Novel isosteric charge-deficient spermine analogue-1,12-diamino-3,6,9- triazadodecane: Synthesis, pKa measurement and biological activity

Article abstract of DOI:10.1007/s00726-009-0409-6

Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2-11.0 using two-dimensional ¹H-¹⁵N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic pK_avalues of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes. Springer-Verlag 2009.

Full text of DOI:10.1007/s00726-009-0409-6

Amino Acids (2010) 38:501–507
DOI 10.1007/s00726-009-0409-6
ORIGINAL ARTICLE
Novel isosteric charge-deficient spermine analogue—1,12-
diamino-3,6,9-triazadodecane: synthesis, pKa measurement
and biological activity
•
Janne Weisell Mervi T. Hyvonen Jouko Vepsalainen
•
•
¨
Leena Alhonen Tuomo A. Keinanen
¨ ¨
•
•
¨
Alex R. Khomutov Pasi Soininen
•
Received: 18 August 2009 / Accepted: 20 October 2009 / Published online: 2 December 2009
Ó Springer-Verlag 2009
Abstract Ionic interactions are essential for the biologi-
cal functions of the polyamines spermidine and spermine in
mammalian physiology. Here, we describe a simple gram
scale method to prepare 1,12-diamino-3,6,9-triazadodecane
(SpmTrien), an isosteric charge-deficient spermine ana-
logue. The protonation sites of SpmTrien were determined
at pH range of 2.2–11.0 using two-dimensional 1H-15N
NMR spectroscopy, which proved to be more feasible than
conventional methods. The macroscopic pKa values of
SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly
lower than those of 1,12-diamino-4,9-diazadodecane
(spermine). The effects of SpmTrien and its parent mole-
cule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth
and polyamine metabolism were investigated in DU145
prostate carcinoma cells. SpmTrien downregulated the
biosynthetic enzymes ornithine decarboxylase (ODC) and
S-adenosyl-L-methionine decarboxylase and decreased
intracellular polyamine levels, whereas the effects of Trien
alone were minor. Interestingly, both SpmTrien and Trien
were able to partially overcome growth arrest induced by
an ODC inhibitor, a-difluoromethylornithine, indicating
that they are able to mimic some functions of the natural
polyamines. Thus, SpmTrien is a novel tool to influence
polyamine interaction sites at the molecular level and
offers a new means to study the contribution of the pro-
tonation of spermine amino group(s) in the regulation of
polyamine-dependent biological processes.
Keywords Polyamines ꢀ Spermine analogues ꢀ
pKa ꢀ NMR spectroscopy ꢀ Cell growth
Abbreviations
AdoMetDC S-adenosyl-L-methionine decarboxylase
AG
Aminoguanidine
N1,N11-diethylnorspermine
DENSpm
DFMO
a-Difluoromethylornithine (2,5-diamino-2-
(difluoromethyl)pentanoic acid)
Heteronuclear multiple bond correlation
Heteronuclear single quantum correlation
Ornithine decarboxylase
HMBC
HSQC
ODC
Pu
Putrescine (1,4-diaminobutane)
Spermine (1,12-diamino-4,9-diazadodecane)
(1,12-Diamino-3,6,9-triazadodecane)
Spermidine (1,8-diamino-4-azaoctane)
Spermidine/spermine N1-acetyltransferase
Triethylenetetramine (1,8-diamino-3,6-
diazaoctane)
Spm
SpmTrien
Spd
SSAT
Trien
¨ ¨
J. Weisell (&) ꢀ J. Vepsalainen ꢀ P. Soininen
TSP
Sodium 3-trimethylsilyl[2,2,3,3-
d4]propionate
Laboratory of Chemistry, Department of Biosciences,
University of Kuopio, Kuopio, Finland
e-mail: Janne.Weisell@uku.fi
¨
¨
M. T. Hyvonen ꢀ L. Alhonen ꢀ T. A. Keinanen
Department of Biotechnology and Molecular Medicine,
A. I. Virtanen Institute, Biocenter Kuopio, University of Kuopio,
Kuopio, Finland
Introduction
The polyamines spermine (Spm) and spermidine (Spd), and
their diamine precursor putrescine (Pu) are present at high
concentrations in practically all cell types and are crucial
A. R. Khomutov
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Moscow, Russia
123

