Synthesis and conformation of deuterated spermidine for investigating weak interaction with polyanionic biomolecules

Article abstract of DOI:10.1016/j.tet.2004.04.056

Polyamines such as spermidine and spermine are known to interact with polyanionic biomolecules under physiological conditions. Conformation analysis of these highly flexible acycles is virtually unprecedented due to the lack of appropriate methodologies.

Full text of DOI:10.1016/j.tet.2004.04.056

Tetrahedron 60 (2004) 5163–5170
Synthesis and conformation of deuterated spermidine for
investigating weak interaction with polyanionic biomolecules
Keisuke Maruyoshi, Tetsuo Demura, Takeshi Sagane, Nobuaki Matsumori, Tohru Oishi
*
and Michio Murata
Department of Chemistry, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
Received 23 March 2004; revised 22 April 2004; accepted 23 April 2004
Abstract—Polyamines such as spermidine and spermine are known to interact with polyanionic biomolecules under physiological
conditions. Conformation analysis of these highly flexible acycles is virtually unprecedented due to the lack of appropriate methodologies.
1
Spin–spin coupling constants, which are often utilized for acyclic systems, are rather uninformative for polyamines due to unresolved H
NMR signals of their tri- and tetra-methylene moieties. We attempted to solve this problem by synthesizing diastereospecifically deuterated
p
p
1
1
spermidine, (1S ,2S )-1,8-diamino-4-azaoctane-1,2,3,3,5,5,6,6,7,7,8,8-d12. To evaluate its utility, H– H spin coupling constants were
measured as trihydrochloride or tripolyphosphate salt. The relevant coupling constant markedly decreased upon complexation to
tripolyphosphate. Under neutral pH, where the net charge of tripolyphosphate became tetravalent, the coupling constant was small; as pH was
lowered to 1.4, where the charge was divalent, the constant increased significantly. These data clearly indicate that, when interacting with
polyanions, spermidine undergoes conformational changes to increase bent conformation, which could be effectively monitored by the
deuterated spermidine.
q 2004 Elsevier Ltd. All rights reserved.
1
. Introduction
understanding of molecular recognitions among bio-
molecules, the structure basis of weak interactions is
essential information. In particular, conformation of flexible
molecules upon complexation should be the first question to
address.
The interaction between a bioactive compound and its target
molecule is one of the most intriguing subjects in
biochemistry and pharmacology. Recently, highly specific
recognitions between ligand and receptor have been
extensively studied in structural biology, particularly in
association with drug–target interactions. The crystal
structures of a ligand–protein complex are solved by
X-ray crystallography, and partial structures near a ligand
binding site are elucidated by NMR-based methodologies.
Pieces of the structural information have deepened our
understanding about the molecular basis of physiological
events such as cellular signal transductions, endocrine
systems, and immune reactions. Besides these strict
intermolecular recognitions, weak interactions between
biomolecules sometimes play a crucial role. For example,
neuronal receptors mostly have relatively low affinity for
their intrinsic neurotransmitter, which enables the rapid
on/off switching of nervous signal transmission. Structural
requirements for these compounds to elicit weak inter-
actions, however, remain mostly unelucidated; they are
mostly flexible molecules and undergo rapid association/
dissociation, which makes the elucidation of 3D structures
of a complex exceptionally difficult. To gain a better
Thus, we aimed at establishing a methodology toward
conformation analysis of flexible small molecules impli-
cated in weak interactions. As a model compound, we
became interested in polyamines such as putrescine,
spermidine and spermine because they distribute ubiqui-
tously over eukaryotes and prokaryotes as major con-
stituents and are considered to regulate diverse cellular
events through weak interactions; e.g. polyamines are
known to possess affinity to various negatively charged
biopolymers, such as nucleic acids, phospholipids, and
1
,2
proteins,
and regulate their functions, particularly in
association with cell growth, differentiation, and prolifer-
ation. In contrast to nanomolar/subnanomolar dissociation
constants as seen for drug-target and hormone-receptor
interactions, the values of spermidine with DNA, 16S
rRNA, phospholipid, and ATP are reported to be 15.6, 2.66,
3
5.56 and 2.24 mM, respectively. Among polyamines, we
have focused on spermidine (SPD), which bears three amino
groups (Fig. 1) and comparable affinity with polyanions to
spermine, a tetramine congener. We are also interested in
the difference in conformation between a butanylene
(C2–C5) and propanylene (C7–C9) parts. In this study,
the conformation change of SPD upon weakly complexating
Keywords: Spermidine; TPP; Weak interaction; Conformation analysis.
*
Corresponding author. Tel./fax: þ81-6-6850-5774;
e-mail address: murata@ch.wani.osaka-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.04.056

5164
K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
3
The conformation analysis of SPD by using JH,H, however,
is hampered by its acyclic structure, where the absence of
1
asymmetric centers causes unresolved H NMR methylene
signals. If the spin system can be simplified by the diastereo-
specific labeling of each one of two geminal protons with
deuterium, this problem should be addressed. In compound
1
, the rest of methylene protons are mostly deuterized to
1
avoid the overlap of H NMR signals. In this study, we
attempted to synthesize a molecular probe (1) for measure-
ment of the dihedral angles with respect to the C8–C9 bond
and evaluate its conformational alteration when forming a
salt with monovalent or multivalent anion.
Figure 1. Structure of spermidine, ATP, TPP.
to tripolyphosphoric acid (TPP) was examined to obtain a
clue as to how spermidine recognize polyanionic bio-
molecules (Fig. 2).
2. Result and discussion
2.1. Synthesis
The deuterated SPD (1) would be synthesized through the
coupling of bromide (2) and 2-nitrobenzenesulfonamide (3)
6
,7
by means of Fukuyama method (Fig. 3). The crucial steps
of the present synthesis involve the diastereoselective
construction of the erythro-1,2-dideuterio-1,3-diamino-
propane moiety, which commenced with 5,6-dihydro-2H-
pyran-2-one (4) as shown in Scheme 1. Although catalytic
cis-deuteration of alkenes sometimes resulted in scrambled
addition of deuterium and polydeuteration through the
Figure 2. Hypothetical model when spermidine interacts with TPP.
Spin–spin coupling constants are a useful NMR parameter
for conformational studies of biomolecules. In particular,
3
8
proton–proton vicinal coupling constants ( JH,H) are most
frequently used for this purpose because of their high
b-hydride elimination of the intermediate, that of 4 using
Wilkinson catalyst [RhCl(PPh ) ] in benzene successfully
3
3
9
sensitivity and a well-established theory known as the
3
afforded 5 as a single diastereomer. The purity of 5 was
Karplus equation. J
possesses several advantages over
H,H
NOE in conformation studies of acyclic and flexible
systems. For compounds undergoing a conformational
alteration, the coupling constants are observed as a
weighted average of those due to each conformer, thereby
greatly facilitating the determination of population of
4
,5
conformers.
