6
Y. Amino and Y. Suzuki
(3S)-3-(tert-Butoxycarbonyl)amino-4-iodobutanoic
acid tert-butyl ester (13). NMR (CDCl3) δH: 1.46
(9H, s), 1.48 (9H, s), 2.55 (1H, dd, J = 6.1, 15.8 Hz),
2.64 (1H, dd, J = 6.1, 15.7 Hz), 3.37–3.48 (2H, m),
3.89 (1H, brs), 5.10 (1H, brd, J = 8.4 Hz). ESI-MS m/z:
385.9 (M + H)+, 408.1 (M + Na)+.
DMF (20 mL) at room temperature and stirred for
20 h. The mixture was then quenched with a citric acid
solution (10%, 10 mL) and extracted with AcOEt
(35 mL, twice). The combined organic layers were
washed with a citric acid solution (10%, 10 mL) and
brine (10 mL), dried over anhydrous MgSO4, and
concentrated in vacuo. The residue (2.46 g) was
purified by column chromatography (silica gel 50.0 g,
n-hexane:AcOEt = 4:1–7:3) to afford 14 as a colorless
oil (998 mg, 1.87 mmol); NMR (CDCl3) δH: 1.46
(27H, s), 1.49 (9H, s), 2.56 (2H, m), 2.77 (2H, m),
2.99 (2H, d, J = 5.1 Hz), 4.04 (1H, brs), 4.42 (1H,
brs), 5.23 (1H, brs), 5.40 (1H, brs). NMR (CDCl3) δC:
28.0, 28.2, 28.3, 28.4, 35.8, 37.5, 38.7, 47.5, 53.9,
79.5, 79.9, 81.2, 82.5, 155.1, 155.2, 169.9, 170.6. ESI-
MS m/z: 535.1 (M + H)+, 556.8 (M + Na)+. FAB-MS
m/z 535.3055 (M + H) (Calcd for C25H46N2O8S:
535.3053).
S-[(2R)-3-(tert-Butoxy)-2-(tert-butoxycarbonyl)amino-
3-oxypropyl]-N-tert-butoxycarbonyl-L-homocysteine
tert-butyl ester (9). CsCO3 (34.7 g, 106.4 mmol) was
added to a solution of 4 (31.0 g, 111.8 mmol) and 8
(41.4 g, 106.7 mmol) in anhydrous DMF (600 mL) at
room temperature. The mixture was stirred for 18 h,
quenched with a citric acid solution (10%, 750 mL)
and brine (400 mL), and extracted with AcOEt
(750 mL, twice). The combined organic layers were
washed with a citric acid solution (10%, 500 mL) and
brine (200 mL), dried over anhydrous MgSO4, and
concentrated in vacuo. The residue (2.31 g) was puri-
fied by column chromatography (silica gel 1500 g,
n-hexane:AcOEt = 9:1–2:1) to afford 9 as a white solid
(45.4 g, 84.9 mmol); NMR (CDCl3) δH: 1.46 (18H, s),
1.49 (18H, s), 1.81–1.95 (1H, m), 2.03–2.12 (1H, m),
2.58–2.60 (2H, m), 2.90–3.04 (2H, m), 4.25–4.30 (1H,
m), 4.38–4.45 (1H, m), 5.08–5.16 (1H, m), 5.33–5.38
(1H, m). NMR (CDCl3) δC: 28.0, 28.3, 28.8, 32.9,
34.8, 53.3, 53.9, 79.8, 79.9, 82.2, 82.6, 155.2, 155.4,
170.0, 171.3. ESI-MS m/z: 557.4 (M + Na)+. FAB-MS
m/z 535.3077 (M + H) (Calcd for C25H46N2O8S:
535.3053). mp: 85–87 °C.
4-[(2R)-2-Amino-2-carboxyethyl]thio-(3S)-3-amino-
butanoic acid (15).
