Determination of the structure and its absolute configuration of 2″-hydroxynicotianamine, an inhibitor against Angiotensin-I converting enzyme in buckwheat, through the total synthesis

Article abstract of DOI:10.3987/COM-10-11931

Nicotianamine is known as an inhibitor against Angiotensin-I Converting Enzyme (ACE). We synthesized a new nicotianamine derivative with an additional hydroxy group isolated from buckwheat (Fagopyrum esculentum Moench) powder and determined its regio and stereochemistry unambiguously by the enantioselective synthesis of diastereomers.

Full text of DOI:10.3987/COM-10-11931

HETEROCYCLES, Vol. 81, No. 6, 2010
1435
HETEROCYCLES, Vol. 81, No. 6, 2010, pp. 1435 - 1444. © The Japan Institute of Heterocyclic Chemistry
Received, 25th February, 2010, Accepted, 8th April, 2010, Published online, 9th April, 2010
DOI: 10.3987/COM-10-11931
DETERMINATION OF THE STRUCTURE AND ITS ABSOLUTE
CONFIGURATION
OF
2”-HYDROXYNICOTIANAMINE,
AN
INHIBITOR AGAINST ANGIOTENSIN-I CONVERTING ENZYME IN
BUCKWHEAT, THROUGH THE TOTAL SYNTHESIS
Keisuke Yoshikawa,a,* Hidenori Watanabe,b Yasuo Aoyagi,c and Takeshi
Kitaharab,d
a
Technical Research Institute, R&D Center, T. Hasegawa Co., Ltd., 29-7
Kariyado, Nakahara-ku, Kawasaki city, 211-0022, Japan
E-mail: keisuke_yoshikawa@t-hasegawa.co.jp
b
Department of Applied Biological Chemistry, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan
c Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado, Saitama 350-0288, Japan
d
School of Pharmacy, Teikyo Heisei University, 2289 Uruido, Ichihara city,
Chiba 290-0436, Japan
Abstract – Nicotianamine is known as an inhibitor against Angiotensin-I
Converting Enzyme (ACE). We synthesized a new nicotianamine derivative with
an additional hydroxy group isolated from buckwheat (Fagopyrum esculentum
Moench) powder and determined its regio and stereochemistry unambiguously by
the enantioselective synthesis of diastereomers.
INTRODUCTION
In 2006, Aoyagi (one of the authors) reported the isolation of a compound with ACE inhibitory activity
from buckwheat (Fagopyrum esculentum Moench) powder.1 The compound was supposed to be
hydroxylated nicotianamine from its analytical data and its proposed structure is shown in Figure 1.
Although its MS spectral data suggested the position of hydroxy group to be C-2” as depicted below, it
has not been completely clear and its absolute stereochemistry has also been unidentified yet.

1436
HETEROCYCLES, Vol. 81, No. 6, 2010
CO2H
N
CO2H
CO2H
NH2
2"
OH
N
H
Figure 1. Proposed structure of the compound identified from buckwheat powder
During the course of our continuing study on phytosiderophores,2 we have been interested in
structure-activity relationship on nicotianamine and related analogs. Therefore, we tried to synthesize
both diastereomers of the proposed structure not only to confirm the position of the hydroxy group of this
compound including the stereochemistry but also to study biological activities of this natural product and
congeners.
Our synthetic plan is shown below. The protected product 2 could be obtained from amine 3 and aldehyde
4 via reductive amination. Preparation of amine unit 3 must be obtainable according to the procedure
previously reported by us.2a Both diastereomers of aldehyde 4 could be synthesized from methyl
cinnamate 5 or cinnamyl alcohol 6 by Shioiri's methods.3,4
CO2H
CO2H
CO2But
N
CO2H
CO2But
CO2But
*
*
N
N
H
NH2
N
H
NHBoc
OMOM
OH
1
2
CO2Me
OH
CO2But
NHBoc
CO2But
NH2
5
6
CHO
ButO2C
*
N
OMOM
3
4
Scheme 1. Retro-synthetic plan of 2”-hydroxynicotianamine
RESULTS AND DISCUSSION
The amine unit (3) was synthesized as shown in Scheme 2. We have already reported a preparation of the
similar unit with different protective groups in 1998.2a In the present work, protective group of the amine
moiety was replaced with Cbz and the series of reactions were performed smoothly to give amine unit 3
in a similar manner as before.