Figure 3. The coupling by Fukuyama’s method.
Scheme 1. Reagents and conditions. (a) D
3
2
, RhCl(PPh
0 min; (d) toluene, reflux, 1 h then HCl; (e) (Boc) O, Et
.5 h then NaBH , 0 8C, 2 h; (g) (Boc) O, Et N, CH Cl , rt, 2 h (87% from 7); (h) SO
O, rt, 1 h (91% for two steps); (j) CH I, NaHCO , DMF, rt, 14 h (85%); (k) CH
2) benzylalcohol, reflux, 22 h (76% for two steps); (m) H , 20% Pd(OH) -C, MeOH, rt, 14 h (97%); (n) NsCl, Et
3
)
3
, benzene, rt, 40 h (b) hydrazine, MeOH, reflux, 2 h (98% for two steps); (c) NaNO
N, H O–THF, rt, 10 h (15% from 6); (f) (1) TFA–CH Cl , rt, 10 min, 2) C CHO, Et
-py, DMSO, Et N, rt, 30 min; (i) NaClO , NaH PO , 2-methyl-2-butene,
OD, CH ONa, reflux, 50 h (94%); (l) (1) DPPA, Et N, reflux, 1 h,
N, CH Cl , rt, 16 h (99%).
2
, 1 M HCl, 0 8C,
2
3
2
2
2
6
H
5
3
N, MeOH, rt,
1
4
2
3
2
2
3
3
2
2
4
t
BuOH–H
2
3
3
3
3
3
(
2
2
3
2
2

K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
5165
1
3
13
confirmed by C NMR measurements. Ring opening
reaction of the lactone 5 with hydrazine in methanol at
reflux for 2 h yielded hydrazide 6 (98% for two steps).
chloride in the presence of triethylamine to afford
propanylene fragment 3 in high yields.
1
0
Curtius rearrangement of 6 under the standard conditions
proceeded with the complete retention of the configuration
p
to afford (3S ,4S )-4-amino-1-propanol-3,4-d , which was
Synthesis of the butanylene fragment 2 and coupling with 3
was achieved as shown in Scheme 2. Commercially
available succinonitrile-d (15) was reduced to 1,4-diami-
4
p
2
isolated as its N-Boc derivative 7 in 15% yield. The low
yield of 7 was due to the competitive intramolecular
nucleophilic attack of the primary alcohol to the acyl azide
intermediate giving lactone 5.
nobutane-d by treatment with PtO in acetic acid-d under
8
the deuterium atmosphere, which was isolated as
hydrochloride salt (16). Mono-bromination of the diamine
2
1
4
16 using 0.9 equiv. of NaNO followed by protection of the
2
remaining amino group with Boc afforded fragment 2 in
12% yield with the recovery of 16 (84%). Then, next task is
the coupling of fragments 2 and 3 and successive removal of
the protecting groups.
Removal of the Boc group of 7, followed by reductive
amination of the resulting primary amine using benzal-
dehyde and NaBH , afforded N-benzylamine 8, which was
4
further protected with Boc group to yield 9 in 87% from 7.
Successive oxidation of the primary alcohol 9 with SO ·py
followed by NaClO led to carboxylic acid 10, which was
2
Treatment of the bromide 2 with 2-nitrobenzenesulfona-
mide 3 in the presence of K CO in DMF resulted in the
3
2
3
6
,7
treated with methyl iodide to afford ester 11. Hydrogen–
deuterium exchange at Ca carbon of 11 was successfully
achieved by treatment with sodium methoxide in methanol-
d at reflux for 50 h, and concomitant hydrolysis of the
methyl ester occurred under the reaction conditions to yield
formation of 18, followed by removal of the Ns group
with thiophenol afforded 19 in 68% yield for two steps.
Although removal of the N-benzyl group of the secondary
amine 19 was sluggish, that of corresponding carbamate 20
proceeded smoothly to give 21 in 41% yield. Removal of the
1
1
p
p
carboxylic acid 12. Modified Curtius rearrangement of 12
using DPPA in benzylalcohol proceeded smoothly to yield
Boc groups of 21 with TFA afforded (1S ,2S )-1,8-
diamino-4-azaoctane-1,2,3,3,5,5,6,6,7,7,8,8-d , 1, which
1
2
1
2
benzylcarbamate 11 in 76% for two steps. Cbz group of 13
was converted to 2-nitrobenzenesulfonyl (Ns) group
through the hydrogenolysis of 13, followed by treatment
of the resulting primary amine with 2-nitrobenzenesulfonyl
was isolated as trihydrochloride salt 22. These synthetic
strategies will be essentially applicable to preparation of
other deuterated spermidine analogues for conformational
study of the rest of four C–C bonds although yields in some
reactions such as Curtius rearrangement from 6 to 7 and the
hydrogenation of 20 should be improved. The deuterium
1
3
contents were estimated by C NMR to be .99% for C2,
1
C3, C4, C5, C8, C9, and by H NMR to be 93% for C7.
2
.2. Measurements of 3JH,H values
3
The spin–spin coupling constant between H-8 and H-9 ( J
8,H-9) was measured for non-labeled SPD and the molecular
H-
3
probe 1. With use of non-labeled one, J values could not
H,H
1
be determined better than 0.5 Hz due to the overlap of H
NMR signals and the equalization of spin coupling
constants, which was further complicated by second order
couplings derived from close chemical shifts of H -7 and
2
H -9. The deuterated SPD–HCl and SPD–TPP at various
2
pH (Fig. 4(B)–(D)) were subjected to NMR measurements
(
Fig. 4).
3
As depicted in Figure 4, J
was clearly determined
H-8,H-9
with the accuracy of 0.1 Hz although signals were
broadened partly by the association/dissociation equili-
brium of complexation. The value of SPD–HCl gave rise to
a large value of 9.8 Hz, which showed that C7–N9
conformation of 1 as a HCl salt was anti-dominant. In
3
acidic pH 1.4 and 3.2, JH-8,H-9 of SPD–HCl was
unchanged. Next, the same measurements were carried out
for the SPD–TPP complex. As reported previously, the
dissociation constant (K ) of SPD–TPP complexes was
d
1
5
calculated to be 1.23^0.07 mM , which indicated that 98%
of SPD formed complex with TPP under the conditions of
NMR measurements, and thus the spin coupling constants
obtained from Figure 4(B)–(D) should be those upon
complexation with TPP (Table 2). The net charge of TPP
was 22, 23, and 24 at pH 1.40, 3.20, and 7.35,
respectively, whereas SPD being a trivalent cation
Scheme 2. Reagents and conditions. (a) D
HCl (42%); (b) NaNO (0.9 equiv.), HBr, H
Et N, CHCl , rt, 3 h (12% for two steps, 84% recovery of 16); (d) 3, K
DMF, 80 8C, 14 h; (e) PhSH, K CO , DMF, rt, 2 h (68% for two steps); (f)
Boc) O, Et N, CH Cl , rt, 1 h (91%); (g) H , 20% Pd(OH) -C, MeOH, rt,
2 h (41%); (h) TFA–CH Cl , rt, 15 min, then HCl (78% for two steps).
