Novel metabolically stable and functionally active mimetic of spermidine

Article abstract of DOI:10.1134/S1068162011020075

Earlier unknown 1,8-diamino-3-methyl-4-azanonane (γ-MeSpd) was synthesized. The analogue was a substrate of neither spermine/spermidine N ¹-acetyltransferase nor spermine synthase, but was capable to support the growth of DU145 cells having depleted polyamine pools. Such a combination of γ-MeSpd properties discloses novel opportunities to study cellular functions of catabolically unstable and easily interconvertible spermine and spermidine.

Full text of DOI:10.1134/S1068162011020075

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 2, pp. 225–230. © Pleiades Publishing, Ltd., 2011.
Published in Russian in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 2, pp. 253–258.
Novel Metabolically Stable and Functionally Active Mimetic
1
of Spermidine
a, 2
b
a
c
b
M. A. Khomutov , M. T. Hyvönen , A. R. Simonian , J. Vepsäläinen , L. Alhonen ,
a
b
S. N. Kochetkov , and T. A. Keinänen
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
b
Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Eastern Finland, Kuopio, Finland
c
Department of Biosciences, Biocenter Kuopio, University of Eastern Finland,
P.O. Box 1627, FINꢀ70211, Kuopio, Finland
Received July 8, 2010; in final form, July 12, 2010
Abstract—Earlier unknown 1,8ꢀdiaminoꢀ3ꢀmethylꢀ4ꢀazanonane (
γ
ꢀMeSpd) was synthesized. The analogue
ꢀacetyltransferase nor spermine synthase, but was capable
to support the growth of DU145 cells having depleted polyamine pools. Such a combination of ꢀMeSpd
1
was a substrate of neither spermine/spermidine
N
γ
properties discloses novel opportunities to study cellular functions of catabolically unstable and easily interꢀ
convertible spermine and spermidine.
1
Keywords
:
polyamines, spermidine analogues,
γ
ꢀMeSpd, spermine synthase, spermine/spermidine N ꢀacetylꢀ
transferase, DU145 cells
DOI: 10.1134/S1068162011020075
2
1
INTRODUCTION
[5]). Application of specific inhibitors of the enzymes
of polyamine metabolism is an efficient approach in
vitro, but the necessity to inhibit several enzymes leads
High intracellular concentrations of biogenic
polyamines spermine (Spm) and spermidine (Spd)
determine their participation in the regulation of
many vitally important processes (for reviews, see [1–
to cumbrous combinations of the inhibitors (for
reviews, see [6, 7]). A relatively simple method to disꢀ
criminate the cellular effects of Spm and Spd is the
depletion of the polyamine pool and subsequent appliꢀ
cation of functionally active metabolically stable
3
]). Investigation of the cellular functions of Spm and
Spd at the molecular level is rather complicated due to
their partial interchangeability and also due to their
feasible interconversion (Fig. 1). Moreover, the activiꢀ
ties and biosyntheses of the key enzymes of polyamine
metabolism are precisely regulated at different levels
of gene expression including the downꢀregulatory
effects exerted by Spm and Spd [1, 2]. To study the celꢀ
lular functions of Spm and Spd, cells and microorganꢀ
isms deficient of the key enzymes of polyamine
metabolism are being used. One of the recent examꢀ
ples of such an approach is the construction of E. coli
strains deficient of all the enzymes of polyamine bioꢀ
synthesis [4]. Different lines of transgenic animals
have been generated, which has given an opportunity
either to activate or to “switch off the targeted steps of
polyamine metabolism even in vivo (for review, see
3
mimetics of Spm and Spd. However, the analogues
having such a set of properties are still unknown
amongst monoꢀCꢀmethylated polyamine derivatives.
NH2
α
ꢀMeSpd
H N
2
N
H
NH2
γ
ꢀMeSpd
H N
N
H
2
In the present paper, we describe synthesis of earꢀ
lier unknown ꢀMeSpd and demonstrate the analogue
not to be a substrate of SSAT which makes ꢀMeSpd
catabolically stable. Under cell culture conditions
MeSpd to the
γ
γ
Abbreviations: AG, aminoguanidine; Ms, methanesulfonylꢀ;
Spd, spermidine(1,8ꢀdiaminoꢀ4ꢀazaoctane);
γ
ꢀMeSpd (1,8ꢀ
diaminoꢀ5ꢀazanonane); Spm, spermine(1,12ꢀdiaminoꢀ4,8ꢀ there was no detectable conversion of
diazadodecane); ꢀMeSpd (1,8ꢀdiaminoꢀ2ꢀmethylꢀ4ꢀazaocꢀ
tane); ꢀeSpd (1,8ꢀdiaminoꢀ3ꢀ methylꢀ4ꢀazanoctane).