HETEROCYCLES, Vol. 81, No. 6, 2010
1437
ButO2C
ButO2C
CO2But
N
O
CO2But
S
CO2But
NHCbz
CO2But
NHCbz
a
b
N
NHCbz
N
7
8
9
CO2But
CO2But
NH2
c
N
3
Scheme 2. Reagents and conditions: a) Lawesson's reagent, toluene, 90 °C, 93.7%; b) Raney-Ni, THF, rt,
75.8%; c) Pd-C, H2, EtOAc, rt, 89.2%.
Synthesis of (2”S)-1 was carried out as shown in Scheme 3. The key intermediate 10 was prepared from
cinnamyl alcohol 6 by using Shioiri’s method.3 Swern oxidation of the alcohol 10 afforded crude
aldehyde (2R)-4, which was submitted to the reductive amination with amine unit 3 to give the product
with whole assembly. Finally, treatment with TFA, followed by Dowex purification and recrystallization
from EtOH-H2O gave pure (2”S)-hydroxynicotianamine [(2”S)-1].
NHBoc
NHBoc
ref. 3
a
2
CHO
ButO2C
OH
ButO2C
OH
OMOM
OMOM
6
10
(2"R)-4
CO2But
CO2H
CO2H
CO2H
N
CO2But
CO2But
c
b
2"
2"
N
H
NH2
N
N
NHBoc
OMOM
H
OH
(2"S)-2
(2"S)-1
Scheme 3. Reagents and conditions: a) (COCl)2, DMSO, CH2Cl2 then Et3N, 78 °C to rt; b) 3,
NaBH3CN, AcOH, THF, 0 °C, 2 steps, 37.6%; c) TFA, 0 °C; Dowex 50W-X8, 30.7%.
Scheme 4 summarizes the synthesis of (2”R)-isomer from methyl cinnamate 5. The known azidoester4
(11) was treated with methanol and potassium bicarbonate to give alcohol 12a. After the
reduction-protection of the azide group by hydrogenolysis in the presence of Boc2O, the product (12b)
was reduced with lithium aluminum hydride to furnish corresponding diol 13a. Treatment of diol 13a
with acetic anhydride gave diacetate 13b, which was oxidized using sodium periodate and catalytic
amount of ruthenium trichloride to afford carboxylic acid 14a. The acid was converted to ester 14b by
treatment with t-butyl alcohol and O-tert-butyl N,N'-diisopropyl isourea (BDIU), and successive

1438
HETEROCYCLES, Vol. 81, No. 6, 2010
deacetylation was achieved with triethylamine and aqueous methanol to give diol 15a. Its primary and
secondary hydroxy groups were successively protected using tert-buthyldimethylsilyl chloride and
chloromethyl methyl ether, respectively. The silyl protecting group was selectively removed with
tetra-n-butylammonium fluoride to give alcohol 15d. Swern oxidation of the primary hydroxy group
afforded crude aldehyde (2R)-4, which was submitted to reductive amination with the amine unit 3 in the
same manner as described above. Finally, treatment with TFA followed by Dowex purification and
recrystallization from EtOH-H2O gave (2”R)-hydroxynicotianamine [(2”R)-1].