2
, PtO
O, 0 8C, 17 h; (c) (Boc)
CO
2
, CH
3
COOD, rt, 48 h then
2
2
2
O,
3
3
2
3
,
2
3
(
1
2
3
2
2
2
2
2
2

5166
K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
these values in hand, we estimated the population of anti
and gauche rotamers as shown in Table 2.
The population of anti conformer was 86% around the C8–
C9 bond of SPD 3HCl. With respect to the C2–C3 bond of
butane, anti is known to be about 70%, while a pair of
gauche comprising about 30%. These findings indicate that
anti conformer of SPD C8/C9 is more populated than that of
alkane. This can be accounted for by the electrostatic
repulsion among three ammonium groups in SPD, which
force these cation sites to separate each other, resulting in
the extended conformation. On the other hand, when TPP
was present, the anti conformer decreased significantly
(Table 2). These results suggest that SPD tends to take a
bent conformation to interact with TPP. Actually, the
distance between nitrogen atoms in the propanylene part
(N6–N10) is about 0.5 nm in the extended conformation,
which is larger than the distance between neighbouring
oxygens of TPP (ca. 0.3 nm). Therefore, SPD needs to bend
its structure to arrange each ammonium group close to an
anionic site of TPP. Furthermore, when pH was raised from
1.40 to 7.35, the population of gauche rotamers markedly
increased. This may be accounted for by a notion that SPD
more firmly interacts with TPP as a tetravalent anion than a
divalent or trivalent form.
1
Figure 4. Partial H NMR spectra (500 MHz) of 1. NMR experiments were
measured with 25 mM of 1 as 3HCl salt in D O at pH 7.30 (A), and in the
presence of 100 mM tripolyphosphate (TPP) at pH 1.40 (B), and 100 mM
2
TPP at pH 3.20 (C), and 100 mM TPP at pH 7.35 (D). The spectra of 1 as
3HCl salt at pH 1.4 and 3.2 were virtually identical with (A).
1
6
throughout experiments. When compared with the HCl
3
salt, the spin coupling constant J
of the TPP salt
notably decreased. In neutral pH, where the net charge of
H-8,H-9
3
TPP becomes tetravalent, J
was the smallest, which
H-8,H-9
clearly indicated that SPD undergoes conformational
changes to increase the bent conformation (equal to gauche
rich).
2
.3. Conformation analysis
In flexible or acyclic molecules like SPD, staggered
conformers, where H–H dihedral angles are 60, 180 and
3
008, are predominant. These anti and gauche angles are not
4
greatly deviated unless bulky substituents are attached.
Thus, the conformation bearing intermediate angles, such as
skew conformations, is virtually negligible. When falling on
1
Table 2. Populations of rotamers obtained from H NMR experiments
3
J
H-8,H-9 (Hz)
Anti
Gauche (%)
3
intermediate values, JH,H values are not due to non-
staggered rotamers but due to the rapidly interconverting
anti and gauche rotamers. Thus, once the spin coupling
constants for the rotamers are obtained by using the Karplus
relation, the population ratio between anti and gauche
conformers can be determined. We attempted to use a
3þ
SPD –3HCl (pH 1–7)
9.8
9.6
9.0
8.5
86
84
78
73
14
16
22
27
3
SPD –TPP (pH 1.40)
þ
22
3
SPD –TPP (pH 3.20)
þ
32
3
þ
42
SPD –TPP (pH 7.35)
1
7
modified Karplus-type equation for this purpose. How-
ever, since this equation was derived from rigid cyclic
compounds, fluctuation from the staggered angles should be
taken into accounts before applying to flexible acyclic
systems. First, Boltzmann populations of rotamers around
most stable conformation were obtained using force field
3. Conclusions
p
p
We synthesized (1S ,2S )-1,8-diamino-4-azaoctane-
1,2,3,3,5,5,6,6,7,7,8,8-d , as a molecular probe for investi-
1
2
gating weak interaction by SPD and utilized it for
conformation analysis with polyanionic polytriphosphate.
The detailed examinations on proton–proton coupling
constants have disclosed that SPD undergoes conformation
change upon complexation with polyanion and this
interaction becomes firmer as the number of negative
charges in the polyphosphate increases from divalent to
tetravalent. The present results indicate for the first time that
the structure of the flexible molecule with rapidly alternat-
ing conformations can be quantitatively analyzed and
characterized by using diastereospecifically deuterated
analogues. This method would provide a powerful strategy
for investigating the molecular basis underlying weak
interactions among biomolecules. Currently, we are
examining the weak interaction between polyamine and
ATP using this method.
p18
19
calculations (OPLSA
essentially same manner as those in the previous study. The
force field, MacroModel ) in an
4
3
values of J
for flexible SPD thus obtained are shown in
H,H
Table 1. Since the difference between hydrogen and
deuterium is negligible for conformational potentials,
gauche1 and gauche2 should be equally populated. With
3
Table 1. Calculated JH,H for anti and gauche orientations in fluctuating
system corresponding to the C8–C9 bond of SPD
3
C7–N10 dihedral angle
JH-8,H-9 (Hz)
gauche1 (near 608)
anti (near 1808)
gauche2 (near 3008)
0.9
11.2
1.7

K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
5167
4
. Experimental
patterns are indicated as s, singlet; d, doublet; t, triplet; q,
quartet; quin, quintet; m, multiplet; br, broad peak. Mass
spectra (ESI) were recorded on a ThermoQuest LCQ DECA
instrument.
3
4
.1. Method for measuring JH,H
3
1
JH,H values were extracted from 1D H NMR spectra.
NMR spectra were obtained on a JEOL GSX-500
500 MHz) spectrometer. Digital resolution for H NMR
spectra is 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 40 8C. A sample of
p
4.3.1. (2S ,3S )-d-Valerolactone-2,3-d (5). A solution of
p
2
1
(
5,6-dihydro-2H-pyrane-2-one (4) (5.27 g, 53.7 mmol) and
Wilkinson catalyst (500 mg, 0.01 equiv.) in benzene (50 ml)
was stirred under 1 atm of deuterium atmosphere for 40 h.