ꢀMeSpd [8] were cataꢀ
γ
ꢀ
γ
3
γ
Earlier synthesized optical isomers of
bolically stable [9], but in cell culture they were converted into
corresponding analogues of Spm [9]. Furthermore, ꢀMeSpm is
readily metabolized to ꢀMeSpd and Spd by SMO [10].
α
1
2
The article was translated by the authors.
Corresponding author: phone: +7 (499) 135ꢀ60ꢀ65; eꢀmail:
hommaximus@mail.ru.
α
α
225

226
KHOMUTOV et al.
NH2
H N
2
Putrescine
APAO
O
NH2
N1ꢀAcetylspermidine
N
H
N
H
SpdSy
SSAT
NH2
H N
N
H
2
Spermidine
APAO
O
H
SpmSy
SMO
N
NH2
N
H
N
H
N1ꢀAcetylspermine
SSAT
H
N
H
NH2
H N
2
N
H
Spermine
Fig. 1. Interconversions of spermine and spermidine. APAO—acetylpolyamine oxidase; SMO—spermine oxidase; SpdSy—spermiꢀ
1
dine synthase; SpmSy—spermine synthase; SSAT—spermine/spermidine
N ꢀacetyltransferase.
corresponding Spm derivative. These characteristics, backbone (Scheme). [13,14]. NꢀBocꢀaminoalcohol (I)
together with the ability of the analogue to restore the was prepared from commercially available 4ꢀaminoꢀ2ꢀ
growth of DU15 cells with depleted polyamine pool butanol under standard conditions, using Boc O for the
2
renders ꢀMeSpd the first monoꢀCꢀmethylated metaꢀ protection of the amino group. Conversion of the secꢀ
γ
bolically stable functionally active mimetic of Spd that
is suitable to study the cellular effects of easily interꢀ
convertible Spm and Spd.
ondary alcohol (I) into the corresponding methane sulꢀ
fonate was performed with a practically quantitative
yield that gave a possibility to use it at the next stage
without isolation. However, the alkylation of the excess
of Put with the methane sulfonate of the secondary
alcohol was not as successful as in the case of the prepꢀ
RESULTS AND DISCUSSION
aration of
nyl)aminoꢀ1ꢀpropyl methane sulfonate and Put. Interꢀ
mediate 1ꢀ( tertꢀbutyloxycarbonyl)aminoꢀ3ꢀmethylꢀ
ꢀazaꢀ8ꢀaminooctane (II) was isolated by flash chroꢀ
matography on silicagel with only 31% yield. The relaꢀ
tively low yield was partly due to the necessity of comꢀ
α
ꢀMeSpd from 3ꢀ(Nꢀbenzyloxycarboꢀ
Synthesis of γꢀMeSpd (struct.) may be performed
according to different schemes, using the methods
being applied in the chemistry of polyamines to build up
the C–Nꢀbond [11, 12]. The most simple approach
seems to be the addition of putrescine (Put, 1,4ꢀdiamiꢀ
nobutane) to crotononitrile with a subsequent reducꢀ
tion of the nitrile group. However, here we used feasible
Nꢀ
4
plete separation of
ꢀmethyꢀ ninhydrinꢀpositive byꢀproducts. Removal of
lated polyamine analogues, i.e. alkylation of the excess group was performed with HCl in aq. alcohol that
of Put with the methane sulfonate of 4ꢀ(
tertꢀbutyloxyꢀ resulted in target ꢀMeSpd (III) with an overall yield of
carbonyl)aminoꢀ2ꢀbutanole to build up the polyamine 19%, as calculated for 4ꢀaminoꢀ2ꢀbutanol.
NꢀBocꢀtriamine (II) from minor
strategy employed earlier for the synthesis of
α
NꢀBocꢀ
Nꢀ
γ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

NOVEL METABOLICALLY STABLE AND FUNCTIONALLY ACTIVE MIMETIC
227
i
ii
Ms
Boc
Boc
H N
OH
N
H
OH
N
H
O
2
(I)
iv
iii
⋅
3HCl
Boc
NH2
NH2
ꢀMeSpd (III
H N(CH ) NH /TNF;
N
H
N
H N
2
N
H
H
(
II
)
γ
)
Reaction conditions:
i
⎯
Boc O/THF; ii
2
⎯
MsCl/C H /Et N; iii
6
⎯
6
3
2
2 4
2
iv⎯H /Pd.