X
N3
ref. 4
a, b
CO2Me
CO2Me
OH
CO2Me
OAc
5
11
12a (X=N3)
12b (X=NHBoc)
NHBoc
NHBoc
NHBoc
g, h, i, j
c, d
e, f
OR
RO2C
OAc
ButO2C
OR'
OR
OR
OAc
13a (R=H)
13b (R=Ac)
14a (R=H)
15a (R=R'=H)
14b (R=But)
15b (R=TBS, R'=H)
15c (R=TBS, R'=MOM)
15d (R=H, R'=MOM)
CO2But
CO2But
CO2But
NHBoc
k
l
2
CHO
ButO2C
2"
N
N
H
NHBoc
OMOM
OMOM
(2S)-4
(2''R)-2
CO2H
N
CO2H
CO2H
NH2
m
2"
N
H
OH
(2"R)-1
Scheme 4. Reagents and conditions: a) K2CO3, MeOH, 0 °C, 89.2%; b) Boc2O, Pd-C, H2, EtOAc, rt,
93.6%; c) LAH, Et2O, rt, 69.4%; d) Ac2O, pyridine, CH2Cl2, rt, 95.4%; e) RuCl3, NaIO4, EtOAc, MeCN,
H2O, rt; f) BDIU, ButOH, CH2Cl2, 50 °C, 2 steps, 65.3%; g) Et3N, MeOH, H2O, 0 °C, 70.4%; h) TBSCl,
Et3N, DMAP, CH2Cl2, rt; i) MOMCl, Pri2NEt, CH2Cl2, reflux, 2 steps, 92.2%; j) TBAF, THF, 0 °C,
57.6%; k) (COCl)2, DMSO, CH2Cl2 then Et3N, 78 °C to rt; l) 3, NaBH3CN, AcOH, THF, 0 °C, 2 steps,
56.4%; m) TFA, 0 °C; Dowex 50W-X8, 54.3%.
1
With two isomeric 1 in hand, we compared the H NMR spectrum data with that of the natural 1, and
found that (2”S)-1 was identical with the natural product. In addition to that, specific rotation of (2”S)-1
showed almost the same value as that of the natural 1. [synthetic: []24 46.6 (c 0.51, H2O), natural: []24
D
D
40 (c 0.4, H2O)].

HETEROCYCLES, Vol. 81, No. 6, 2010
1439
In conclusion, we have succeeded in the enantioselective synthesis of both the diastereomers of
1
2”-hydroxynicotianamine and by the H NMR comparison, it was proved that the hydroxy group of the
natural hydroxynicotianamine isolated from buckwheat is located at 2”-position and its absolute
configuration is S. ACE inhibitory activities of hydroxynicotianamines and related analogs have been
currently investigated and detailed results will be reported in due course.
EXPERIMENTAL
GENERAL
1
Melting points were measured with a BUCHI B-545. H NMR spectra were recorded by JEOL
JNM-AL300 spectrometer (300 MHz) and by a JEOL JNM-LA400 spectrometer (400 MHz), the peak for
chloroform (at  7.24) being used as the internal standard. MS spectra were obtained with a JEOL
JMS-T100LC AccuTOF, and optical rotation values were measured with a JASCO polarimeter P-1030.
Column chromatography was carried out using MERCK Kieselgel 60 Art 7734.
tert-Butyl
(2S,3’S)-1-(3’-benzyloxycarbonylamino-4’-tert-butoxy-4’-oxobutanethioyl)azetidine-2-
carboxylate (8) To a solution of 7 (3.95 g, 8.5 mmol) in toluene (100 mL) was added Lawesson’s
reagent (1.90 g, 4.7 mmol) and the mixture was stirred at 90 °C for 2 h. The reaction mixture was filtered
through celite and the filtrate was purified by chromatography on silica gel (CH2Cl2/EtOAc=10/1) to give
20
1
8 (3.83 g, 93.7%) as a colorless and amorphous solid; []D 21.8 (c 0.35, CHCl3); H NMR (300 MHz,
CDCl3) 1.45 (9H, S), 1.58 (9H, s), 2.10-2.20 (1H, m), 2.45-2.55 (1H, m), 2.81-3.11 (2H, m), 4.09-4.22
(2H, m), 4.56-4.63 (1H, m), 4.73-4.79 (1H, m), 5.10 (2H, s), 6.02 (0.5H, d, J = 9.3 Hz), 6.32 (0.5H, d, J =