502
J. Weisell et al.
for cell growth and viability. The effects are determined
by the shape of the molecule providing proper spatial
localization of the amino groups. The degree of the pro-
tonation of the amino groups along the spatially flexible
carbon chain is essential for the molecular recognition of
polyamines and their analogues with different cellular
constituents (Basu et al. 1990; Ramirez et al. 2002;
Bergeron et al. 1995). The investigation of the structure–
activity relationships for the Spm and Spd analogues have
focused mainly on the contribution of molecular geome-
try to the biological effects (for the review see Casero and
Woster 2001; Wallace and Niiranen 2007; Casero and
Marton 2007), while the impact of the protonation of the
amino group(s) to the biological activity of polyamines
has not been studied in detail. To decrease the pKa values
of the terminal amino groups in terminally bis-alkylated
derivatives, a family of the analogues bearing piperidine-
or pyridine groups has been synthesized together with the
terminally bis-trifluoroethylated Spm (Bergeron et al.
1988, 1995). Also, a family of 2,2-, 6,6-, and 7,7-diflu-
orospermidines has been designed and investigated as
polyamine surrogates (Baillon et al. 1988). However, a
rational approach to design abnormally protonated Spm
analogues should definitely result in a decrease in the pKa
values of H2N– or –NH-groups with minimal steric
disturbances of the parent polyamine molecule. This
seriously limits the possibility of designing isosteric
charge-deficient analogues of Spm. One approach is the
substitution of terminal H2NCH2-group of Spd or Spm
with H2NO-group. The aminooxy analogues of Spd and
Spm (Khomutov et al. 1996) have been of some interest
becomes, intolerant to the primary medication with pen-
icillamine (Dixon et al. 1972; Schilsky 2001). Recently,
Trien was successfully used to regenerate the diabetic
heart by selective copper chelation (Cooper et al. 2004).
The ability of SpmTrien to induce pH-dependent con-
densation of DNA, which can be converted to isotropic
state upon the addition of Cu2? ions, is the only known
biochemical application of this Spm analogue (Khomutov
et al. 2007). Hence, the knowledge of the protonation
state of SpmTrien at different pH is of crucial importance
for the interpretation of the biological activity of this
unusual Spm analogue. Here, we present a convenient,
gram scale synthesis of SpmTrien, NMR determination of
the pKa values of SpmTrien amino groups, and bio-
chemical data on the effects of SpmTrien and Trien on
cell growth and polyamine metabolism in DU145 prostate
cancer cells.
Materials and methods
Acrylonitrile, Adam’s catalyst, Celite, and triethylenetet-
ramine (Trien) were purchased from Aldrich (United
States); aluminium oxide and the rest of chemicals from
Fluka (Switzerland). TLC was carried out on precoated
Kieselgel 60 F254 plates from Merck (Germany) and
SpmTrien was developed with ninhydrin. Melting points
were determined in open capillary tubes and are
uncorrected.
12-Amino-4,7,10-triazadodecanonitrile (TrienC2CN)
¨
to the polyamine field already (Hyvonen et al. 1995;
Eloranta et al. 1990; Turchanowa et al. 2002). The oxa-
analogues of polyamines containing an –NH–O-group
instead of the central –NH–CH2-group have also been
synthesized (Lin et al. 1994; Khomutov et al. 2005), but
their biochemistry has not yet been investigated.