The resulting precipitates were removed off by filtration
through the florisil pad, and the filtrate containing the
volatile material was concentrated carefully in vacuo to give
lactone 4 as a colorless oil. The purity was estimated to be
1
was dissolved in D O as a solvent. The concentration of
2
SPD and TPP was prepared by 25 and 100 mM,
respectively. The pH was adjusted to 1.4, 3.2, and 7.3 by
deuterium chloride and sodium deuteroxide. The chemical
shifts were recorded from 3-(trimethylsilyl)-1-propane-
sulfonic acid, sodium salt (DSS).
1
3
.95% by C NMR analysis, and this material was
employed without further purification: R 0.16 (hexane–
f
ethyl acetate 3:1); dH (500 MHz; CDCl ) 4.27 (2H, t,
3
J5,4¼5.5 Hz, 5-H), 2.40 (1H, br d, 2-H), 1.82 (1H, br q,
4
.2. Determination of anti/gauche populations
3-H), 1.78 (2H, q, J ¼5.5 Hz, J ¼7.0 Hz, 4-H); d
4
,5
4,3
C
(
125 MHz; CDCl ) 171.1 (C1), 69.2 (C5), 29.3 (t, C2), 22.1
3
3
þ
JH,H for the C8–C9 bond of 1 with conformational
fluctuation were calculated as follows. The energies (Ei)
of 360 conformers (total number, N) were obtained for each
(C4), 18.5 (t, C3); MS (ESI) m/z 103 [MþH] ; calcd for
þ
C H D O [MþH] 103.
5
6 2 2
p
p
1
8 in the dihedral angle along the C8–C9 bond using
4.3.2. (2S ,3S )-5-Hydroxypentanoylhydrazide-2,3-d
2
p
OPLSA force field on MacroModel. The calculations were
performed with taking account of the solvent effect of water
as a dielectric constant. These values were substituted for
(6). Hydrazine monohydrate (3.9 ml, 80.6 mmol) was
added to a solution of the crude lactone 5 (ca. 53.7 mmol)
in methanol (200 ml), and the mixture was heated at reflux
for 2 h. The solvent was removed under reduced pressure
and the residue was purified by recrystallization from
methanol/diethyl ether to give hydrazide 6 (6.92 g,
the formula (1) and Boltzmann population P was obtained.
i
expð2E =RTÞ
i
Pi ¼
ð1Þ
N
X
5
8
1.6 mmol, 96% yield from 4) as colorless prisms: mp
9–91 8C; Rf 0.083 (chloroform–methanol 9:1); dH
expð2E =RTÞ
i
i
(
500 MHz; CD OD) 3.54 (2H, t, J ¼6.5 Hz, 5-H), 2.16
3
5,4
3
A spin coupling constant J in conformer i is given by
i
substituting the dihedral angle between H8 and H9 for the
7
modified Karplus equation.
(1H, d, J2,3¼8.0 Hz, 2-H), 1.64 (1H, q, J3,2¼8.0 Hz,
J
4-H); d
3,4¼7.5 Hz, 3-H), 1.52 (2H, q, J4,5¼6.5 Hz, J3,4¼7.5 Hz,
1
(125 MHz; CD
OD) 174.9 (C1), 62.3 (C5), 34.3 (t,
C
3
þ
C2), 32.9 (C4), 22.8 (t, C3); MS (ESI) m/z 135 [MþH] ;
N
þ
X
calcd for C H D N O [MþNa] 135.
3
3
P J
5
10 2 2 2
JH;H
¼
ð2Þ
i
i
i
p
.3.3. (3S ,4S )-4-(tert-Butoxycarbonylamino)butanol-
p
4
3,4-d
The averaged values for gauche1 608, anti 1808, and
gauche2 3008 in Table 1 were obtained from formula (2) for
the dihedral angles between 0 and 1208, those between 120
and 2408, and those between 240 and 3608, respectively.
2
(7). A solution of sodium nitrite (5.34 g,
77.4 mmol) in water (50 ml) was slowly added to a solution
of the hydrazide 6 (6.92 g, 51.6 mmol) in 0.5 M HCl
(400 ml) at 0 8C over 30 min. The reaction mixture was
extracted with diethyl ether, and the combined organic
4
.3. Synthesis of labeled spermidine
layers were washed with saturated aqueous NaHCO
over Na SO , concentrated in vacuo to give a yellow oil,
, dried
3
2
4
All reactions sensitive to air or moisture were carried out
under argon atmosphere in dry, freshly distilled solvents
under anhydrous conditions, unless otherwise noted. THF
was distilled sodium/benzophenone, diethyl ether (Et O)
2
from LiAlH , and DMF, and DMSO from calcium hydride
4
which was dissolved in toluene (300 ml) and heated at reflux
for 1 h. While still hot, concentrated HCl (10 ml) and
2-propanol (90 ml) were poured into the solution and heated
at reflux for 10 h, then toluene was removed in vacuo.
Ethanol (50 ml) was added to the flask and then distilled off.
This process was repeated twice to obtain crude amino-
alcohol as a cream-yellow oil (2.26 g, 17.7 mmol), which
was dissolved in tetrahydrofuran (150 ml) and water
(50 ml). Triethylamine (4.5 ml, 32.2 mmol) was added to
the solution, followed by addition of di-tert-butoxy-
dicarbonate (6.3 ml, 26.6 mmol). After stirring for 10 h,
the reaction mixture was extracted with diethyl ether, and
the combined organic layers were washed with brine, dried
under reduced pressure. Dichloromethane (CH Cl ) and
2
2
toluene was dried over activated MS4A. All other reagents
were used as supplied unless otherwise stated. Analytical
thin-layer chromatography (TLC) was performed using
E. Merck Silica gel 60 F254 pre-coated plates. Column
chromatography was performed using 100–210 mm Silica
Gel 60N (Kanto Chemical), and for flash column chroma-
1
tography 40–63 mm Silica Gel 60N (Merck) was used. H
1
and C NMR spectra were recorded on a JEOL GSX-500
3
over Na
chromatography on silica gave 7 (1.48 g, 7.74 mmol, 15%
from 6) as a colorless oil: R 0.21 (hexane–ethyl acetate
1:1); d (500 MHz; CDCl ) 4.57 (1H, br s, NH), 3.64 (2H, t,
SO , filtered, and concentrated in vacuo. Column
2 4
(
(
500 MHz) spectrometer. Chemical shifts are reported in d
ppm) using residual CHCl as an internal standard of d 7.26
3
13
f
1
and d 77.00 for H and C NMR, respectively. Signal
H
3

5168
K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
J1,2¼6.0 Hz, 1-H), 3.13 (1H, br d, 4-H), 1.55–1.46 (3H, m,
-H, 2-H), 1.41 (9H, s, Boc); d (125 MHz; CDCl ) 156.0
solution of the crude aldehyde in tert-butanol (10 ml) and
water (5 ml), subsequently sodium chlorite (990 mg,
9.96 mmol) was added in portionwise. After stirring for
1 h, the reaction mixture was poured into saturated aqueous
NaCl, and extracted with ethyl acetate, dried over Na SO ,
3
C
3
(
(
Boc), 79.2 (Boc), 62.5 (C1), 40.0 (t, C4), 29.7 (C2), 28.5
þ
Boc), 26.2 (t, C3); MS (ESI) m/z 214 [MþNa] ; calcd for
þ
C H D NO [MþNa] 214.