9.3 Hz), 7.28-7.37 (5H, m); HRMS m/z (M+Na)+: calcd. for C24H34N2NaO6S, 501.5913; found, 501.2037.
tert-Butyl
(2S,3’S)-1-(3’-benzyloxycarbonylamino-4’-tert-butoxy-4’-oxobutyl)azetidine-2-
carboxylate (9) To a suspension of Raney–Ni (W-2) (38 g) in THF (50 mL) was added 8 (3.83 g) in
THF (5 mL) at 0 °C and the mixture was stirred for 10 min at rt. The reaction mixture was filtered
through celite by washing with THF and the filtrate was concentrated under reduced pressure. The residue
was purified by chromatography on silica gel (hexane/EtOAc=1/1) to give 9 (2.72 g, 75.8%) as a yellow
20
1
oil; []D 44.9 (c 0.09, CHCl3); H NMR (300 MHz, CDCl3) 1.42 (9H, S), 1.45 (9H, s), 1.60-1.86 (2H,
m), 2.02-2.39 (3H, m), 2.69-2.75 (2H, m), 3.34 (1H, t, J = 7.2 Hz), 3.44 (1H, t, J = 8.4 Hz), 4.23 (1H, dd,
J = 2.7, 7.8 Hz), 5.09 (2H, s), 6.20 (1H, d, J = 7.8 Hz), 7.28-7.37 (5H, m); HRMS m/z (M+Na)+: calcd.
for C24H36N2NaO6, 471.2471; found, 471.2487.

1440
HETEROCYCLES, Vol. 81, No. 6, 2010
tert-Butyl (2S,3’S)-1-(3’-amino-4’-tert-butoxy-4’-oxobutyl)azetidine-2-carboxylate (3) The mixture
of 9 (115 mg, 0.26 mmol) and 10% Pd-C (20 mg) in EtOAc (5 mL) was stirred for 2 h under hydrogen
atmosphere at rt. The reaction mixture was filtered through celite and the filtrate was concentrated under
reduced pressure. The residue was purified by chromatography on silica gel (hexane/EtOAc=1/1) to give
3 (71.9 mg, 89.2%) as a colorless oil; []2D0 12.9 (c 0.35, CHCl3); 1H NMR (300 MHz, CDCl3) 1.35 (9H,
s), 1.40 (9H, s), 1.39-1.47 (1H, m), 1.69 (2H, br), 1.72-1.85 (1H, m), 2.05-2.29 (2H, m), 2.42 (1H, m),
2.71 (2H, m), 3.29-3.50 (3H, m); HRMS m/z (M+Na)+: calcd. for C16H30N2NaO4, 337.2103; found,
337.2081.
tert-Butyl
(2S,3’S,2”S,3”S)-1-(3’-amino-(2”-methoxymethyloxy-3”-tert-butoxycarbonylamino-4”-
tert-butoxy-4”-oxobutyl)-4’-tert-butoxy-4’-oxobutyl)azetidine-2-carboxylate [(2”S)-2] To a cooled
solution of (COCl)2 (466 mg, 3.67 mmol) in CH2Cl2 (30 mL) was added DMSO (382 mg, 4.90 mmol) at
–78 °C. After the mixture was stirred for 10 min, 10 (0.82 g, 2.45 mmol) and Et3N (742 mg, 7.34 mmol)
was added successively. The mixture was stirred at the same temperature for 1 h and then at the ambient
temperature for 2 h. The reaction mixture was washed with brine, dried over Na2SO4 and concentrated
under reduced pressure to give crude (2R)-4 (1.17 g) as an oil. This was used for the next step without
further purification.
To a solution of (2R)-4 (crude, 1.17 g) and 3 (1.15 g, 3.67 mmol) and AcOH (147 mg, 2.45 mmol) in
THF (10 mL) was added NaBH3CN (154 mg, 2.45 mmol) at 0 °C. The mixture was stirred overnight at rt
and saturated aqueous NaHCO3 was added. The mixture was extracted with CH2Cl2, dried over Na2SO4
and concentrated under reduced pressure. The residue was purified by chromatography on silica gel
(hexane/EtOAc= 4/1) to give (2”S)-2 (582 mg, 2 steps 37.6%) as a colorless oil; []2D0 17.0 (c 0.58,
CHCl3); 1H NMR (300 MHz, CDCl3) 1.23 (36H, s), 1.21-1.81 (5H, m), 2.03-3.13 (7H, m), 3.30-3.49 (3H,
m), 3.84 (1H, br), 4.20-4.34 (1H, m), 4.58-4.79 (2H, m), 5.93 (1H, d, J = 8.1 Hz), 7.24 (1H, m); HRMS
m/z (M+Na)+: calcd. for C31H57N3NaO10, 654.7882; found, 654.3919.