A mixture of Trien (5.14 g, 35 mmol) and acrylonitrile
(0.627 g, 12 mmol) was stirred at 20°C for 16 h and then
all volatile material was evaporated in vacuo that resulted
in crude TrienC2CN, which was purified by flash chro-
matography on aluminium oxide using 1% NH3 in meth-
anol as an eluent, to give TrienC2CN (1.3 g, 55%) as
The present paper shows an alternative approach to
design an isosteric charge-deficient analogue of Spm
consisting of replacement of the Spd fragment of Spm
molecule with a triethylenetetramine (Trien) that gives
rise to SpmTrien (Table 1). This compound can be con-
sidered as an isoster of Spm with non-symmetrical
structure. Very limited biochemical data related to the
effects on polyamine metabolism is available even for the
parent Trien, which in certain cellular processes seems to
be capable of mimicking the natural polyamines (Ko-
bayashi et al. 1999). This property is partly interpreted in
terms of the protonation status of this Spd analogue. Trien
forms a very stable complex with Cu2? [pKuns 20.4 at pH
14 and pKuns 14.2 at pH 7 (Schwarzenbach 1950)] and is
used as an alternative treatment of Wilson’s disease
1
viscous oil. H NMR (D2O): d 3.19–3.16 (4H, m), 3.10
(2H, t, J 6.1 Hz), 2.99–2.91 (4H, m), 2.69 (2H, t, J 6.7 Hz);
13C 20.34, 41.58, 46.47, 46.70, 47.19, 48.09, 49.27, 49.64,
51.77, 123.30.
1,12-Diamino-3,6,9-triazadodecane pentahydrochloride
(SpmTrien)
A mixture of TrienC2CN (0.7 g, 3.5 mmol) and Adams
catalyst (0.08 g) in 75% aqueous AcOH (20 ml) was
hydrogenated at 3 bar overnight. The catalyst was filtered
off through Celite and combined filtrates were evaporated
to dryness in vacuo. To the residue 6 M HCl (5 ml) was
added and after evaporation to dryness in vacuo the residue
was crystallized from minimal volume of 1 M HCl and
(hepatolenticular degeneration) if
a patient is, or
123

Novel isosteric charge-deficient spermine analogue
503
Table 1 The structures and the pKa values of Spd, Spm, and their analogues—Trien and SpmTrien
Abbreviation
Spd
Structure
pKa 1
pKa 2
pKa 3
pKa 4
pKa 5
8.24
9.81
10.89
NH2
H2N
N
H
Spm
7.96
3.27
3.3
8.85
6.56
6.3
10.02
9.07
8.5
10.80
9.74
9.5
H
N
NH2
H2N
N
H
Trien
H
N
NH2
H2N
N
H
SpmTrien
10.3
H
N
NH2
H2N
N
H
N
H
pKa Values of Spd, Spm, and Trien are from the review (Bencini et al. 1999)
dried over P2O5/KOH in vacuo to give SpmTrien penta-
chlorohydrate (0.96 g, 71%) as colourless solid, mp 263–
265°C (dec.), Rf 0.22 (n-butanol–AcOH–pyridine–H2O;
in the phase-sensitive mode using the Echo/Antiecho-TPPI
gradient selection. The data matrix was 128 9 1 K and the
typical spectral width was 4.7 kHz for proton and 15 kHz
for carbon. An evolution time of 1/(4JCH) = 1.72 ms was
used. For each FID, 4 transients were accumulated. A pure
squared cosine window function was applied in both
1
4:2:1:2). H NMR (D2O, at pH* 4.5): d 3.375 (2H6, m, J
6.66), 3.363 (2H1, m, J 7.52; 6.31), 3.348 (2H2, m, J 6.31;
7.52), 3.270 (2H3, m, J 6.36), 3.266 (2H5, m, J 6.66), 3.236
(2H7, m, J 5.62; 10.25), 3.209 (2H4, m, J 6.36), 3.133(2H9,
m, J 9.81; 5.83), 2.142 (2H8, m, J 5.62; 5,83; 9.81; 10.25);
13C NMR (D2O, at pH* 4.5): d 45.72 (C3), 45.20 (C6),
45.04 (C4, C7), 44.68 (C2), 43.87 (C5), 36.74 (C9), 36.48
(C1), 23.84 (C8). [M ? H]? calc for C9H26N5: 204.2188.
