Conformational change of spermidine upon interaction with adenosine triphosphate in aqueous solution

Article abstract of DOI:10.1002/chem.200801961

Endogenous polyamines, represented by putrescine, spermidine, and spermine, are known to exert their physiological functions by interacting with polyanionic biomolecules such as DNA, RNA, adenosine triphosphate (ATP), and phospholipids. Very few examples

Full text of DOI:10.1002/chem.200801961

DOI: 10.1002/chem.200801961
Conformational Change of Spermidine upon Interaction with Adenosine
Triphosphate in Aqueous Solution
Keisuke Maruyoshi, Kaori Nonaka, Takeshi Sagane, Tetsuo Demura,
Toshiyuki Yamaguchi, Nobuaki Matsumori, Tohru Oishi, and Michio Murata*[a]
Abstract: Endogenous polyamines, rep-
resented by putrescine, spermidine, and
spermine, are known to exert their
physiological functions by interacting
with polyanionic biomolecules such as
DNA, RNA, adenosine triphosphate
(ATP), and phospholipids. Very few ex-
amples of conformation analysis have
been reported for these highly flexible
polymethylene compounds, mainly due
to the lack of appropriate methodolo-
gies. To understand the molecular basis
of the weak interaction between poly-
amines and polyanions that underlies
their physiological functions, we aimed
to elucidate the solution conformation
of spermidine by using diastereospecifi-
cally deuterated and 13C-labeled deriv-
atives (1–7), which were designed to di-
agnose the orientation of seven confor-
mationally relevant bonds in spermi-
1
dine. H–1H and 13C–1H NMR coupling
3
constants (3JH,H and JC,H) were success-
fully determined for a spermidine–ATP
complex. The relevant coupling con-
stants markedly decreased upon com-
plexation. The results reveal that sper-
midine, when interacting with ATP, un-
dergoes changes that make the confor-
mation more bent and forms the com-
plex with the triphosphate part of ATP
in an orientation-sensitive manner.
Keywords: biomolecular
interactions · conformation analysis ·
coupling constants · polyamines ·
polyanions
Introduction
compounds involved are largely flexible and rapidly reach
an association–dissociation equilibrium, which makes the
conformation analysis of a bound ligand extremely difficult.
Thus, there are very few examples of conformation studies
of highly flexible linear biomolecules.[3] For complexes be-
tween flexible ionic compounds, almost nothing has been re-
ported on their 3D structures. As a first step toward under-
standing the structure requirements for these interactions,
we aimed to establish a basic strategy for the conformation
analysis of weakly interacting biomolecules in aqueous envi-
ronments. For example, endogenous polyamines such as pu-
trescine, spermidine, and spermine interact with polyanionic
biomolecules and provide an ideal model for this purpose.
Their interactions with adenosine triphosphate (ATP),
DNA, RNA, and phospholipids are assumed to be involved
in essential biological events such as cell growth, differentia-
tion, and proliferation,[4–6] despite the fact that their affinity
to polyanions is usually low, with dissociation constants in
the millimolar range.[7] Among the polyamines, we focused
on spermidine (SPD) because its affinity to biological poly-
anions is comparable with that of tetravalent spermine and
its asymmetric structure allows us to deduce the functional
difference between the tetramethylene (butanylene) and tri-
methylene (propanylene) parts. Batista de Carvalhoꢀs group
has reported some conformation studies of polyamines[8] on
The interaction between a bioorganic small molecule and its
target biopolymer plays an important role in cell physiology.
Highly specific bimolecular recognition, and thus high affini-
ty, such as that between a ligand and its receptor, has been
extensively studied; this has often resulted in the elucidation
of receptor-bound conformations[1] of hormones, drugs, sec-
ondary messengers, and toxins. Besides these strict molecu-
lar recognitions with nanomolar or sub-nanomolar dissocia-
tion constants, weak interactions among biomolecules often
play a crucial role. For example, neurotransmitters usually
possess relatively low affinity for their intrinsic receptors,
which facilitates the rapid on–off switching of nervous signal
transmission.[2] The molecular basis responsible for weak in-
teractions, however, remains mostly unknown because the
[a] Dr. K. Maruyoshi, K. Nonaka, T. Sagane, T. Demura, T. Yamaguchi,
Dr. N. Matsumori, Prof. Dr. T. Oishi, Prof. Dr. M. Murata
Department of Chemistry
Graduate School of Science, Osaka University
1-1 Machikaneyama, Toyonaka, Osaka 560-0043 (Japan)
Fax : (+81)66850-5774
E-mail: murata@ch.wani.osaka-u.ac.jp
Supporting information for this article is available on the WWW
under 10.1002/chem.200801961.
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FULL PAPER
Results
Molecular design of 2H- and 13C-labeled spermidines: We
have previously reported that stereoselectively deuterated
SPD may be used for conformation analysis by measure-
1
ment of its H–1H coupling constants.[10] In this study, we ex-
tended this approach to all of the conformationally relevant
bonds in SPD, and we designed molecular probes 1–7 for
3
measurement of the spin–spin coupling constants JH,H and
3JC,H. SPD 1, deuterated in an erythro manner on the C2 and
the basis of molecular orbital calculations and Raman spec-
troscopy, and these studies have partly provided the struc-
ture basis for polyamine interactions with biomolecules.
Computer simulations cannot be applied for polyamine–
polyanion complexes in aqueous environments because the
coexistence of the multivalent cation and anion within a
short distance causes hydration problems that make energy
calculation and conformation analysis virtually impossible.
Among NMR methodologies,[9] NOEs often play an es-
sential role in the conformation analysis of biomolecules.
For highy flexible systems, however, NOEs are rather unin-
formative because minor conformers sometimes elicit dis-
proportionally large NOE values. Instead, 1H NMR spin–
spin coupling constants are often utilized for conformation
analysis of flexible compounds, including acyclic molecules.