(2”S)-Hydroxynicotianamine [(2”S)-1] A solution of (2”S)-2 (582 mg, 0.92 mmol) in TFA (5 mL) was
stirred overnight at 0 °C. The reaction mixture was concentrated and purified by ion exchange resin
(Dowex 50W-X8, H2O then 1N aq. NH3) to give (2”S)-hydroxynicotianamine [(2”S)-1] (360 mg) as a
yellow solid. The solid was recrystallized from water (7.2 g) and EtOH (trace amount) to give (90 mg,
30.7%) of the product as a colorless solid; mp 320 ºC (decomp.); []2D4 46.6 (c 0.51, H2O); 1H NMR (400
MHz, D2O) 2.00-2.10 (2 H, m), 2.3-2.6 (2H, m), 3.13-3.35 (4H, m), 3.71(1H, dd, J = 4.4, 8.4 Hz),
3.79-4.0 (2H, m), 3.86 (1H, d, J = 2.8 Hz), 4.28 (1H ,m), 4.63 (1H, t, J = 9.6 Hz); HRMS m/z (M+Na)+:
calcd. for C12H21N3NaO7, 342.1277; found, 342.1283.

HETEROCYCLES, Vol. 81, No. 6, 2010
1441
Methyl (2S,3R)-3-azido-2-hydroxy-3-phenylpropanoate (12a) To a solution of 11 (1.63 g, 6.2 mmol)
and MeOH (50 mL) was added K2CO3 (trace amount) at 0 °C. The mixture was stirred for 1 h at 0 °C and
concentrated under reduced pressure. The residue was purified by chromatography on silica gel
(hexane/EtOAc=4/1) to give 12a (1.22 g, 89.2%) as a white solid; mp 130.3 ºC; []2D0 +7.8 (c 1.09,
1
CHCl3); H NMR (300 MHz, CDCl3) 3.20 (1H, d, J = 6.3 Hz), 3.81 (3H, s), 4.37 (1H, dd, J = 3.0, 6.3
HZ), 4.86 (1H, d, J = 3.0 Hz), 7.24-7.35 (5H, m); HRMS m/z (M+Na)+: calcd. for C10H11N3NaO3,
244.2024; found, 244.0715.
Methyl (2S,3R)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropanoate (12b) A mixture of
12a (3.2 g, 14.5 mmol), Boc2O (12.6 g, 57.9 mmol) and 10% Pd-C (1.6 g) in EtOAc (50 mL) was stirred
for 2 h under hydrogen atmosphere (0.15 MPa) at rt. The reaction mixture was filtered through celite and
the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on
silica gel (hexane/EtOAc=1/1) to give 12b (4.0 g, 93.6%) as a white solid; mp 131.9 ºC; []2D0 +7.9 (c
0.74, CHCl3); 1H NMR (300 MHz, CDCl3) 1.44 (9H, s), 3.12 (1H, s), 3.82 (3H, s), 4.45 (1H, s), 5.20 (1H,
d, J = 9.0 Hz), 5.37 (1H, d, J = 9.0 Hz), 7.24-7.35 (5H, m); HRMS m/z (M+Na)+: calcd. for C15H21NNaO5,
318.1317; found, 318.1309.
tert-Butyl (1R,2S)-2,3-dihydroxy-1-phenylpropylcarbamate (13a) To a suspension of lithium
aluminum hydride (1.60 g, 42.2 mmol) in dry Et2O (20 mL) was added 12b (12.5 g, 42.2 mmol) and the
mixture was stirred for 2 h under argon atmosphere at rt. To the reaction mixture was added a few drops
of water, the mixture was filtered through celite and the filtrate was concentrated under reduced pressure.