Obs: 204.2186.
1
dimensions prior to Fourier transform. H–15N heteronu-
clear multiple bond correlation (HMBC) experiments were
carried out in the magnitude mode. The typical data matrix
was 128 9 2 K. The typical spectral width was 3 kHz for
proton and 2 kHz for nitrogen. An evolution time of 1/
(2JNH) = 71 ms was used. For each FID, 4–10 transients
were accumulated depending on the concentration of the
sample. A pure sine window function was applied in both
dimensions prior to Fourier transform.
Determination of pKa values
Determination of the macroscopic pKa values of SpmTrien
was performed with a Sirius PC-200 automatic titrator
according to the manufacturer’s protocol. One-dimensional
Cell culture
1H and two-dimensional H-15N and H-13C NMR spec-
troscopy were used to discover the protonation order of
SpmTrien. NMR samples were prepared by dissolving
SpmTrien to H2O (100 mg/ml) and adjusting pH gradually
from 2.1 to 10.9 with NaOH. Samples were taken
approximately at intervals of 0.3 pH units. NMR spectra
were recorded with a Bruker AVANCE DRX spectrometer
operating at 500.13 MHz using a double-tube system
facilitating locking and chemical shift referencing. The
external reference tube (o.d. 2 mm, supported by a
Teflon adapter) containing the reference substance (sodium
3-trimethylsilyl[2,2,3,3-d4]propionate (TSP) 40 mmol/l,
MnSO4 0.6 mmol/l in 99.8% D2O) was placed coaxially
into the NMR sample tube (o.d. 5 mm) containing 400 ll
The human prostate carcinoma cell line DU145 (American
Type Culture Collection) was cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM, Sigma) supplemented
with 10% heat-inactivated foetal bovine serum (Sigma) and
50 lg/ml gentamycin (Sigma). The cells were incubated in
humidified atmosphere ?37°C, 10% CO2. The cells were
seeded at density of 2 9 106 cells/10 cm plate and let to
attach overnight. Fresh medium was then changed, con-
taining 50 lM Trien (Sigma), or SpmTrien with or without
5 mM a-difluoromethylornithine (DFMO, ILEX oncology
Inc.) or 1 mM aminoguanidine (AG, Sigma). The cells
were detached using solution containing 0.25% trypsin and
1 mM EDTA in phosphate-buffered saline. The cell num-
ber was measured electronically with Coulter Counter
model Z1. The cells were washed, pelleted and stored at
-70°C prior to analysis. The pellets were lysed in lysis
buffer [50 mM potassium phosphate buffer pH 7.2, 1 mM
1
1
1
of each sample. H spectra were measured using standard
1
protocol. H–13C gradient-enhanced heteronuclear single
quantum correlation (HSQC) experiments were carried out
123

504
J. Weisell et al.
EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, Complete
EDTA-free protease inhibitor cocktail (Roche)] and incu-
bated for 20 min on ice. Samples for polyamine measure-
ment were taken and mixed 9:1 with 50% sulphosalicylic
acid containing 100 lM diaminoheptane. The rest of the
lysate was centrifuged for 12,0009g 20 min at ?4°C. The
supernatant fraction was used for enzymatic assays of
spermidine/spermine N1-acetyltransferase (SSAT), orni-
thine decarboxylase (ODC), and S-adenosyl-L-methionine
decarboxylase (AdoMetDC).
H
N
NH2
Trien
H2N
N
H
CN
H
N
H
N
H2N
H2N
N
CN
TrienC2CN
H
PtO2/H2
Enzyme activities, polyamines and analogs
H
N
H
N
NH2
Intracellular polyamines were measured with HPLC
¨
according to the published method (Hyvonen et al. 1992).