We needed to prepare isotope-labeled SPD derivatives in
order to obtain the conformationally relevant coupling con-
stants because of unresolved signals due to equivalent and/
or overlapping signals of the methylene protons. In this
study, we synthesized diastereoselectively 2H/13C-labeled
SPDs 1–7 and determined their coupling constants in the
presence of ATP. With these data in hand, we aimed to elu-
cidate the conformational change in SPD upon complexa-
tion with ATP (Figure 1) in order to gain a better under-
standing of the interactions between polyionic biomolecules.
C3 atoms, was used for evaluating the populations of gauche
ꢀ
and anti conformers around the C2 C3 bond on the basis of
the JH2,H3 value (carbon atom numbering follows the tradi-
3
tional pattern for polyamine derivatives).[10] An overlap of
the signals for the methylene protons adjacent to the amino
group, which hampered accurate measurement of the spin–
spin coupling constants, was solved by introducing deuteri-
um atoms at the C5, C7, C8, and C9 positions. In an analo-
gous strategy, we carried out the conformation analysis for
ꢀ
ꢀ
ꢀ
the C4 C5, C7 C8, and C8 C9 bonds by using labeled
SPDs 3, 6, and 7, respectively. However, this strategy was
ꢀ
ꢀ
not applicable to the C5 N6 or N6 C7 bonds. To examine
3
3JC,H values instead of JH,H values, SPDs labeled with 13C
atoms at the C7 (4) and C5 (5) positions were synthesized;
in these compounds, the methylene hydrogen atoms at the
C2 and C9 positions were deuterated to avoid overlap with
the methylene signals from the C5 and C7 positions, respec-
tively. For the analysis of the remaining dihedral angle
ꢀ
about the C3 C4 bond, labeled SPD 2, in which the C3 and
C4 positions were deuterated in the erythro configuration
and C4 was 13C-labeled, was designed. The close proximity
of the chemical shifts of the H3 and H4 protons, which pre-
Figure 1. Hypothetical images of the SPD–ATP complex. The molecular
surface is colored to indicate atom charges. The conformation of the
complex on the right is that shown in Figure 4c.
3
vented measurement of the JH3,H4 value, was resolved by in-
troducing a 13C atom at the C4 position to make the adja-
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1619

M. Murata et al.
1
cent methylene signals split by the JC,H value (see Figure 2).
ence of deuterium substitution in SPD on the interaction
with ATP. The association constant of decadeuterated SPD
1 was determined to be 540mꢀ1, which is virtually equivalent
with that of nondeuterated SPD (553mꢀ1) and, thus, indi-
cates that deuterium introduction hardly affects the affinity
of SPD for ATP. Moreover, the Ka value is comparable with
those reported by De Stefano et al, if the ionic strength is
taken into account.[12] The stoichiometry of the SPD–ATP
complex was determined by Jobꢀs method,[13] in which the
induced shift (DdꢂnSPD; see the Experimental Section), was
plotted against the mole fraction of SPD to ATP (Figure S2
in the Supporting Information). One-to-one stoichiometric
complexation was indicated by the maximum value at the
host mole fraction of 0.5. As an alternative polyanion to
ATP, we picked up tripolyphosphate (TPP) and determined
its affinity with SPD, which turned out to be particularly
high with a Ka value of 3960mꢀ1. On the other hand, SPD
showed much lower affinity for ATP–Mg2+ with a Ka value
of 22.4mꢀ1. Under the same conditions as those for the SPD
experiments, the Ka values of the diaminopropane–ATP and
putrescine–ATP complexes were determined to be 105.5
and 11.6mꢀ1, respectively (Table S2 in the Supporting Infor-
mation). Note the much higher affinity of diaminopropane
to ATP than putrescine despite a difference of only one
methylene moiety in their structures.
All of the erythro-labeled SPDs (1–3, 6, and 7) were racemic
mixtures; this was better for determining the ratio of anti/
gauche rotamers for the following reasons. ATP is optically
pure so there are two ways for diastereomeric interactions
to occur between ATP and racemic SPD. In the anti orienta-
3
tion, the JH,H values should be the same for both enantio-
meric SPDs. In the gauche orientation, the populations of
the two diastereomeric SPD–ATP complexes should be
equal because the substitution effect of deuterium is negligi-
ble, as described later. The relevant JH,H value in this case is
different between the enantiomers upon interaction with
3
3
ATP because the JH,H value is dependent on the orientation
of a nitrogen atom; the JH2,H3 value of 1 for the gauche (+)
3
rotamer is smaller than that for gauche (ꢀ) rotamer (as
shown in Table 1). Even if there is a major rotamer with re-
spect to one relevant bond upon interaction with ATP, the
gauche (+) rotamer from one enantiomer and the gauche
(ꢀ) rotamer from the other enantiomer equally contribute
3
to the JH,H value. The same holds true with a minor rota-
mer. Thus, the total population of the gauche (+) and
gauche (ꢀ) conformers can be determined from the average
3JH,H (or JC,H) values (equal to the observed values) derived
3
from two diastereomeric interactions.
3
Table 1. Calculated JH,H values for anti and gauche orientations in the
SPD system.