2
Scheme.
Investigation of biochemical properties of itself, can participate in maintaining of polyamine
ꢀMeSpd was initiated from the study of its substrate homeostasis in DU145 cells.
γ
properties in SSAT reaction. It turned out that
ꢀMeSpd, like ꢀMeSpd [15], was a very poor subꢀ
strate of mouse recombinant SSAT. Practically, the
γ
ꢀMeSpd, like Spd and ꢀMeSpd (this analogue
α
γ
α
fulfills the cellular functions of Spd both in vitro and in
vivo [18]), effectively restored the growth of DU145
cells with decreased levels of Put and Spd as the result
of 72 h incubation with DFMO (Fig. 4). The ability of
ꢀMeSpd to neutralize efficiently the acute deficiency
of polyamines and to regulate polyamine homeostasis
ꢀMeSpd a functionally active
14
incorporation of [ C]ꢀAcꢀgroup into
about threeꢀfold as compared to ꢀMeSpd (Fig. 2).
Thus, ꢀMeSpd was a competitive inhibitor of the
SSAT with of Кi 50 M ( ꢀMeSpd has Кi 140 M [10]).
γ
ꢀMeSpd was
α
γ
γ
μ
γ
μ
Catabolism of Spd is initiated by N1ꢀacetylation by (see above) makes
SSAT and the use of ꢀ/ ꢀmethylꢀsubstituent renders mimetic of Spd.
γ
α
γ
the analogue stable against SSAT/APAOꢀmediated
degradation. However, chemically synthesized
Finally, we investigated the intracellular conversion
ꢀMeSpd to ꢀMeSpm. As depicted at table, the forꢀ
mation of ꢀMeSpm in DU145 cells was not detected
of
γ
γ
1
1
N
ꢀacetylꢀ
α
ꢀMeSpd, like
N
ꢀAcꢀSpd, can be splitted
γ
by APAO [16, 17]. Finally,
γ
ꢀMeSpd proved to be nonꢀ
toxic to cultured cells without aminoguanidine (AG)
supplementation under conditions were Spd was
toxic, thus showing stability against serum aminooxiꢀ
Puls/min
000
9
dases (data not shown). Hence,
γ
ꢀMeSpd is a new catꢀ
abolically stable analogue of Spd.
At the next step, we investigated the effects of
ꢀMeSpd on the level of polyamines in DU145 cells
γ
7000
and its ability to restore the growth of these cells with
depleted Spd and Put pools as the result of DFMO
(
suicide inhibitor of ornithine decarboxylase) treatꢀ
5
000
000
ment. To prevent nonspecific oxidation of Spd by
serum aminooxidases, the experiments were carried
out in the presence of AG.
γꢀMeSpd efficiently penetrated into DU145 cells,
3
competing for the transport with Spd (data not
shown), and its intracellular content was about the
same as that of
ꢀMeSpd and ꢀMeSpd decreased the levels of Put and
Spd in DU145 cells (table) suggesting that they downꢀ
regulated polyamine synthesis. The effect of ꢀMeSpd
on the level of Spm was comparable with the effect of
Spd, but was much less than the effect of ꢀMeSpd
table), which may be due to the fact that in DU145
α
ꢀMeSpd and Spd (table). Both
γ
γ
1000
0
γ
Spd
α
ꢀMeSpd
γ
ꢀMeSpd
α
0.25 / 0.5 / 1.0 / 2.0
2.0 / 10.0 / 20.0
ng of SSAT
(
cells about 20% of
γ
ꢀMeSpd is converted to
14
α
ꢀMeSpm, which is a metabolically competent anaꢀ
Fig. 2. Incorporation of [ C]ꢀacetyl group in Spd,
or ꢀMeSpd as a function of the amount of SSAT.
γ
ꢀMeSpd,
logue of Spm. Thus,
γ
ꢀMeSpd, like
α
ꢀMeSpd and Spd
γ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

228
KHOMUTOV et al.
Effect of the analogs on growth and polyamine levels in DU145 cells after 72 h of culture
Polyamines
Spm
Cells DU145
Growth, %
Put
Spd
MeSpd
MeSpm
6
(
pmol/10 cells)
AG
AG
AG
AG
AG
AG
AG
AG
100
94
±
±
±
±
±
±
±
±
4
93
±
±
±
±
±
±
±
±
25
1065
2416
115
136
55
±
±
±
±
±
±
±
58
103*** 976
12*** 549
14*** 1026
6*** 1197
196*** 831
1513
±
±
±
±
±
±
±
±
78
36***
+
+
+
+
+
+
+
Spd
MeSpd
MeSpd
3
48
34
20
59
35
21
18
21
α
ꢀ
97
3
14*
4**
25
20*** 3053
59*** 3030
57***
±
±
47
858
±
95
γ
ꢀ
101
49
4
160
n.d.