9
17
2
3
2
4
and concentrated in vacuo. Column chromatography on
silica (hexane–ethyl acetate 3:1) gave 10 (670 mg, 91%
p
.3.4. (3S ,4S )-4-(Benzylamino)butanol-3,4-d (8). TFA
p
4
2
(
CH Cl (16 ml). After stirring for 10 min, the solvent was
8 ml) was added to a solution of 7 (860 mg, 4.50 mmol) in
from 9) as a colorless oil: R 0.080 (hexane–ethyl acetate
f
3:1); d (500 MHz; CDCl ) 7.25–7.15 (5H, m, Bn), 4.40
3
2
2
H
removed in vacuo. Benzaldehyde (551 ml, 5.4 mmol) and
triethylamine (1.9 ml, 13.5 mmol) were added sequentially
to a solution of the residue in methanol (10 ml). After
(2H, br, Bn), 3.23–3.16 (1H, br d, 4-H), 2.31 (2H, br, 2-H),
1.79 (1H, br, 3-H), 1.48–1.43 (9H, br, Boc); d (125 MHz;
CDCl ) 178.3 (C1), 155.8 (Boc), 138.1 (Bn), 128.3 (Bn),
3
C
stirring for 10 min, anhydrous MgSO was added and the
4
127.6 (Bn), 127.1 (Bn), 80.1 (Boc), 50.1 (br d, Bn), 45.2 (t,
C4), 31.1 (C2), 28.4 (Boc), 22.6 (t, C3); MS (ESI) m/z 318
resulting suspension was stirred for 1 h. The reaction
mixture was cooled to 0 8C, and treated with sodium
borohydride (2.04 g, 54 mmol). After stirring for 2 h, the
reaction was quenched with water, and methanol was
removed in vacuo. The residue was extracted with diethyl
ether, and combined organic layers were washed with water,
dried over Na SO , filtered, and concentrated in vacuo to
þ
þ
[MþNa] ; calcd for C H D NO [MþNa] 318.
1
6
21
2
4
p
p
4.3.7. Methyl(3S ,4S )-4-(benzyl-tert-butoxycarbonyl-
amino)butanoate-3,4-d2 (11). Methyl iodide (226 ml,
3.63 mmol) was added to a mixture of 10 (670 mg,
2.27 mmol) and NaHCO (381 mg, 4.54 mmol) in DMF
3
2
4
give 8 as a colorless oil. The purity was estimated to be
1
(10 ml). After stirring for 14 h, the reaction mixture was
poured into water, and extracted with ethyl acetate. The
combined organic layers were washed with saturated
aqueous NaCl, dried over Na SO , and concentrated in
3
.
employed without further purification: R 0.18 (chloro-
95% by C NMR analysis, and this material was
f
form–methanol–isopropylamine 45:5:1); dH (500 MHz;
CDCl ) 7.28–7.13 (5H, m, Bn), 3.71 (2H, s, Bn), 3.52
2
4
vacuo. Column chromatography on silica (hexane–ethyl
acetate 3:1) gave 11 (597 mg, 85%) as a yellow oil: R 0.39
3
(
(
(
2H, t, J1,2¼4.8 Hz, 1-H), 2.63 (1H, br d, 4-H), 1.61–1.57
f
3H, m, 2-H, 3-H); d (125 MHz; CDCl ) 140.4 (Bn), 129.4
3
(hexane–ethyl acetate 3:1); dH (500 MHz; CDCl ) 7.25–
3
C
Bn), 129.3 (Bn), 128.0 (Bn), 62.7 (C1), 54.4 (Bn), 49.8 (t,
þ
7.15 (5H, m, Bn), 4.37 (2H, br d, Bn), 3.59 (3H, s, OMe),
3.19–3.11 (1H, br d, 4-H), 2.21 (2H, br, 2-H), 1.73 (1H, br,
3-H), 1.48–1.43 (9H, br d, Boc); dC (125 MHz; CDCl3)
173.2 (C1), 155.8 (Boc), 138.1 (Bn), 128.2 (Bn), 127.4 (Bn),
126.9 (Bn), 79.6 (Boc), 51.4 (OMe), 50.0 (br d, Bn), 45.2 (t,
C4), 31.7 (C2), 27.2 (t, C3); MS (ESI) m/z 182 [MþH] ;
þ
calcd for C H D NO [MþH] 182.
1
1 15 2
p
p
.3.5. (3S ,4S )-4-(Benzyl-tert-butoxycarbonylamino)
4
butanol-3,4-d (9). Triethylamine (1.9 ml, 13.5 mmol) and
C4), 31.0 (C2), 28.3 (Boc), 22.7 (t, C3); MS (ESI) m/z 332
þ
2
þ
di-tert-butoxy-dicarbonate (1.2 ml, 4.95 mmol) was added
sequentially to a solution of 8 (ca. 4.50 mmol) in CH Cl
[MþNa] ; calcd for C H D NO [MþNa] 332.