The residue was purified by chromatography on silica gel (hexane/EtOAc=1/1) to give 13a (7.82 g,
69.4%) as a colorless oil; []2D0 3.2 (c 0.19, CHCl3); 1H NMR (300 MHz, CDCl3) 1.36 (9H, s), 3.39 (2H,
m), 3.65-3.82 (3H, m), 4.63 (1H, br), 5.49 (1H, br), 7.18-7.37 (5H, m); HRMS m/z (M+Na)+: calcd. for
C14H21NNaO4, 290.1368; found, 290.1356.
(2S,3R)-3-tert-Butoxycarbonylamino-3-phenylpropane-1,2-diyl diacetate (13b) To a solution of 13a
(7.82 g, 29.3 mmol) and pyridine (9.26 g, 117 mmol) in CH2Cl2 (70 mL) was added Ac2O (8.96 g, 87.9
mmol) and the mixture was stirred overnight at rt and and saturated aqueous NaHCO3 was added. The
mixture was extracted with CH2Cl2, dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel (hexane/EtOAc=4/1) to give 13b (9.81 g, 95.4%) as
20
1
a colorless oil; []D 9.0 (c 0.34, CHCl3); H NMR (300 MHz, CDCl3) 1.37 (9H, s), 2.00 (3H, s), 2.02
(3H, s), 3.92 (1H, dd, J = 6.3, 12.0 Hz), 4.20 (1H, dd, J = 4.2, 12.0 Hz), 4.95 (1H, br), 5.16 (1H, d, J = 9.6
Hz), 5.32 (1H, dd, J = 6.3, 9.6 Hz), 7.21-7.33 (5H, m); HRMS m/z (M+Na)+: calcd. for C18H25NNaO6,

1442
HETEROCYCLES, Vol. 81, No. 6, 2010
374.1580; found, 374.1570.
tert-Butyl (2S,3S)-3,4-diacetoxy-2-tert-butoxycarbonylaminobutanoate (14b) To a solution of 13b
(9.8 g, 27.9 mmol) in EtOAc (100 mL), MeCN (100 mL) and H2O (500 mL) was added NaIO4 (149.4 g,
0.7 mol) and RuCl3 (0.3 g, 1.4 mmol) at 0 °C and the mixture was stirred at rt for 24 h. The reaction
mixture was extracted with EtOAc. The extract was concentrated under reduced pressure to give crude
14a (8.49 g) as a brown oil. The crude product was dissolved in CH2Cl2 (70 mL) and to this was added
t-BuOH (140 mL) and BDIU (22.4 g, 112 mmol). After being stirred for 18 h at 50 °C, the reaction
mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica
gel (hexane/EtOAc=4/1) to give 14b (6.84 g, 2 steps 65.3%) as a colorless oil; []2D0 +27.3 (c 1.09,
CHCl3); 1H NMR (300 MHz, CDCl3) 1.42 (9H, s), 1.44 (9H, s), 2.02 (3H, s), 2.04 (3H, s), 4.04 (1H, dd, J
= 7.5, 11.4 Hz), 4.27 (1H, dd, J = 5.4, 11.4 Hz), 4.51 (1H, dd, J = 2.7, 9.6 Hz), 5.15 (1H, d, J = 9.6 Hz),
5.57 (1H, m); HRMS m/z (M+Na)+: calcd. for C17H29NNaO8, 398.1791; found, 398.1753.
tert-Butyl (2S,3S)-2-tert-butoxycarbonylamino-3,4-dihydroxybutanoate (15a) To a solution of 14b
(6.7 g, 17.9 mmol) in MeOH (100 mL) and H2O (50 mL) was added Et3N (5.4 g, 53.6 mmol) at 0 °C and
the mixture was stirred for 5 h at the same temperature. The reaction mixture was concentrated under
reduced pressure. The residue was purified by chromatography on silica gel (hexane/EtOAc=1/1) to give
15a (3.66 g, 70.4%) as a colorless oil; []2D0 +27.9 (c 0.14, CHCl3); 1H NMR (300 MHz, CDCl3) 1.46 (9H,
s), 1.48 (9H, s), 1.84 (1H, br), 3.43-3.66 (3H, m), 4.17 (1H, m), 4.38 (1H, m), 5.39 (1H, d, J = 6.6 Hz);
HRMS m/z (M+Na)+: calcd. for C13H25NNaO6, 314.1580; found, 314.1590.