Determination of NMR coupling constants: The spin–spin
3
coupling constants (3JH,H and JC,H) were measured for the
Labeled SPD gauche (+)[a] [Hz], anti[a] [Hz],
gauche (ꢀ)[a] [Hz],
near 3008
labeled SPDs. The SPDs as HCl salts (Figure 2A) and in
ATP complexes were subjected to NMR measurements at
various pH values (Figure 2B–D). When 1 and 3 were used
near 608
near 1808
SPD-2,3-d10
(1)
1.2
11.5
2.8
2.3
1.2
1.8
1.0
3
1
SPD-4-13C,
3,4-d2 (2)
SPD-4,5-d10
(3)
2.3
2.8
1.0
1.7
12.9
11.5
11.4
11.4
for determination of the JH,H values, the relevant H NMR
signals were simulated by the spectral-simulation software
gNMR (Adept Scientific), because they were complicated
by second-order couplings of the AB2X type due to the
close chemical shifts of the H3 and H4 signals. Upon mea-
surement of 2, the H4 signal was decoupled to a doublet by
irradiation at the H5 protons. For the labeled SPDs 1–7,
SPD-7,8-d12
(6)
SPD-8,9-d12
(7)
3
3JH,H and JC,H values could be determined with an accuracy
3
of 0.1 Hz. The JH,H values of 1–3, 6, and 7 as 3HCl salts of
[a]
SPD ranged between 9.7 and 10.5 Hz, which revealed that
the N1–C4, C2–C5, C3–N6, N6–C9, and C7–N10 sections
predominantly take anti conformations (Table 2). With pH
3
values of 3.3, 5.6, and 7.3, the JH,H values of the 3HCl salts
ꢀ
Vicinal H H orientations for gauche (+), anti, and gauche (ꢀ) are shown
for 1. Similar orientations occur for 2, 3, 6 and 7, despite the fact that the
interaction between the nitrogen and hydrogen atoms is inverted for 2, 3,
and 7. This leads to the reversed sizes of the JH,H values for gauche (+)
and gauche (ꢀ).
of the SPDs were virtually unchanged. Next, we carried out
the same measurements for the SPD–ATP and SPD–TPP[10]
complexes (see the Supporting Information for the SPD–
TPP data). The association constant of the SPD–ATP com-
plex indicated that 99% of SPD formed a complex with
ATP under the conditions of the NMR measurements. The
spin coupling constants in Table 2 should, therefore, be
those of the SPD–ATP complex. Whereas SPD is a trivalent
cation throughout the experiments, the net charge of ATP
depends on the pH value, so ATP is present as divalent
(ꢀ2), trivalent (ꢀ3), and tetravalent (ꢀ4) anions at pH 3.3,
5.6, and 7.3, respectively. At pH 3.3, the charges are ꢀ3 on
the triphosphate and +1 on adenine; the charge is ꢀ3 on
3
Association constants of SPD–ATP, SPD–ATP–Mg, SPD–
TPP, diaminopropane–ATP, and putrescine–ATP com-
plexes: The association constant (Ka) and binding stoichiom-
etry of the SPD–ATP complex were determined by NMR ti-
tration experiments, which were carried out with 25 mm
SPD at pH 7.3 and various concentrations of ATP (see Fig-
ures S2–S5 in the Supporting Information for the experi-
mental procedures and data).[11] We first examined the influ-
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Chem. Eur. J. 2009, 15, 1618 – 1626

Conformation Analysis of Spermidine–ATP Complexes
FULL PAPER
(Table 2). These results indicate that complexation with
ATP does not significantly alter the average conformation
ꢀ
ꢀ
ꢀ
with respect to the C3 C4, C5 N6, or N6 C7 bonds. In the
presence of Mg2+ ions, which form a complex with ATP
with a higher affinity than SPD, the conformational changes
of SPD are much attenuated (Table 3).
Conformation analysis: In flexible or acyclic molecules, stag-
gered conformers are predominant.[8,15] Ab initio calcula-
tions demonstrated that this holds true for alkyl amines[8]
and further confirmed that the energy gap between the stag-
gered and eclipsed rotamers in polyamines is comparable
with that in alkane systems. The dihedral angles in the
stable conformers are known to represent either the anti ori-
entation at approximately 1808 or the gauche orientation at
approximately 608, unless a bulky substituent is attached.
Thus, we assumed that those conformations bearing inter-
mediate angles or skew conformations hardly occur. When
3
ꢀ
the JH,H value falls on an intermediate value, the H H ori-
entation is supposed to belong not to a nonstaggered rota-
mer but to interconverting anti and gauche rotamers. It is,
therefore, possible to determine the population ratio be-
tween the anti and gauche conformers on the basis of spin–
spin coupling constants,[10] provided that JH,H values typical
3
of the anti and gauche conformers are in hand. The Karplus
equation, which is used to determine dihedral angles from
3JH,H values, was originally derived from rigid cyclic com-
pounds, so fluctuation from staggered rotamers should be
taken into account for these acyclic compounds.[10] In this
study, the modified Karplus equation[16] was applied for each
3
rotamer in 18 steps. The weighted average of the JH,H
values, which were obtained from the Boltzmann population
and the coupling constant for each rotamer,[17,18] was calcu-
lated for the gauche (+), anti, and gauche (ꢀ) conformers
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
around the C2 C3, C3 C4, C4 C5, C7 C8, and C8 C9
bonds (Table 1). The difference between hydrogen and deu-
terium is negligible for conformation potentials, so gauche
(+) and gauche (ꢀ) should be equally populated, although
their J values are different due to the hydrogen orientation
with respect to a nitrogen atom (Table 1).
Figure 2. Partial 1H NMR spectra (500 MHz, 408C) of the labeled SPDs
1, 2, and 5. NMR spectra were measured with 25 mm labeled SPD as the
3HCl salt in D2O at pH 7.3 (A), and in the presence of 100 mm ATP at
pH 3.3 (B), 100 mm ATP at pH 5.6 (C), and 100 mm ATP at pH 7.3 (D).
The spectra of 1–7 as the 3HCl salt at pH 3.3 and 5.6 were virtually iden-
tical with those shown in (A). Resolution enhancement was carried out
for the spectra of compounds 4 and 5 in the presence of ATP. The spectra
of other labeled SPDs are provided in the Supporting Information.