1
7
23
2
4
2
2
p
4.3.8. (3S ,4S )-4-(Benzyl-tert-butoxycarbonylamino)
p
(
20 ml). After stirring for 2 h, the reaction mixture was
poured into water, and extracted with diethyl ether. The
combined organic layers were washed with saturated
aqueous NaCl, dried over Na SO , and concentrated in
butanoic acid-2,2,3,4-d (12). A solution of 11 (550 mg,
2
1.78 mmol) sodium methoxide (144 mg, 2.67 mmol) in
methanol-d (10 ml, 246 mmol) was heated at reflux for
2
4
1
vacuo. Column chromatography on silica (hexane–ethyl
acetate 1:1) gave 9 (1.10 g, 87% from 7) as a colorless oil:
R 0.69 (chloroform–methanol 9:1); d (500 MHz; CDCl )
50 h. The solvent was removed in vacuo and the residue was
diluted with 1 M deuterium chloride in D O (18 ml, 99 atom
2
% D), extracted with ethyl acetate, dried over Na SO , and
4
f
H
3
2
7
1
2
.25–7.15 (5H, m, Bn), 4.34 (2H, br, Bn), 3.53 (2H, br,
-H), 3.16–3.08 (1H, br d, 4-H), 1.41–1.36 (12H, br m,
-H, 3-H, Boc); d (125 MHz; CDCl ) 155.8 (Boc), 138.3
concentrated in vacuo to afford 12 (496 mg, 94%) as a
colorless oil. The material was employed without further
purification: Rf 0.080 (hexane–ethyl acetate 3:1); dH
C
3
(
(
Bn), 128.3 (Bn), 127.2 (Bn), 127.0 (Bn), 79.8 (Boc), 62.1
C1), 50.5 (Bn), 46.0 (t, C4), 29.6 (C2), 28.5 (Boc), 23.8 (t,
(500 MHz; CDCl ) 7.25–7.15 (5H, m, Bn), 4.40 (2H, br,
3
Bn), 3.23–3.16 (1H, br d, 4-H), 1.79 (1H, br, 3-H), 1.48–
1.43 (9H, br, Boc); d (125 MHz; CDCl ) 178.3 (C1), 155.8
þ
C3); MS (ESI) m/z 304 [MþNa] ; calcd for C H D NO
1
6
23
2
3
C
3
þ
[
MþNa] 304.
(Boc), 138.1 (Bn), 128.3 (Bn), 127.6 (Bn), 127.1 (Bn), 80.1
(Boc), 50.1 (br d, Bn), 45.2 (t, C4), 30.6 (quin, C2), 28.4
p
p
þ
4.3.6. (3S ,4S )-4-(Benzyl-tert-butoxycarbonylamino)
butanoic acid-3,4-d (10). Pyridine sulfur trioxide complex
(Boc), 22.6 (t, C3); MS (ESI) m/z 320 [MþNa] ; Calcd for
þ
C H D NO [MþNa] 320.
2
16 19
4
4
(
2
3.17 g, 19.9 mmol) was added to a solution of 9 (700 mg,
.49 mmol) and triethylamine (5.2 ml, 37.4 mmol) in
dimethyl sulfoxide (14 ml, 199 mmol). After stirring for
0 min, the reaction mixture was poured into water, and
p
p
4.3.9. (1S ,2S )-1-(Benzyl-tert-butoxycarbonylamino)-
3-(benzyloxycarbonylamino)propane-1,2,3,3-d4 (13).
3
Triethylamine (279 ml, 2.00 mmol) and diphenyl phosphor-
azidate (431 ml, 2.00 mmol) were added to a solution of 12
(496 mg, 1.67 mmol) in anhydrous toluene (10 ml), and
heated at reflux for 1 h. Benzylalcohol (259 ml, 2.51 mmol)
was added to the mixture, and heated at reflux for 22 h. The
solvent was removed in vacuo, and the residue was
dissolved in ethyl acetate. The solution was washed
sequentially with 1 M HCl, water, saturated aqueous
extracted with diethyl ether. The combined organic layers
were washed sequentially with saturated aqueous NH Cl,
saturated aqueous NaHCO , saturated aqueous NaCl, dried
3
4
over Na SO , and concentrated in vacuo to give a yellow
4
2
oil. The crude aldehyde was immediately subjected to the
next reaction. 2-Methyl-2-butene (1.3 ml, 12.5 mmol) and
NaH PO –2H O (775 mg, 4.98 mmol) was added to a
2
4
2

K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
5169
NaHCO , saturated aqueous NaCl, dried over Na SO , and
3
colorless prisms: R 0 (chloroform–methanol 9:1); dC
f
2
4
concentrated in vacuo. Column chromatography on silica
hexane–ethyl acetate 3:1) gave 13 (511 mg, 76%) as a
yellow oil: Rf 0.31 (hexane–ethyl acetate 3:1); dH
(125 MHz; D O) 39.3 (quin, C1, C4), 23.9 (quin, C2, C3);
2
þ
(
MS (ESI) m/z 97 [M22Cl–H] ; calcd for C H D Cl N
4
6
8
2
2
þ
[M22Cl–H] 97.
(
500 MHz; CDCl ) 7.31–7.14 (10H, m, Cbz, Bn), 5.05
3
(
1
2H, s, Cbz), 4.40 (2H, br, Bn), 3.23–3.11 (1H, br, 1-H),
.55–1.42 (10H, br, 2-H, Boc); d (125 MHz; CDCl ) 156.4
4.3.13. 4-(tert-Butoxycarbonylamino)butylbromide-
1,1,2,2,3,3,4,4-d (2). Diammonium chloride (16) (4.91 g,
C
3
6
(
Cbz), 155.9 (Boc), 138.0 (Bn), 136.4 (Cbz), 128.3 (Bn,
Cbz), 127.3 (Cbz), 127.0 (Bn), 126.9 (Bn), 79.8 (Boc), 66.7
24.8 mmol) was dissolved in 1.5 M HBr (100 ml) at 0 8C.
After short stirring, a solution of sodium nitrite (1.54 g,
22.3 mmol) in water (25 ml) was added in dropwise to the
reaction mixture with stirring at 0 8C over a period of 1 h.
Stirring was continued at room temperature for 16 h.
Evaporation of the solvent gave crude bromide 17 as a
yellow solid which was used in the next step without further
purification. A solution of 17 in methanol (50 ml) was added
dropwise to a solution of di-tert-butoxy-dicarbonate (7.1 ml,
29.7 mmol) and triethylamine (34 ml, 248 mmol) in CHCl3
(100 ml) over a period of 1 h at 0 8C. After stirring for 2 h at
room temperature, the solvent was removed in vacuo. Water
(
(
Cbz), 50.0 (br d, Bn), 43.7 (t, C1), 37.1 (quin, C3), 28.2
þ
Boc), 26.9 (t, C2); MS (ESI) m/z 425 [MþNa] ; calcd for
þ
C H D N O [MþNa] 425.