N
H
SpmTrien
ODC, SSAT and AdoMetDC activities were measured as
¨
described earlier (Janne and Williams-Ashman 1971;
Bernacki et al. 1992; Hyvonen et al. 2009). 14C-labelled
Scheme 1 Synthesis of SpmTrien
¨
substrates used in the assays were obtained from GE
Healthcare.
types of molecules ranging from small molecules to large
proteins (Szakacs et al. 2004). However, in some cases the
overlap of signals in the spectrum prevents the signal
assignment, and thus two-dimensional methods must be
Results and discussion
1
used. The complexity of the H spectrum of SpmTrien at
pH 4.5 with somewhat separated proton chemical shifts is
shown in Fig. 1. Different NMR methods have been
compared for the pKa determination of Spm, where two-
Synthesis of SpmTrien
SpmTrien was prepared at gram scale by a cyanoethylation
of Trien following a known method (Jones et al. 1995) with
a subsequent catalytic reduction of the intermediate nitrile
(TrienC2CN) with hydrogen over PtO2 (Scheme 1). The
key point of this synthetic strategy was the purification of
the TrienC2CN by flash chromatography on aluminium
oxide. Compared to other methods, such as high vacuum
distillation, or flash chromatography on silica gel, the use
of Al2O3 chromatography was superior in separating Tri-
enC2CN from both the remaining starting material and the
side-formed bis-nitrile derivative. The required (Tri-
enC2CN) was obtained with 98% purity and 50–60%
yields. The hydrogenation over Adam’s catalyst in 75%
aq.AcOH, which dissolves completely the TrienC2CN,
afforded target SpmTrien with practically quantitative
yield. Subsequent crystallization from 1 M aq.HCl resulted
in required SpmTrien pentahydrochloride.
1
dimensional H-13C HMQC gave the most precise values
1
when compared to one-dimensional H, 13C or 15N tech-
niques (Frassineti et al. 1995).
In the case of SpmTrien, the protonation pattern could
not be studied by these conventional NMR methods due to
the seriously overlapping signals. In addition, the effect of
pH on the chemical shift differences in 15N NMR spectra
was superior compared to 1H NMR spectra. Assignment of
(N-II)-(N-III)-(N-IV) was apparent but trials to assign
1
required CH2 signals unambiguously in H NMR spectra
were laborious when pH was above 3.5. As an example
1
(see Fig. 1), we analysed the H NMR spectrum at pH 4.5
using PERCH NMR software (Laatikainen et al. 1996). At
this pH value, the spectral lines were relatively sharp (half
width ca. 0.3 Hz) and the chemical shifts of different N–
CH2-protons were resolved. The main problems with these
similar N–CH2-protons are the second order effects which
appear when protons having close chemical shifts (here
0.2 ppm or 100 Hz) are coupled to each other. Typically,
second order effects give rise to distorted intensities of the
chemical shift multiplets and also the number of peaks
might increase dramatically. The calculated and observed
spectra (Fig. 1) were almost identical and the differences
were mostly due to the minor impurities, distinctions in the
line widths or long range couplings, which were not
NMR determination of pKa values of SpmTrien
Traditionally, pKa determination is performed using
potentiometric titration. However, the titration method
cannot be used to identify individual protonation sites, and
therefore a molecular method must be used. With NMR
spectroscopy the precise site of the protonation can be
1
determined and H, 13C and 15N NMR methods for the
3
included in the system. The calculated vicinal JHH
determination of pKa values have been used for various
123

Novel isosteric charge-deficient spermine analogue
505
Fig. 1 Observed versus calculated NMR spectra of SpmTrien at pH*
4.5. Figures above signals indicate exact chemical shifts of the
assigned –CH2– protons of SpmTrien
couplings were in the normal range and typically each
proton has two different couplings to neighbouring protons
due to hindered rotation of C–N- or C–C-bonds in the
2
NMR time scale but germinal JHH (H–C–H) coupling
constants were not resolved due to identical or almost
Fig. 2 15N Chemical shifts (ppm) as a function of pH. The dashed
lines illustrate the titrated pKa values of SpmTrien. The chemical
shifts evidence that the charge is delocalized into the different amines
and the protonation does not occur in classical way
identical chemical shifts of the CH2-protons.