tert-Butyl
(2S,3S)-2-tert-butoxycarbonylamino-4-tert-butyldimethylsilyloxy-3-hydroxybutanoate
(15b) To a solution of 15a (253 mg, 0.87 mmol), Et3N (351 mg, 26.0 mmol) and DMAP (21.2 mg, 0.17
mmol) in CH2Cl2 (5 mL) was added TBSCl (393 mg, 2.61 mmol) at rt and the mixture was stirred
overnight. The reaction mixture was concentrated under reduced pressure. The residue was purified by
chromatography on silica gel (hexane/EtOAc=4/1) to give 15b (365 mg) as a colorless oil. This product
20
1
was contaminated with a small amount of TBSOH; []D +2.5 (c 0.19, CHCl3); H NMR (300 MHz,
CDCl3) 0.07 (6H, s), 0.89 (9H, s), 1.43 (9H, s), 1.47 (9H, s), 2.62 (1H, br), 3.52 (1H, d, J = 8.7 Hz), 3.67
(1H, dd, J = 4.5, 9.9 Hz), 4.09-4.19 (2H, m), 5.31 (1H, d, J = 8.7 Hz); HRMS m/z (M+Na)+: calcd. for
C19H39NNaO6Si, 428.2444; found, 428.2466.
tert-Butyl
(2S,3S)-2-tert-butoxycarbonylamino-4-tert-butyldimethylsilyloxy-3-methoxymethoxy-
butanoate (15c) To a solution of 15b (365 mg, 0.90 mmol) and Pri2NEt (700 mg, 5.4 mmol) in CH2Cl2

HETEROCYCLES, Vol. 81, No. 6, 2010
1443
(5 mL) was added MOMCl (290 mg, 3.6 mmol) at rt and the mixture was stirred overnight under reflux.
The reaction mixture was washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by chromatography on silica gel
(hexane/EtOAc=4/1) to give 15c (360 mg, 2 steps 92.2%) as a colorless oil; []20 7.5 (c 0.53, CHCl3);
D
1H NMR (300 MHz, CDCl3) 0.04 (6H, s), 0.88 (9H, s), 1.43 (9H, s), 1.45 (9H, s), 3.30 (3H, s), 3.55-3.68
(2H, m), 4.09 (1H, m), 4.35 (1H, d, J = 9.3 Hz), 4.57-4.67 (2H, m), 5.17 (1H, d, J = 9.3 Hz); HRMS m/z
(M+Na)+: calcd. for C21H43NNaO7Si, 472.2707; found, 472.2688.
tert-Butyl (2S,3S)-2-tert-butoxycarbonylamino-4-hydroxy-3-methoxymethoxybutanoate (15d) To a
solution of 15c (4.19 g, 9.3 mmol) in THF (30 mL) was added 1.0 M TBAF in THF (9.3 mL, 9.3 mmol)
at 0 °C and the mixture was stirred for 1 h under the same condition. The reaction mixture was
concentrated under reduced pressure. The residue was diluted with Et2O, washed with saturated aqueous
NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified
by chromatography on silica gel (hexane/EtOAc=4/1) to give 15d (1.80 g, 57.6%) as a colorless oil; []2D0
+85.1 (c 0.18, CHCl3); 1H NMR (300 MHz, CDCl3) 1.42 (9H, s), 1.44 (9H, s), 3.30 (3H, s), 3.43 (1H, m),
3.66 (1H, m), 3.84 (1H, m), 4.05 (1H, m), 4.44 (1H, dd, J = 1.8, 8.7 Hz), 4.57 (2H, dd, J = 6.6, 11.7 Hz),
5.36 (1H, d, J = 8.7 Hz); HRMS m/z (M+Na)+: calcd. for C15H29NNaO7, 358.1842; found, 358.1825.