2
3 26 4 2 4
p
p
4
3
.3.10. (1S ,2S )-1-(Benzyl-tert-butoxycarbonylamino)-
-aminopropane-1,2,3,3-d (14). 20% Pd(OH) on char-
4
2
coal (100 mg) was added to a solution of 13 (511 mg,
.27 mmol) in methanol (5 ml), and the mixture was stirred
1
under 1 atm of hydrogen atmosphere for 14 h. The catalyst
was removed by filtration through a celite pad, and the
filtrate was concentrated in vacuo. Column chromatography
on silica (chloroform–methanol–isopropylamine 45:5:1)
gave 14 (331 mg, 97%) as a colorless oil: Rf 0.18
was added to the residue, and extracted with CHCl , dried
3
over Na SO , and concentrated in vacuo. Column chroma-
2
4
tography on silica (hexane–ethyl acetate 3:1) gave 2
(774 mg, 12% for two steps, 84% recover of 16) as a
colorless amorphous: R 0.59 (hexane–ethyl acetate 3:1); dH
(
chloroform–methanol–isopropylamine
500 MHz; CDCl ) 7.25–7.15 (5H, m, Bn), 4.34 (2H, br,
45:5:1);
dH
(
Bn), 3.19–3.10 (1H, br d, 3-H), 1.52–1.37 (10H, br m, 2-H,
3
f
(500 MHz; CDCl ) 1.39 (9H, s, Boc); d (125 MHz; CDCl )
3
C
3
Boc); d (125 MHz, CDCl ) 178.3 (C1), 155.7 (Boc), 138.1
C
155.8 (Boc), 79.3 (Boc), 38.8 (quin, C4), 32.4 (quin, C1),
28.7 (quin, C3), 28.4 (Boc), 27.6 (quin, C2); MS (ESI) m/z
3
(
(
(
Bn), 128.2 (Bn), 127.4 (Bn), 126.9 (Bn), 79.7 (Boc), 49.8
br d, Bn), 43.0 (t, C1), 37.9 (quin, C3), 30.6 (t, C2), 28.3
þ
þ
282 [MþNa] ; calcd for C H D BrNO [MþNa] 282.
9
10
8
2
Boc); MS (ESI) m/z 269 [MþH]þ; calcd for
þ
p
p
C H D N O [MþH] 269.
4.3.14. (1S ,2S )-1-(Benzyl-tert-butoxycarbonylamino)-
-(tert-butoxycarbonylamino)-4-azaoctane-1,2,3,3,5,
1
5 20 4 2 2
8
p
.3.11. (1S ,2S )-1-(Benzyl-tert-butoxycarbonylamino)-
p
4
5,6,6,7,7,8,8-d12 (19). Bromide 2 (180 mg, 0.692 mmol)
was added to a mixture of diamine 3 (209 mg, 0.461 mmol)
and K CO (319 mg, 2.31 mmol) in DMF (5 ml), and the
3-[(2-nitrobenzenesulfonyl)amino]-1,3-propane-1,2,3,3-
d4 (3). Triethylamine (78 ml, 0.559 mmol) and 2-nitro-
benzenesulfonyl chloride (124 mg, 0.559 mmol) were
2
3
mixture was stirred for 14 h at 80 8C. After cooling, K CO
2 3
sequentially added to
0
a
solution of 14 (125 mg,
.466 mmol) in CH Cl (5 ml). After stirring for 16 h, the
(127 mg, 0.922 mmol) and thiophenol (141 ml, 1.38 mmol)
were added to the reaction mixture, and stirred for 2 h. The
reaction mixture was poured into water, and extracted with
CHCl . The combined organic layers were washed with
3
saturated aqueous NaHCO , saturated aqueous NaCl, dried
3
2
2
reaction mixture was poured into water, and extracted with
CH Cl . The combined organic layers were washed with
saturated aqueous NaCl, dried over Na SO , and concen-
2
2
2
4
trated in vacuo. Column chromatography on silica (hexane–
ethyl acetate 3:1) gave 3 (209 mg, 99%) as a yellow
amorphous: Rf 0.24 (hexane–ethyl acetate 3:1); dH
over Na SO , and concentrated in vacuo. Column chroma-
2
4
tography on silica (chloroform–methanol–isopropylamine
45:5:1) gave 19 (140 mg, 68% for two steps) as a colorless
oil: Rf 0.14 (chloroform–methanol–isopropylamine
(
500 MHz; CDCl ) 8.01 (1H, m, Ns), 7.80 (1H, m, Ns),
3
7
.65 (2H, m, Ns), 7.24–7.14 (5H, m, Bn), 5.36 (1H, br d,
NH), 4.30 (2H, br, Bn), 3.19 (1H, br, 1-H), 1.55–1.36 (10H,
45:5:1); dH (500 MHz; CDCl ) 7.25–7.16 (5H, m, Bn),
3
4.37 (2H, br, Bn), 3.20–3.09 (1H, br d, 1-H), 1.46–1.35
(19H, br, 2-H, Boc); d (125 MHz; CDCl ) 155.8 (Boc),
br, 2-H, Boc); d (125 MHz; CDCl ) 156.0 (Boc), 147.6
C
3
C
3
(
(
(
Ns), 137.8 (Bn), 133.2 (Ns), 132.4 (Ns), 130.3 (Ns), 128.3
Bn), 127.0 (Bn), 126.9 (Bn), 124.8 (Ns), 80.1 (Boc), 50.3
br d, Bn), 42.6 (t, C1), 40.1 (quin, C3), 28.2 (Boc), 27.6 (t,
155.7 (Boc), 138.1 (Bn), 128.2 (Bn), 127.4 (Bn), 126.9 (Bn),
78.7 (Boc), 78.0 (Boc), 50.0 (br d, Bn), 48.3 (quin, C3), 44.9
(quin, C5), 43.4 (t, C1), 39.5 (quin, C8), 28.3 (Boc), 27.9
(quin, C6), 26.8 (t, C2), 25.8 (quin, C7); MS (ESI) m/z 448
C2); MS (ESI) m/z 476 [MþNa]þ; calcd for
þ
þ
þ
C H D N O S [MþNa] 476.
[MþH] ; calcd for C H D N O [MþH] 448.
2
1
23
4
3
6
24 29 12 3 4
p
p
4
.3.12. 1,4-Butyldiammonium chloride-1,1,2,2,3,3,4,4-d8
16). Platinum (IV) oxide (200 mg, 0.881 mmol) was added
4.3.15. (1S ,2S )-1-(Benzyl-tert-butoxycarbonylamino)-
8-(tert-butoxycarbonylamino)-4-(tert-butoxycarbonyl)-
4-azaoctane-1,2,3,3,5,5,6,6,7,7,8,8-d12 (20). Compound 20
was obtained (91%) as a colorless oil from 19 by an
(
to a solution of succinonitrile-d (15) (5.00 g, 59.4 mmol) in
4
acetic acid-d (20 ml, 350 mmol, 99.5 atom%), and stirred
under 1 atm of deuterium atmosphere for 48 h. The reaction
mixture was filtered through a celite pad, and the filtrate was
concentrated in vacuo. Concentrated HCl was added to the
residue, and then the solvents were distilled off. This
process was repeated twice to obtain crude diamine.