ꢀ
ꢀ
For the C5 N6 and N6 C7 bonds, however, the same
strategy was not applicable due to the exchangeable protons
3
of the ammonium groups. Instead, JC,H values were used for
3
these bonds. The JC,H values of the anti and gauche orienta-
13
ꢀ ꢀ ꢀ
tions for a C N C H system were not available, so
HMBC experiments were carried out for a conformationally
restricted system, 1-deoxynojirimycin.[19] These values were
converted into those for the flexible system by the same
the triphosphate at pH 5.6 and it is ꢀ4 on the triphosphate
at pH 7.3.[14] When compared with those of the HCl salt, the
3JH,H values of the ATP complex were notably lower for deu-
method as described above to give a JCNCH anti value of
,
3
3
7.34 Hz and a JCNCH gauche
,
value of 1.59 Hz. The JC,H values
3
terated SPDs 1, 3, 6, and 7. The JH,H value was the smallest
for H5/13C7 and H7/13C5 can be used to determine the anti/
ꢀ
ꢀ
at a neutral pH value, at which the charge of ATP was ꢀ4;
this clearly indicated that SPD undergoes conformational
changes to increase the amount of the bent conformation
(equivalent to gauche rich). On the other hand, only mini-
mal changes in the JH,H
served for 2, 4, and 5, respectively, in the presence of ATP
gauche populations with respect to the C5 N6 and N6 C7
bonds, respectively.
The NMR spectra of labeled SPDs 1–7 (Figure 2 and Fig-
ure S1 in the Supporting Information) revealed that, for
SPD as the HCl salt, the populations of the anti conformers
3
3
,
3JC,H, and JC,H values were ob-
ꢀ
around the C C bonds were about 76–85% and those
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1621

M. Murata et al.
Table 2. Populations of anti/gauche rotamers in the presence of ATP as obtained from 1H NMR experiments.
was raised from 3.3, to 5.6, and
then to 7.3. These data indicat-
ed that the conformation of
SPD is significantly influenced
by the number of charges on
the counterions.
Labeled SPD
Bond (torsion
angle)
Observed Counterion
J
3J
[Hz]
gauche[a]
[%]
Increment[b]
[%]
SPD-2,3-d10 (1)
C2 C3 (N1/C4)
3JH,H
3HCl
9.7
9.0
18
26
–
8
ꢀ
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
)
)
)
8.4
7.9
32
37
24
24
25
25
14
19
–
Discussion
SPD-4-13C, 3,4-d2
(2)
C3 C4 (C2/C5)
3JH,H
10.5
10.4
10.2
10.3
ꢀ
Conformation of SPD as the
HCl salt: To our knowledge,
the present study provides the
first experimental basis for the
conformation of acyclic multi-
valent ions in solution. Before
dealing with an SPD–ATP com-
plex, we discuss the conforma-
tion of SPD as the HCl salt.
The J values in Table 2 reveal
that the population of the anti
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
3HCl
pH 3.3
(ATP2ꢀ
pH 5.6
(ATP3ꢀ
pH 7.3
(ATP4ꢀ
0
)
)
)
1
1
SPD-4,5-d10 (3)
SPD-7-13C (4)
SPD-5-13C (5)
SPD-7,8-d12 (6)
SPD-8,9-d12 (7)
C4 C5 (C3/N6)
3JH,H
3JC,H
3JC,H
3JH,H
3JH,H
10.0
9.1
15
25
–
10
ꢀ
)
)
)
8.5
8.5
31
31
16
16
ꢀ
ꢀ
C5 N6 (C4/C7)
2.7
2.6
39
35
–
ꢀ4
conformer about each C C
bond was 76–85%, whereas a
significantly higher population
of gauche conformer was ob-
)
)
)
2.7
2.7
39
39
0
0
ꢀ
served for the central C5 N6
ꢀ
and N6 C7 bonds. With respect
ꢀ
N6 C7 (C5/C8)
2.5
2.5
32
32
–
0
ꢀ
to a C2 C3 bond of butane, the
population of the anti confor-
mer is about 56% in the liquid
phase.[20] The findings indicate
that the anti conformer of SPD
is more populated than that of
alkanes, which can be account-
ed for by the electrostatic re-
pulsion among the three ammo-
nium groups of SPD. The
energy difference between the
N1–C4 anti (82%) and gauche
(9%) conformers, despite the
fact that the distance between
N1 and N6 should be signifi-
cantly shorter in the gauche
conformer, is 1.3 kcalmolꢀ1,
which implies that the counter-
ions and hydration greatly at-
tenuate the electrostatic repul-
sion. A similar tendency was
)
)
)
2.5
2.6
32
35
0
3
ꢀ
C7 C8 (N6/C9)
9.9
9.5
15
19
–
4
)
)
)
9.2
8.8
22
26
7
11
ꢀ
C8 C9 (C7/N10)
9.8
9.0
16
24
–
8
)
)
)
9.0
8.2
24
32
8
16
[a] The percentage is a sum of gauche (+) and gauche (ꢀ) populations. [b] The increment of the gauche popu-
lation relative to that of the 3HCl salt of SPD.
ꢀ
ꢀ
ꢀ
around the C5 N6 and N6 C7 bonds were 61–68%
(Table 2). When interacting with ATP, SPD significantly
observed for the other C C bonds of SPD (Table 2). The
ꢀ
highly populated gauche conformers for the C5 N6 and
ꢀ
ꢀ
ꢀ
ꢀ
changes its conformation. For the C2 C3, C4 C5, C7 C8,
and C8 C9 bonds, the gauche rotamer particularly increases
N6 C7 bonds are explainable by the notion that the dis-
ꢀ
tance between the neighboring ammonium groups (N1/N6
or N6/N10) is hardly affected by the conformation changes
around these bonds. At acidic pH values of 3.3 and 5.6, the
populations of rotamers around all the relevant bonds of the
SPD HCl salt were essentially the same as those at pH 7.3
(data not shown), which indicates that SPD forms a trivalent
ꢀ
ꢀ
by 11–19%, whereas the rest of the C C and C N bonds
show insignificant changes of less than 4% (Table 2). These
conformational alterations are dependent on the pH value
and, thus, on the number of negative charges on ATP; 1, 3,
6, and 7 showed higher gauche populations as the pH value
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Chem. Eur. J. 2009, 15, 1618 – 1626

Conformation Analysis of Spermidine–ATP Complexes
FULL PAPER
Table 3. Populations of anti/gauche rotamers of SPD in the presence of ATP and Mg2+
.
the distance was significantly
Labeled SPD
Bond (torsion
angle)
Observed
J
Counterion
3J
[Hz]
gauche[a]
[%]
Increment[b]
[%]
ꢀ
reduced to 0.53 nm in the C2
ꢀ
C3 gauche (GAA) and C4 C5
ꢀ
SPD-2,3-d10
(1)
SPD-4,5-d10
(3)
C2 C3 (N1/C4)
3JH,H
3JH,H
3JC,H
3JC,H
3JH,H
3JH,H
pH 7.3
9.3
9.9
2.7
2.5
9.7
9.6
23
+5
+1
0
gauche (AAG) conformers.