1
Thus, a 2D H-15N heteronuclear multiple bond corre-
lation (HMBC) method was used to determine the proton
coordination of SpmTrien. Furthermore, the 1H–15N
method is an excellent choice for the analysis of proton-
ation of the polyamines, since the binding site of a proton is
directly detectable giving more precise way to look at
the binding of the proton in detail. Here, we report rela-
tively low pKa values of the aliphatic amino groups of
SpmTrien.
Effects of SpmTrien on cell growth and polyamine
metabolism in prostate carcinoma cells
DU145 cells could be cultured with Trien and SpmTrien
(50 lM) in the absence of aminoguanidine (AG), an
inhibitor of amine oxidases present in culture media
serum (Fig. 3). By contrast, under the same conditions the
natural polyamines Spd and Spm were toxic to the cells
(data not shown). Thus, Trien and SpmTrien seemed to be
resistant to serum amine oxidases. This may be partly due
to their copper chelating properties, since many serum
amine oxidases are Cu2? dependent (Agostinelli et al.
1998; Nocera et al. 2003). Cells treated with Trien for
72 h grew as well as control cells, whereas SpmTrien
exposure resulted in a slightly cytostatic effect (Fig. 3).
This difference is likely to be attributable to their effects
on intracellular polyamine pools (Table 2). After 72 h
incubation, Trien exerted only minor effects on the
activities of the polyamine-metabolizing enzymes. By
contrast, treatment with SpmTrien inhibited ODC activity
more efficiently than DFMO (Table 2), the commonly
used irreversible inhibitor of ODC (Wallace and Fraser
2004). SpmTrien also inhibited the activity of another
important biosynthetic enzyme, AdoMetDC, and slightly
induced the catabolic enzyme SSAT. However, the extent
of SSAT induction was low compared to the superin-
ducers, such as N1,N11-diethylnorspermine (DENSpm),
which can induce SSAT activity several 1,000-fold
The macroscopic pKa values of the amino groups of
SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) determined with
potentiometric titration (Fig. 2; Table 1) were compara-
ble to those of similar but symmetric molecules summa-
rized in a review (Bencini et al. 1999). SpmTrien is not a
symmetrical molecule and its protonation behaviour dif-
fers from other similar, but symmetrical, compounds. The
protonation of SpmTrien first occurs at both the ends of
the molecule. According to our results, the central N-III
amino group started to protonate third, but then the proton
migrated from the central N-III amino group to either N-
II or N-IV amino groups when pH decreased (Fig. 2).
Hence, there was equilibrium in the protonation of the
secondary amino groups of SpmTrien (Fig. 2) when the
pH value was between 7 and 8. This caused a ‘‘wrong
way’’ evolution in the 15N chemical shifts. In general, the
microscopic pKa values of polyprotic acids with more
than three binding sites for protons and no symmetry are
impossible to obtain due to the proton transfers (Ullmann
2003). Similar behaviour is also seen in diethylene-
triaminepentaacetic acid, and is explained by the change
in the proton-binding site (Ullmann 2003; Onufriev et al.
2001).
123

506
J. Weisell et al.
Fig. 3 DU145 cell growth
(72 h) with 50 lM analogs with
or without 5 mM DFMO, or
with or without 1 mM AG.
Results are mean SD
(n = 3). ***P values of \0.001
as analysed by one-way analysis
of variance with Tuckett’s post
hoc test
Table 2 Effect of 50 lM Trien and SpmTrien (72 h) on polyamine metabolism in DU145 cells
Treatment
ODC
(pmol/h
AdoMetDC
(pmol/h
SSAT
(pmol/h
Pu
(pmol/h
Spd
(pmol/h
Spm
(pmol/h
Analogue
(pmol/h
Total PA
(pmol/h
per 106 cells) per 106 cells) per 106 cells) per 106 cells) per 106 cells) per 106 cells) per 106 cells) per 106 cells)
Control
1,401 193
772 65
71
68
23
89
85
30
3
4
2
7
5
2
756 12
720 18
1,128 42
720 24
744 12
1,116 36
630 12
666 24
1,152 66
164
165
n.d.