DFMO
2***
DFMO + Spd
89
4
4
5
3*** 2567
21***
DFMO
DFMO
+
+
α
ꢀ
MeSpd
86
2***
1***
47
5***
246
486
26*** 4223
36*** 5131
±
±
509
159
959
±
119
γ
ꢀMeSpd
91
n.d.
n.d.
Note: Cells were cultured for 72 h in the presence of 100 μM Spd, or its analogues and 1 mM AG in the presence or absence of 5 mM DFMO.
Results are means SD, n = 3. n.d., not detectable; *, ** and *** refer to statistical significance of <0.05, 0.01 and 0.001, respectively,
as compared to AGꢀtreated control.
by HPLC. Moreover, HPLC analysis did not show the ppm, and spin–spin coupling constants in Hz. Meltꢀ
presence of any acetylated
even when MDL72,527 (20
γ
ꢀMeSpm intermediate ing points were determined in open capillary tubes
μM) was used to prevent using Electrotermals MelꢀTemp 1202D apparatus and
APAO/SMOꢀmediated backconversion (data not are uncorrected. Elemental analysis was performed in
shown). Hence,
was not a substrate of spermine synthase.
The obtained data proves
metabolically stable and functionally active mimetic
γ
ꢀMeSpd, in contrast to
γ
ꢀMeSpd, Nesmeyanov Institute of Organoelement Comꢀ
pounds, RAS, Moscow, using CHNꢀanalyser Carlo
Erba 1106.
γꢀMeSpd to be the first
DU145 prostate carcinoma cells were grown as
of Spd among monoꢀCꢀmethylated polyamine anaꢀ described earlier [9]. The effects of Spd analogues on
logues. This analogue is a promising tool for the invesꢀ the intracellular content of polyamines, as well as the
tigation of individual cellular functions of readily investigation of the ability of
ꢀMeSpd to restore the
γ
interconvertible Spd.
growth of DU145 cells with depleted polyamine pool
were performed as described earlier [19]. Intracellular
polyamines were measured with HPLC according to
the published method [20]. Substrate properties of
EXPERIMENTAL
4ꢀAminoꢀ2ꢀbutanol was obtained from Acros (Belꢀ
α
ꢀMeSpd and ꢀMeSpd in SSAT reaction were deterꢀ
γ
14
gium); 1,4ꢀdiaminobutane from Aldrich (United mined following the incorporation of [ C]ꢀgroup of
ꢀMeSpd or ꢀMeSpd in comparison with
ꢀMeSpd was synthesized following the earlier pubꢀ that for Spd, in accordance with the published protoꢀ
States); Boc O and MsCl from Fluka (Switzerland), AcꢀCoA in
α
γ
2
α
lished method [13].
col for polyamines [21], but the incubation time was
0 min. Concentrations of Spd and its ꢀmethylated
derivatives in the substrate mixture were 2.5 M, while
the amounts of mouse recombinant SSAT were 0.25,
.50, 1.0 and 2.0 ng of the protein/per probe in the
case of Spd; and 2.0,10.0 and 20.0 ng of the proꢀ
tein/per probe in the case of ꢀMeSpd and ꢀMeSpd.
1
C
Flash chromatography was carried out on Kieselgel
40–63 m, Merck), systems for elution are specified
μ
(
μ
in the text. TLC was carried out on precoated Kieselꢀ
gel 60 F254 plates (Merck) in systems: CHCl –MeOH,
0
3
1
n
00 : 2 (A); dioxane–25% NH OH, 8 : 2 (B);
ꢀBuOH–AcOH–pyridine–H O, 4 : 2 : 1 : 2 (C).
4
α
γ
2
The experiments were performed twice in triplicates.