(ATP4ꢀ
pH 7.3
(ATP4ꢀ
pH 7.3
(ATP4ꢀ
pH 7.3
(ATP4ꢀ
pH 7.3
(ATP4ꢀ
pH 7.3
(ATP4ꢀ
)
)
)
)
)
)
The same set of coupling
constants were obtained for the
complex with tripolyphosphate
(Figure 3). The close similarity
between the results for SPD–
TPP and SPD–ATP demon-
strates that SPD chiefly recog-
nizes the triphosphate group of
ATP, a conclusion that is fur-
ther supported by the high af-
finity between SPD and TPP.
ꢀ
C3 C4 (C3/N6)
16
39
32
17
18
SPD-7-13C (4) C5 N6 (C4/C7)
ꢀ
SPD-5-13C (5) N6 C7 (C5/C8)
0
ꢀ
ꢀ
SPD-7,8-d12
(6)
SPD-8,9-d12
(7)
C7 C8 (N6/C9)
+2
+2
ꢀ
C8 C9 (C7/N10)
[a] The percentage is a sum of gauche (+) and gauche (ꢀ) populations. [b] The increment of the gauche popu-
lation relative to that of the 3HCl salt of SPD.
cation in this pH range. Under alkaline conditions (pH 11),
in which case SPD was not ionized, the gauche conformer
ꢀ
comprised 32% and 31% with respect to the C2 C3 and
ꢀ
C4 C5 bonds, respectively, with increments of 14 and 10%
from those of its trivalent cation at pH 7.3. These conforma-
tional changes support the conclusion that electrostatic re-
pulsion between the ammonium groups moderately but sig-
nificantly affects the conformation of SPD.
Conformation of SPD upon interaction with ATP: Upon in-
teraction with ATP, the gauche orientation of SPD greatly
increased (Table 2), which indicates that SPD tends to take
a bent conformation in its ATP complex. The increments in
ꢀ
ꢀ
ꢀ
the gauche rotamer are larger for the C2 C3, C4 C5, C7
ꢀ
C8, and C8 C9 bonds, for which the gauche populations are
roughly doubled (Table 2). These conformational alterations
are significant; the internal rotations with respect to the
ꢀ
ꢀ
ꢀ
C2 C3, C3 C4, and C4 C5 bonds alter the distance be-
tween N1 and N6 in the butanylene part of SPD, in which
the populations of gauche-containing conformers are esti-
mated to comprise over 90%, whereas those for the 3HCl
salt of SPD comprise approximately 57%. For the propany-
lene part, the gauche–anti and anti–gauche (GA and AG)
Figure 3. Increments of gauche conformers in SPD–ATP (a) and SPD–
TPP (b) at pH 7.3 as compared with those in the 3HCl salt of SPD. De-
tailed data for the SPD–TPP complex are provided in the Supporting In-
formation. : The increment percentage was less than the detection limit
*
of 1.5% in measurements of C H coupling constants.
ꢀ
ꢀ
conformers of the C7 C8 and C8 C9 bonds, which affect
the distance between N6 and N10, are estimated to comprise
over 50% in the ATP complex as compared with approxi-
mately 30% in the SPD HCl salt. By contrast, the other
ꢀ
The distances between the nitrogen atoms in the butanylene
and propanylene parts (N1/N6 and N6/N10) are about 0.63
and 0.50 nm, respectively, in the extended conformation;
these values are larger than the distance between neighbor-
ing oxygen atoms in the triphosphate moiety of ATP (0.41–
0.43 nm). Therefore, SPD needs to change conformation so
as to arrange each ammonium group close to an anionic
point of ATP. The gauche conformer in the butanylene part
was more populated than that in the propanylene section
(Table 2), which indicates that the butanylene part needs to
bend more upon interaction with the triphosphate group of
ATP due to its longer alkyl chain.
ꢀ
ꢀ
ꢀ
bonds, C3 C4, C5 N6, and N6 C7, gave rise to only mini-
mal changes upon complexation with ATP. These differences
can be accounted for by the notion that a rotation around
C5 N6 (or N6 C7) does not alter the distance between N1
and N6 (or N6 and N10). The rotational conformation
around these bonds should, therefore, have little influence
on the electrostatic repulsion among the ammonium groups
in SPD. Similarly, the rotation with respect to the C3 C4
bond changes the distance between N1 and N6 to a smaller
extent than that of the other C C bonds. The interatomic
distance between N1 and N6 was calculated to be 0.63 nm
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
for the C3 C4 anti conformer (AAA for C2 C3/C3 C4/C4
The next question to be addressed is “what does the gross
conformation of SPD in the ATP complex look like?” As
ꢀ
C5) and 0.59 nm for the C3 C4 gauche one (AGA), whereas
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1623