Recrystallized from ethanol gave 16 (4.91 g, 42%) as
analogous procedure described for 9: R 0.77 (chloroform–
f
methanol–isopropylamine 45:5:1); dH (500 MHz; CDCl3)
7.25–7.15 (5H, m, Bn), 4.35 (2H, br, Bn), 3.10–3.01 (1H,
br d, 1-H), 1.42–1.36 (28H, br, 2-H, Boc); d (125 MHz;
C
CDCl ) 155.8 (Boc), 138.0 (Bn), 128.1 (Bn), 127.2 (Bn),
3
126.8 (Bn), 78.7 (Boc), 50.1 (br d, Bn), 44.4 (t, C1), 44.2

5170
K. Maruyoshi et al. / Tetrahedron 60 (2004) 5163–5170
(
(
[
quin, C3), 43.0 (quin, C5), 39.5 (quin, C8), 28.2 (Boc), 27.3
quin, C7), 27.0 (quin, C6), 26.4 (t, C2); MS (ESI) m/z 570
References and notes
þ
þ
MþNa] ; calcd for C H D N O [MþNa] 570.
1. Igarashi, K.; Kashiwagi, K. J. Biol. Chem. Biophys. 1991, 266,
2
2
9 37 12 3 6
0803–20809.
p
p
0
4
.3.16. (1S ,2S )-1,8-Bis(N,N -tert-butoxycarbonyl-
2. Igarashi, K.; Kashiwagi, K. Biochem. Biophys. Res. Commun.
2000, 271, 559–564.
amino)-4-(tert-butoxycarbonyl)-4-azaoctane-1,2,3,3,5,
,6,6,7,7,8,8-d12 (21). Compound 21 was obtained (41%)
from 20 by an analogous procedure described for 14: R 0.17
5
3. McCormack, S. A.; Johnson, L. R. J. Physiol. Pharmacol.
2001, 52, 327–349.
f
(
(
(
(
(
hexane–ethyl acetate 3:1); dH (500 MHz; CDCl ) 3.12
3
4. Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J. Org. Chem. 1999, 64, 866–876.
5. Murata, M.; Matsumori, N.; Tachibana, K. Nippon Kagaku
Kaishi 1997, 11, 749–757.
1H, br d, 1-H), 1.45–1.36 (28H, br, 2-H, Boc); dC
125 MHz; CDCl ) 47.2 (quin, C5), 44.9 (quin, C3), 39.1
3
quin, C8), 37.3 (t, C1), 25.1 (quin, C7), 24.0 (t, C2), 22.6
þ
quin, C6); MS (ESI) m/z 480 [MþNa] ; calcd for
6
7
8
9
. Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T.
þ
C H D N O [MþNa] 480.
2
2 31 12 3 6
Tetrahedron Lett. 1997, 38, 5831–5834.
. Fukuyama, T.; Cheung, M.; Kan, T. Synlett 1999, 8,
p
p
4
5
.3.17. (1S ,2S )-1,8-Diamino-4-azaoctane-1,2,3,3,5,
,6,6,7,7,8,8-d12 (SPD-2,2,3,3,4,4,5,5,7,7,8,9-d12) (22).
1
301–1303.
. Gungor, M.; Jardine, F. H.; Wheatley, J. D. Polyhedron 1988,
, 1827–1829.
. Djerassi, D.; Gutzwiller, J. Am. Chem. Soc. 1966, 88,
TFA (1 ml) was added to a solution of 21 (53 mg,
.117 mmol) in CH Cl (3 ml). After stirring for 15 min, the
7
0
2
2
solvent was removed in vacuo. The residue was dissolved in
M HCl, and the solvent was distilled off. This process was
repeated twice to obtain crude product as a yellow solid.
4
537–4538. Morandi, J.; Jensen, H. B. J. Org. Chem. 1969,
4, 1889–1891.
1
3
1
0. Reddy, V. K.; Valansinas, A.; Sarkar, A.; Basu, H. S.; Marton,
L. J.; Frydman, B. J. Med. Chem. 1998, 41, 4723–4732.
1. MacMillan, J.; Willis, C. L. J. Chem. Soc. Perkin Trans. 1984,
Recrystallization from ethanol gave 22 (24 mg, 78%) as a
colorless powder: mp 252–254 8C; R 0 (chloroform–
methanol 9:1); dH (500 MHz; D O) 3.12 (1H, d,
2
f
1
3
51–355. Beale, M. H.; MacMillan, J. J. Chem. Soc. Perkin 1
980, 877–883.
2. Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974, 30,
J1,2¼9.8 Hz, 1-H), 2.11 (1H, d, J ¼9.8 Hz, 2-H); d
2,1
C
1
(
(
(
125 MHz; D O) 47.2 (quin, C5), 44.8 (quin, C3), 39.2
2
1
quin, C8), 37.3 (t, C1), 24.7 (quin, C7), 24.1 (t, C2), 22.6
þ
2151–2157.
quin, C6); MS (ESI) m/z 158 [M23Cl–2H] ; calcd for
þ
13. Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett.
995, 36, 6373–6374.
4. Smith, R. G.; Daves, G. D., Jr. J. Org. Chem. 1978, 43,
178–2183.
C H D Cl N [M23Cl–2H] 158. Anal. calcd for C7-
7
10 12
3 3
1
H D Cl N ·H O: C, 29.53; HþD, 7.96; N, 14.76. Found:
1
0
12
3
3
2
1
1
1
1
1
1
C, 29.12; HþD, 7.75; N, 14.24.
2
5. Di Paolo, M. L.; Corazza, A.; Scarpa, M.; Stevanato, R.; Rigo,
A. Biochim. Biophys. Acta 1995, 1247, 246–252.
6. Labadi, I.; Jenei, E.; Lahti, R.; Lonnberg, H. Acta Chemica
Scand. 1991, 45, 1055–1059.
Acknowledgements
We are grateful to Prof. Kazuei Igarashi of Chiba University
for creating an opportunity for this polyamine study, to Prof.
Tohru Fukuyama, The University of Tokyo, for suggestions;
to Yukihiro Yamada and Kaori Nonaka in our laboratory,
for their help in synthesizing labeled SPD; and to Prof.
Koichi Ute and Iichiro Fukuda, Osaka University, for their
help in NMR experiments at Venture Business Laboratory;
and to Mr. Seiji Adachi, Osaka University, for measure-
ments of NMR spectra.
7. Haasnoot, C. A. G.; De Leeuw, F. A. A. M.; Altona, C.
Tetrahedron 1980, 36, 2783–2792.
8. Jorgensen, W. L.; Tirado-Rives, J. Am. Chem. Soc. 1988, 110,
1
657–1666.
9. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C.
J. Comput. Chem. 1990, 11, 440.