H, m,
CH2NH), 3.42 (2 H, t, J 6.9, CH2NH), 3.26 (2 H, t, J
8.0, CH2NH), 3.13 (2 H, t, J 7.5, CH2NH), 2.14 (2 H,
m, CH2CH2CH2); 13ë NMR (D2é): 48.09, 47.58,
46.85, 46.73, 46.51, 39.44, 38.50, 26.69. Found, %: C
27.87, H 7.97, N 18.10. C9H30N5Cl5. Calculated, %: C
28.03, H 7.84, N 18.16.
Interaction of SpmTrien (IV) and Spm with
DNA-containing molecular constructs. DNA choles-
teric liquid-crystalline dispersion was prepared in
accordance with the procedure [26, 27], mixing an
equal volume of DNA solution (11.2 µg/ml) in 0.01 M
sodium phosphate buffer (pH 7) containing 0.3 M NaCl
(buffer B) with a PEG4000 solution (340 mg/ml) in the
same buffer B. Then, 4 mM solution of daunomycin in
buffer B (13.7 µl per 2 ml of DNA cholesteric liquid-
crystalline dispersion) was added at an effective stir-
ring. A cholesteric liquid-crystalline dispersion of
DNA–daunomycin complex formed. This dispersion
was supplemented with 1 mM CuCl2 (20 µl) in buffer B
at effective stirring, which gave rise to the required
three-dimensional molecular construct (detailed proto-
cols for the preparation of DNA-containing molecular
construct are described in [26, 27]). Neighboring DNA
molecules in it are crosslinked with polymeric Cu2+-
containing polymer chelate bridges (Fig. 2b). The CD-
spectra of this molecular construct were registered at
the area of daunomycin absorption (Fig. 3a, curve 2).
The above solution of molecular construct (2 ml) was
mixed with 1-µl portions of aqueous 4.9 mM SpmTrien
solution or 4.9 mM Spm solution (1-, 2-, 5-, 10- or
Method B. Approximately 0.5 ml of a suspension of
Pd-black in methanol was added to a solution of (VIII)
(0.42 g, 0.7 mmol) in 1 : 1 AcOH–methanol mixture
(10 ml), and hydrogenation at atmospheric pressure 20-µl portions) and, CD-spectra were measured after
was carried out until the end of CO2 evolution (~1.5 h).
each addition (Fig. 3a, curves 3–5). The increase in the
The catalyst was filtered off and washed with methanol, volume of starting solution was neglected.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

278
KHOMUTOV et al.
13. Tanabe, R., Kobayashi, M., Sugawara, M., Iseki, K., and
ACKNOWLEDGMENTS
Miyazaki, K., J. Pharm. Pharmacol., 1996, vol. 48,
We thank Dr. H.B.F. Dixon (Department of Bio-
chemistry, University of Cambridge, UK) who attracted
our attention to triethylenetetramine (Trien) and for
fruitful discussion.
This work was supported by the Russian Foundation
for Basic Research, project nos. 03-04-49080 and
03-04-48212, and NCI grant R01 CA 85509.
pp. 517–521.
14. Schwarzenbach, G., Helv. Chim. Acta, 1950, vol. 33,
pp. 974–985.
15. Dixon, H.B.F., Gibbs, K., and Walshe, J.M., Lancet,
1972, pp. 853–854.
16. Renault, J., Lebranchu, M., Anne, Lecat, A., and Uriac,
P., Tetrahedron Lett., 2001, vol. 42, pp. 6655–6658.
17. Edwards, M.L., Prakash, N.J., Stemerick, D.M., Sun-
kara, S.P., Bitonti, A.J., Davis, G.F., Dumont, J.A., and
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