M. Murata et al.
the first step, we estimated the distances of N1/N6 and N6/
N10 for each conformer. In the propanylene part, the
ꢀ
ꢀ
gauche C7 C8/gauche C8 C9 (GG or GG’) conformation is
negligible because the distance between N6 and N10 in the
GG conformer is too close (0.36 nm) to accommodate the
ꢀ
ꢀ
negative charges of ATP. Therefore, the C7 C8/C8 C9 con-
formation relevant for the N6/N10 distance takes either the
AA (42%), AG (32%), or GA (26%) orientation; the N6/
N10 distance in these conformers is 0.50, 0.44, and 0.44 nm,
respectively. For the butanylene part, the story is a little
more complicated. There are three bonds relevant for the
ꢀ
ꢀ
ꢀ
N1/N6 distance, namely C2 C3, C3 C4, and C4 C5. If
these bonds take the anti or gauche conformation independ-
ently, the AAA conformer occurs with 33% population. The
GGG conformation is very unlikely because of the short
N1/N6 distance. The GG’ conformers that have the 1,3-syn
orientation are also negligible for the same reason. There-
fore, the butanylene part can be represented by 8 conform-
ers with relative stereochemistry: AAA, AAG, AGA, GAA,
AGG, GAG, GAG’, and GGA. The N1/N6 distance of these
conformers ranges from 0.47 (GAG) to 0.63 nm (AAA).
Provided that each bond takes the anti/gauche rotation inde-
pendently of the other bonds, the all-anti conformer occurs
in 32.6%, single-gauche ones in 44.6%, and double-gauche
ones in 19.9%. Since the double-gauche conformers are
more unstable than the single-gauche ones due to electro-
static repulsion and steric hindrance, the single-gauche con-
formers may actually be more populated, probably in excess
ꢀ
ꢀ
of 50%. In ATP, the distances of Oa Ob and Ob Og are 0.41
and 0.43 nm, respectively, which can be regarded as the dis-
tances between neighboring anionic sites. Therefore, in the
ꢀ
propanylene part, the AG and GA conformers for the C7
Figure 4. Hypothetical images of the SPD shapes accounting for the pH-
dependent increase in gauche orientation. Three of the many possible
ꢀ
C8/C8 C9 bonds are suitable to arrange the N6 and N10
close to the neighboring phosphate groups, and in the buta-
nylene part, one (or two) gauche conformers for the C2 C3/
C3 C4/C4 C5 bonds fit the distance between the phosphate
anions. These notions may account for the increase in the
gauche orientation in each C C bond that was described
earlier. The interaction between the butanylene and propa-
nylene parts in the SPD–ATP complex remains unclear; the
aforementioned accounts of the distances between N1 and
N6 or N6 and N10 imply that these two parts change their
conformations in a rather independent manner.
ꢀ
ꢀ
conformations, a–c, are depicted. The orientations of the C2 C3, C3 C4,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
C4 N5, N5 C6, C6 C7, C7 C8, and C8 C9 bonds are AAGAAAA in
(a), GAGAAAA in (b), and GAGAG’AG in (c). The conformation of
ATP was obtained by calculations with the OPLS force field. When the
pH value was raised from 3.3, the orientation of the adenine group of
ATP switches to the syn conformation at pH 5.6, which leads to the bent
butanylene part (B). The conformation of the propanylene part (P) is fur-
ther altered by the second deprotonation of the g-phosphate group at
pH 7.3. The relative positioning of SPD and ATP derived from the simu-
lation is not based on experimental data.
ꢀ
ꢀ
ꢀ
pH 5.6, at which point the ATP becomes trivalent, the ade-
nine base takes the syn conformation (Figure 4b). This con-
formational alteration should give rise to a greater affect on
a countercharge group with the a-phosphate group, so the
butanylene part is assumed to interact preferentially with
the a-phosphate side. On the other hand, with the confor-
mation change from pH 5.6 to 7.3 in the presence of ATP,
pH-Dependent conformation change of the SPD–ATP com-
plex: We next examined the SPD–ATP interaction in differ-
ent ionization states with respect to the gauche populations
at various pH values. The optimized structures of ATP with
net charges of ꢀ2, ꢀ3, and ꢀ4 have been deduced by a con-
formation search and the X-ray data for Na+2ATP2ꢀ.[21,22]
The conformational changes with a change from pH 3.3 to
5.6 indicate that the increment in gauche conformers is
ꢀ
ꢀ
the gauche populations of the C7 C8 (4%) and C8 C9
(8%) bonds in the propanylene part increase more signifi-
ꢀ
ꢀ
ꢀ
ꢀ
larger for C2 C3 (6%) and C4 C5 (6%) in the butanylene
cantly (Table 2) than those of the C2 C3 (5%) and C4 C5
(0%) bonds. At pH 7.3, the g-phosphate group becomes di-
valent and thus makes ATP tetravalent, so the propanylene
part is assumed to interact with the g-phosphate terminal.
These findings imply that the interaction between ATP and
ꢀ
ꢀ
part than for C7 C8 (3%) and C8 C9 (0%) in the propany-
lene part (Table 2). At pH 3.3, at which point the average
net charge of ATP is ꢀ2, the anti orientation of the adenine
ring to the ribose is dominant (Figure 4a).[9] However, at
1624
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ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1618 – 1626

Conformation Analysis of Spermidine–ATP Complexes
FULL PAPER
ꢀ
ꢀ
SPD is orientation sensitive and the propanylene part of
SPD has a higher affinity to ATP at the neutral pH value.
Furthermore, we obtained experimental results that show
that the affinity of diaminopropane, corresponding to the
propanylene portion, to ATP is significantly higher than that
of putrescine, corresponding to the butanylene part (see the
Supporting Information). A similar observation that poly-
coupling constant for each C C or C N bond. SPD, com-
prising two alkyl parts, butanylene and propanylene, under-
goes conformational change upon interaction with multiva-
lent anions such as ATP and tripolyphosphate. With respect
ꢀ
to the C C bonds relevant to the distance between the
neighboring pairs of ammonium groups, N1/N6 and N6/N10,
the gauche rotamers were increased by 11–19% in the com-
plexes as compared with those in the 3HCl salt of SPD. On
the other hand, the rest of the bonds, the rotation of which
does not greatly affect the distances between the ammonium
groups, showed only minimal alterations. The pH-dependent
conformation changes of SPD revealed that the interaction
between SPD and ATP is orientation sensitive, in that the
butanylene part of SPD tends to come to the ribose side
and the propanylene resides near the g-phosphate end.