5
9
1,231 54
958 54
522 27
1,709 61
1,730 26
1,683 140a
2,940
3,169
2,205
2,875
2,968
2,092
1,135
2,702
1,520
Trien
481 12
a
SpmTrien
43
5
AG
1,001 75
1,004 50
100 17
1,195 103 1,680 52
AG ? Trien
AG ? SpmTrien
DFMO
132
n.d.
n.d.
n.d.
n.d.
9
777 16
424 46
\10
1,688 57
1,668 83¤
1,125 89
1,120 78
1,328 133a
503
a
8
73
57
46
44
8
8
9
8
682 49
304 19
DFMO ? Trien
DFMO ? SpmTrien
\10
192
1,572 67
a
22
1
7
The cells were cultured with 5 mM DFMO or 1 mM AG and 50 lM analogs for 72 h. ‘‘Total PA’’ indicates the total amount of Spd, Spm and
analogue. Data are mean SD (n = 3). Two different unidentified metabolic products of SpmTrien were also found (not shown)
n.d. not detectable
a
SpmTrien is not separated from Spm by our HPLC system, thus it is included in SPM value
(Seiler 2004). The SpmTrien-induced changes in enzyme
activities were reflected in intracellular polyamine pools,
which were decreased markedly, compared to control
cells or cells treated with Trien (Table 2). No additional
metabolic products for Trien were detectable by our
HPLC system, while two different unidentified products
were observed for SpmTrien (data not shown). The only
metabolites characterized for Trien were terminally
mono- and bis-acetylated Trien’s, thus two different
metabolites originating from SpmTrien are most likely
terminally mono-acetylated SpmTriens, since o-phthalal-
dehyde derivatization requires primary amino groups to
be present (Lu et al. 2007). However, the true nature of
the metabolites should be verified with chemically syn-
thesized reference compounds.
Conclusion
In living cells, pH varies between different cellular
organelles, thus one could expect SpmTrien to have unique
biological properties in comparison to Spm. The proton-
ation of SpmTrien at physiological pH may allow inter-
action with some Spm binding sites due to the proton
migration in-between the (N-II)-(N-III)-(N-IV) secondary
amino groups. This unique property of SpmTrien may be
useful in the field of Spm biochemistry, since it may
facilitate the discrimination of, at least some of the diverse
cellular targets and functions of Spm. The present paper
demonstrates that despite the difference in protonation of
SpmTrien, as compared to Spm, it is recognized as a
polyamine-related regulator of such enzymes as ODC,
AdoMetDC and SSAT as well as a growth-supporting
agent. Thus, the knowledge of the pKa and the protonation
sites of SpmTrien molecule can give more precise answers
to questions arising from the enzymatic and cellular
experiments with SpmTrien and may be helpful in under-
standing some aspects of the Spm biochemistry.
When cytostasis was induced by incubating the DU145
cells for 3 days with DFMO and the analogues, both Trien
and SpmTrien were able to partially restore cell growth
(Fig. 3), indicating that these compounds could at least
partially substitute for natural polyamines in their growth-
supporting functions.
123

Novel isosteric charge-deficient spermine analogue
507
Acknowledgments We would like to thank Ms. Maritta Salm-
inkoski, Ms. Tuula Reponen, Ms. Anne Karppinen, Ms. Arja Ko-
rhonen for their skilful technical assistance and Ph.D. Elina Jarho for
her assistance with the automatic titrator. This work was supported by
Academy of Finland (project nos. 124185 and 128702), Russian
Foundation for Basic Research (project no. 09-04-01272), and the
program Molecular and Cell Biology of the Presidium of the Russian
Academy of Sciences.
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