tert-Butyl (2S,3’S,2”R,3”S)-1-(3’-amino-(2”-methoxymethyloxy-3”-tert-butoxycarbonylamino-4”-
tert-butoxy-4”-oxobutyl)-4’-tert-butoxy-4’-oxobutyl)azetidine-2-carboxylate [(2”R)-2] A solution of
(COCl)2 (59.1 mg, 0.47 mmol) in CH2Cl2 (3 mL) was cooled to –78 °C and to this was added DMSO (48
mg, 0.62 mmol). After the mixture was stirred for 10 min, 15d (104 mg, 0.31 mmol) and Et3N (94 mg,
0.93 mmol) was added successively. The mixture was stirred at the same temperature for 1 h and at
ambient temperature for 2 h. The reaction mixture was diluted with CH2Cl2, washed with brine, dried over
Na2SO4 and concentrated under reduced pressure to give (2S)-4 (253 mg). This was used for the next step
without further purification.
To a solution of crude (2S)-4 (Crude, 253 mg), 3 (146 mg, 0.47 mmol) and AcOH (18 mg, 0.31 mmol) in
THF (5 mL) was added NaBH3CN (19.6 mg, 0.31 mmol) at 0 °C. The mixture was stirred overnight at rt
and saturated aqueous NaHCO3 was added. The mixture was extracted with CH2Cl2, dried over Na2SO4
and concentrated under reduced pressure. The residue was purified by chromatography on silica gel
(hexane/EtOAc=4/1) to give (2”R)-2 (110 mg, 2 steps 56.4%) as a colorless oil; []2D0 69.6 (c 0.52,
CHCl3); 1H NMR (300 MHz, CDCl3) 1.42 (36H, s), 1.69-1.81 (1H, m), 2.01-2.67 (4H, m), 2.51-2.80 (5H,
m), 3.08 (1H, t, J = 4.8 Hz), 3.28 (3H, s), 3.43 (1H, t, J = 7.8 Hz), 3.99 (1H, br), 4.34 (1H, d, J = 9.0 Hz),
4.57 (2H, br), 5.29 (1H, d, J =8.4 Hz), 7.51-7.68 (1H, m); HRMS m/z (M+Na)+: calcd. for C31H57N3NaO10,

1444
HETEROCYCLES, Vol. 81, No. 6, 2010
654.3942; found, 654.3990.
(2”R)-Hydroxynicotianamine [(2”R)-1] A solution of (2”R)-2 (110 mg, 0.174 mmol) in TFA (3 mL)
was stirred overnight at 0 °C. The reaction mixture was concentrated and purified by ion exchange resin
(Dowex 50W-X8, H2O then 1N aq. NH3) to give (2”R)-hydroxynicotianamine (2”R)-1 (60.3 mg) as
yellow solid. The solid was recrystallized from water (180 g) and EtOH (trace amount) to give a colorless
24
1
solid (30.2 mg, 54.3%); mp: 320 ºC (decomp.); []D 134.9 (c 0.10, H2O); H NMR (400 MHz, D2O)
1.98-2.1 (2H, m), 2.3-2.6 (2H, m), 3.08-3.34 (4H, m), 3.65 (1H, d, J = 4.8 Hz), 3.73 (1H, dd, J = 4.0, 8.8
Hz), 3.79-3.99 (2H, m), 4.30 (1H, m), 4.63 (1H, t, J = 9.0 Hz); HRMS m/z (M+Na)+: calcd. for
C12H21N3NaO7, 342.1277; found, 342.1283.
ACKNOWLEDGEMENTS
We are most grateful to Mr. Y. Ohkubo of T. Hasegawa Co., Ltd., for the MS analysis.
REFERENCES
1. a) Y. Aoyagi, JP2004-331556; b) Y. Aoyagi, Phytochemistry, 2006, 67, 618 and references cited
therein.
2. a) S. S. Klair, H. R. Mohan, and T. Kitahara, Tetrahedron Lett., 1998, 39, 89; b) K. Miyakoshi, J.
Oshita, and T. Kitahara, Tetrahedron, 2001, 57, 3355 and references cited therein.
3. F. Matsuura, Y. Hamada, and T. Shioiri, Tetrahedron, 1993, 49, 8211 and references cited therein.
4. J. Deng, Y. Hamada, and T. Shioiri, Synthesis, 1998, 627.