Substances on chromatograms were detected by broꢀ
mophenol blue solution (Bocꢀderivatives) and the
Statistical analysis: values are means SD. Oneꢀway
compounds with free amino group were developed analysis of variance (ANOVA) with Tuckey’s postꢀhoc
using color reaction with ninhydrin.
test was used for multiple comparisons with the aid of
a software package, GraphPad Prism 4.03 (GraphPad
NMR spectra were registered on a Bruker Avance
Software Inc.). *, ** and *** refer to
<0.01 and <0.001, respectively.
p values of <0.05,
5
00 DRX (Germany) instrument with working freꢀ
1
quency of 500.1 MHz for H nuclei and 125.8 MHz for
13
C nuclei. Tetramethylsilane was used as an internal
4ꢀ(NꢀtertꢀButyloxycarbonyl)aminoꢀ2ꢀbutanol (I).
standard (CDCl ) and sodium 3ꢀtrimethylꢀ1ꢀproꢀ Вос О (2.4 g, 11 mmol) in THF (10 ml) was added
3
2
panesulfonate (D O). Chemical shifts are given in slowly with stirring to a cooled (+4 ) solution of
°С
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 2
2011

NOVEL METABOLICALLY STABLE AND FUNCTIONALLY ACTIVE MIMETIC
229
4
(
ꢀaminoꢀ2ꢀbutanol (1.04 g, 11.6 mmol) in THF 20
10 ml). Stirring was continued for 1 h at +4°C and 16 in vacuo, the residue was coꢀevaporated with abs.
h at 20°С and the reaction mixture was evaporated to EtOH ( 15 ml), the resulted semiꢀsolid residue was
dryness in vacuo. The residue was taken into CHCl3 recrystallized from abs. EtOH that after drying in
°
C. The reaction mixture was evaporated to dryness
4
×
(
(
30 ml), washed with 10% citric acid (
5 ml), 1 M NaHCO3 (5 ml), Н О (5 ml), brine (10 ml) 71.5%), mp 231–232
4
×
7
ml), Н О vacuo over Р О /КОН gave ꢀMeSpd (III) (0.48 g,
γ
2
2
5
1
°
С, Rf 0.24 (C). H NMR (D O):
2
2
and dried (MgSO4). Solvent was distilled off in vacuo 3.48–3.41 (1H, m, CH(CH )); 3.20–3.03 (6H, m,
3
and the residue was dried in vacuo over Р2О5 to give (
I
)
H NCH (CH ) CH NH + NH CH ); 2.24–2.17
2 2 2 2 2 2 2
1
(
(
(
1.82 g, 87.5%) as a viscous oil; Rf 0.29 (A). H NMR (1H, m, CHCH ); 2.00–1.91 (1H, m, CHCH );
2
2
CDCl ): 4.91–4.76 (1H, bs, NHBoc), 3.88–3.80 1.83–1.74 (4H, m, C–CH CH –C); 1.37 (3H, d,
3
2
2
13
1H, m, CHOH), 3.50–3.37 (1H, m, CH NHBoc),
J 6.7, CH(CH )). C NMR (D O): 54.88, 47.10,
2
3 2
3
.14–3.04 (1H, m, CH NHBoc), 3.00–2.90 (1H, bs, 41.68, 38.75, 33.07, 26.84, 25.83, 17.96. Found, %:
2
OH), 1.64–1.55 (1H, m, CH CHOH), 1.54–1.46 C 35.61; H 9.07; N 15.59. C H Cl N . Calculated, %:
2
8
24
3
3
(
d,
1H, m, CH CHOH), 1.43 (9H, s, (CH ) ), 1.22 (3H, C 35.77; H 9.00; N 15.64.
2
3 3
13
J 1.2, CHCH ). C NMR (CDCl ): 157.04, 79.48,
3 3
6
5.20, 39.50, 37.51, 28.48, 23.36. Found, %: C 56.95;
ACKNOWLEDGMENTS
H 10.27; N 7.14. C H NO . Calculated, %: C 57.12;
9
19
3
H 10.12; N 7.40.
This work was supported by Russian Foundation
for Basic Research (grants nos. 09ꢀ0401272 and 08ꢀ
N1ꢀ(tertꢀButyloxycarbonyl)ꢀ1,8ꢀdiaminoꢀ3ꢀmethylꢀ
0
4ꢀ91777), the Academy of Finland, and the program
Molecular and Cell Biology of the Presidium of the
Russian Academy of Sciences.
4
ꢀazaoctane (II). Methanesulfonyl chloride (1.03 g,
9
.0 mmol) in dry benzene (10 ml) was added dropwise
with stirring to a cooled (+4
°С) solution of (I) (1.75 g,
9
.26 mmol) and Et N (2.08 ml, 15 mmol) in dry benꢀ
3
zene (20 ml). Stirring was continued for 1 h at +4 С
and 4 h at 20 , then the precipitated triethylamine
°
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2011