These results may provide a clue for a better understanding
of weak and soft interactions between polyamines and
anionic biomolecules such as DNA, RNA, and nucleotides.
ꢀ
amines with shorter N N distances show higher affinity for
ATP has been also reported by De Stefano et al.[12]
Conformation of SPD in SPD–ATP–Mg2+ ternary complex:
Under physiological conditions, SPD is supposed to partly
form a ternary complex with ATP–Mg2+, which plays an im-
portant role in ATP-dependent reactions and in the physio-
logical effects of endogenous polyamines.[7–9] We aimed to
investigate the conformation of the SPD–ATP–Mg2+ ternary
complex by using the same strategy. To evaluate the confor-
mational change of SPD upon interaction with ATP–Mg2+
,
the relevant J values were measured under conditions in
which 60–80% of SPD was complexed with ATP–Mg2+
(Table 3). An increment in the gauche conformers was not
apparent compared with those in the absence of Mg2+
(Table 2); respective 5, 2, and 2% increases were observed
Experimental Section
Preparation of labeled SPDs: Labeled SPDs 1–7 were prepared by three
key steps: a) erythro-selective hydrogenation of a a,b-unsaturated lac-
tone, b) Curtius rearrangement to provide a primary amino group, and
c) N-alkylation by using the method of Fukuyama et al.[24] Details of the
synthesis are provided in the Supporting Information. For the following
experiments, including the Jobꢀs plot, NMR titrations, and measurements
of coupling constants, spermidine trihydrochloride, ATP dipotassium salt,
and tripolyphosphate tetrapotassium salt (K5P3O10) were used.
ꢀ
ꢀ
ꢀ
for the C2 C3, C7 C8, and C8 C9 bonds. These observa-
tions suggest that the ATP–Mg2+–SPD ternary complex pos-
sesses a different SPD–ATP interaction. The ATP–Mg2+
complex is present as a divalent ion at the neutral pH
value.[23] This small alteration in the SPD conformation may,
therefore, be ascribable to the interaction with divalent
anionic ATP–Mg2+, an idea that is supported by the fact
Measurements of stoichiometry and association constants in the SPD–
ATP complex: The Jobꢀs plot was performed in D2O at 408C. The total
concentration of SPD plus ATP was maintained at 10 mm. The pH value
was adjusted to 7.3 with deuterium chloride and sodium deuteroxide. The
DdꢂnSPD values (Dd: the change in the chemical shift of H8 of SPD in-
that SPD shows small conformational changes in the pres-
ence of the divalent phosphate ion HPO4
2ꢀ
(data not
shown). The physiological significance of this weaker inter-
action of SPD with ATP–Mg2+ is currently unknown. Yet,
its role in the dehydration of ATP–Mg2+ upon enzymatic
hydrolysis may provide a plausible account because the
SPD–ATP–Mg2+ complex is thought to occur in significant
concentrations under cellular conditions.[7]
The conformations discussed in this study provide time-
average images of an SPD–ATP complex. Combinations of
these conformers may be able to explain the molecular basis
for the weak biomolecular interactions. We think that these
conformational changes of SPD are important for its inter-
actions with biological polyanions, interactions that are plau-
sibly implicated in the physiological functions of SPD, such
as cell proliferation and differentiation. A similar conforma-
tion study for spermine to gain a better understanding of
the role differentiation between SPD and spermine is now
under way and will be reported in due course.
duced by addition of ATP; nSPD: molar fraction of SPD, [SPD]/
ACHTUNGTRENNUNG([ATP]+-
AHCTUNGTRENNUNG
were performed in D2O at 408C with constant SPD concentrations of
25 mm and ATP concentrations of approximately 6.25, 9.38, 12.5, 18.75,
25, 37.5, and 50 mm. The pH value was adjusted to 7.3 with deuterium
chloride and sodium deuteroxide. In these experiments, the downfield
shift of the H8 signal of SPD was monitored as a function of the SPD–
ATP ratio. The revised data were fitted to a theoretical titration curve to
obtain the association constants by using Origin 6.1 software provided by
OriginLab Corporation.
3
3
Method for determining spin–spin coupling constants: JH,H and JC,H
values were extracted from 1D 1H NMR spectra. NMR spectra were ob-
tained on a Jeol GSX-500 500 MHz spectrometer. The digital resolution
for the 1H NMR spectrum was 0.076 Hz/point. Hence, measurement of
coupling constants can be carried out with an accuracy of ꢁ0.1 Hz. The
temperature of the probe was kept at 408C. Samples of 1–7 were dis-
solved in D2O. The concentrations of SPD trichloride salt, ATP dipotassi-
um salt, and MgCl2 were 25, 100, and 100 mm, respectively. The pH value
was adjusted to 3.3, 5.6, or 7.3 with deuterium chloride and sodium deu-
teroxide. The chemical shifts were recorded from 3-(trimethylsilyl)-1-pro-
3
panesulfonic acid sodium salt (DSS). The optimizations of the JH,H
Conclusion
values containing second-order couplings were performed by the simula-
1
tion application gNMR (Adept Scientific). The H NMR spectra of 4 and
3
5 were processed with a shifted SinBell window to measure the JC,H
The present study proposed a method for the conformation
analysis of flexible compounds in aqueous solutions. We syn-
thesized the diastereoselectively 2H-labeled and/or 13C-la-
beled spermidines (SPDs 1–7) and determined the spin–spin
value. HMBC measurements were performed at 258C for a solution of 1-
deoxynojirimycin (approximately 10 mg) in D2O (0.2 mL) in a Shigemi
sample tube on a Jeol ECA 500 MHz spectrometer. Details of the
HMBC experiments will be published elsewhere.
Chem. Eur. J. 2009, 15, 1618 – 1626
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1625

M. Murata et al.
Determination of anti/gauche populations and conformation energy:
3JH,H values with conformational fluctuation were calculated as reported
previously.[10] Briefly, the energies (Ei) of 360 conformers (total number
[4] K. Igarashi, K. Kashiwagi, Biochem. Biophys. Res. Commun. 2000,
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[5] a) H. Tabor, C. W. Tabor, Adv. Enzymol. 1972, 36, 203–268; b) H.
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Jersey, 1971.
ꢀ
N) were obtained for each 18 in the dihedral angle along the C C bonds
by using the OPLSA[17] force field in the MacroModel software.[18] The
calculations were performed with the solvent effect of water. These
values were substituted into Equation (1) and the Boltzmann population
(Pi) was obtained.
[7] S. Watanabe, K. Kusama-Eguchi, H. Kobayashi, K. Igarashi, J. Biol.
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[8] a) L. A. E. Batista de Carvalho, A. M. Amorim da Costa, J. J. C.
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30939–30944.
[10] K. Maruyoshi, T. Demura, T. Sagane, N. Matsumori, T. Oishi, M.
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[11] C. J. Morton, D. J. Pugh, E. L. Brown, J. D. Kahmann, D. A. Renzo-
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[12] a) C. De Stefano, C. Foti, A. Gianguzza, O. Giuffre, S. Sammartano,
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[13] a) Y. Shibata, B. Inoue, Y. Nakatsuka, Nippon Kagaku Kaishi 1921,
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113.
expðꢀEi=RTÞ
Pi ¼
N
P
ð1Þ
expðꢀEi=RTÞ
i
3
A spin coupling constant Ji in conformer i is given by substituting the di-
hedral angle between vicinal protons for the modified Karplus equa-
tion.[16] The averaged values for gauche (+) (608), anti (1808), and gauche
(ꢀ) (3008) in Table 1 were obtained from Equation (2) for the dihedral
angles between 0 and 1208, between 120 and 2408, and between 240 and
3608, respectively. The energies of the SPD conformers were calculated
by using the OPLS2001 force field in the MacroModel software. These
calculations were performed with consideration of the solvent effect of
water and the charges of the ammonium groups. To build each SPD con-
former, constrained torsion angles were defined (gauche (+) 608, anti
1808, and gauche (ꢀ) 3008). Determination of the gauche rotamers based
3
on the JC,H values was carried out in a similar manner by using 7.34 and
1.59 Hz as the static anti and gauche values, respectively.
[14] R. A. Alberty, R. M. Smith, R. M. Bock, J. Biol. Chem. 1951, 193,
425–434.
[15] N. Matsumori, D. Kaneno, M. Murata, H. Nakamura, K. Tachibana,
J. Org. Chem. 1999, 64, 866–876.
N
X
3
3JH,H
¼
Pi ꢂ Ji
ð2Þ
i
[16] C. A. G. Haasnoot, F. A. A. M. De Leeuw, C. Altona, Tetrahedron
1980, 36, 2783–2792.
[17] W. L. Jorgensen, J. Tirado-Rives, J. Am. Chem. Soc. 1988, 110,
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Lipton, C. Caufield, G. Chang, T. Hendrickson, W. C. Still, J.
Comput. Chem. 1990, 11, 440–467.
Acknowledgement
We are grateful to Professor Kazuei Igarashi for encouraging us to
pursue this study. This work was supported by Grants-In-Aid for Scientif-
ic Research (A) (no. 15201048) and (S) (no. 18101010), for Priority
Area (A) (no. 16073211), and for Young Scientists (A) (no. 17681027)
from MEXT, Japan, by the Naito Foundation, by the Yamada Science
Foundation, and by a grant from CREST, the Japan Science and Technol-
ogy Corporation.
[19] T. Yamaguchi, K. Maruyoshi, N. Matsumori, M. Murata, Chem. Lett.
2009, 1172–1173.
[20] E. L. Eliel, S. H. Wilen, L. N. Mander in Stereochemistry of Organic
Compounds, Wiley, New York, 1994, Chapter 10, p. 601.
[21] Y. Huse, Y. Iitaka, Acta Crystallogr. 1969, 25, 498–509.
[22] a) O. Kennard, N. W. Isaacs, J. C. Coppola, A. J. Kirby, S. Warren,
W. D. S. Motherwell, D. G. Watson, D. L. Wampler, D. H. Chenery,
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[23] For example: D. Lꢃthi, D. Gꢃnzel, J. A. S. McGuigan, Exper. Physi-
ol. 1999, 84, 231–252.
[24] a) T. Fukuyama, M. Cheung, C.-K. Jow, Y. Hidai, T. Kan, Tetrahe-
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Kan, Synlett 1999, 1301–1303.
[1] For example: J. P. Snyder, J. H. Nettles, B. Cornett, K. H. Downing,
E. Nogales, Proc. Natl. Acad. Sci. USA 2001, 98, 5312–5316.
[2] For example: U. Quast, M. I. Schimerlik, M. A. Raftery, Biochem-
istry 1979, 18, 1891–1901.
[3] a) R. Brasseur, J.-M. Ruysschaert, P. Chatelain, Biochim. Biophys.
Acta 1985, 815, 341–350; b) S. W. Wilson, W. Cui, F. Guarnieri, Data
Handl. Sci. Technol. 1995, 15, 351–367; c) G. Barone, D. Duca, A.
Silvestri, L. Gomez-Paloma, R. Riccio, G. Bifulco, Chem. Eur. J.
2002, 8, 3240–3245. Similar conformational studies have been car-
ried out for synthetic polymers: d) R. G. Snyder, J. Chem. Phys.
1967, 47, 1316–1360; e) A. J. Hopfinger, Conformational Properties
of Macromolecules, Academic Press, New York, 1973, Chapter 5,
p. 297.
Received: September 23, 2008
Published online: January 7, 2009
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Chem. Eur. J. 2009, 15, 1618 – 1626