Compound 14 (802 mg,
1.50 mmol) was dissolved in 4 N HCl/dioxane solution
(16 mL) and stirred at room temperature for 17 h. The
resulting precipitate was collected by filtration to afford
15·2HCl as a hygroscopic white solid (525 mg). A
solution of 15·HCl (500 mg) in ion-exchange water
(12 mL) was applied to a cation-exchange resin (DOW-
EXTM MONOSPHERETM 650C, H+ form) column
(75 mL) for purification. The column was then washed
with ion-exchange water (300 mL). Compound 15 was
successively eluted with aqueous pyridine (1 N,
300 mL) and aqueous NH3 (1 N, 300 mL). The eluates
containing 15 were pooled and concentrated in vacuo.
The residue was dissolved in water (18 mL), and acti-
vated carbon (20 mg) was added to the solution. After
stirring for 1 h, the solution was filtered and the filtrate
was concentrated in vacuo to give 15 as a white solid
(245 mg, 1.10 mmol); NMR (CDCl3) δH: 2.45 (1H, dd,
J = 7.9, 16.8 Hz), 2.57 (1H, dd, J = 5.1, 16.8 Hz), 2.76
(1H, dd, J = 7.6, 14.4 Hz), 2.90 (1H, dd, J = 5.6,
14.4 Hz), 3.03 (1H, dd, J = 6.5, 14.6 Hz), 3.08 (1H,
dd, J = 4.7, 14.6 Hz), 3.59 (1H, m), 3.87 (1H, dd,
J = 4.8, 6.5 Hz). NMR (D2O) δC: 32.7, 33.9, 37.9,
48.7, 53.6, 172.7, 177.2. ESI-MS m/z: 223.5 (M + H)+,
221.1 (M – H)−. FAB-MS m/z 221.0604 (M – H)
(Calcd for C7H13N2O4S: 221.0596). mp: 142–145 °C
(decomp.).
L-Cystathionine (S-[(2R)-2-Amino-2-carboxyethyl]-L-
homocysteine) (1). Compound 9 (38.8 g, 72.6 mmol)
was dissolved in 4H HCl/dioxane (770 mL) and stirred
at room temperature for 16 h. The resulting precipitate
was collected by filtration to afford 1·2HCl as white
crystals (20.6 g, 69.9 mmol). Aqueous NaOH (2 N,
approximately 70 mL) was added to a solution of
1·2HCl (20.5 g, 69.4 mmol) in ion-exchange water
(200 mL) until the pH reached approximately 7–8, and
the mixture was stirred for 1 h to afford 1 as white
crystals (10.9 g, 49.0 mmol); NMR (D2O) δH: 2.05
(1H, m), 2.10 (1H, m), 2.64 (2H, td, J = 2.3, 7.78 Hz),
3.00 (1H, dd, J = 7.1, 14.8 Hz), 3.07 (1H, dd, J = 4.5,
14.8 Hz), 3.77 (1H, dd, J = 5.6, 6.8 Hz), 3.87 (1H, dd,
J = 4.5, 7.1 Hz); 2HCl salt: 2.12 (1H, m), 2.20 (1H,
m), 2.59 (2H, td, J = 2.9, 7.6 Hz), 3.05 (1H, dd,
J = 7.1, 15.0 Hz), 3.13 (1H, dd, J = 4.5, 15.0 Hz), 4.06
(1H, dd, J = 6.3, 6.5 Hz), 4.11 (1H, dd, J = 4.5,
7.0 Hz). NMR (35% DCl:D2O = 1:10) δC: 26.8, 29.1,
30.6, 51.3, 51.9, 170.0, 171.1. ESI-MS m/z: 223.0
(M + H)+, 244.9 (M + Na)+, 221.1 (M – H)–. FAB-MS
m/z 223.0768 (M + H) (Calcd for C7H15N2O4S:
Amino acid analysis.
Amino acid analysis was
performed with an L-8900 Amino Acid Analyzer (Hita-
chi High-Tech Science Co., Japan) using cation-
exchange chromatography to separate the amino acids,
followed by post-column derivatization with ninhydrin
(Wako Pure Chemical Industries Ltd., Osaka, Japan).
The analytical column (60 mm × 4.6 mm i.d., 3 μm)
and the guard column (5.0 mm × 4.0 mm i.d., 5 mm)
were packed with Hitachi custom ion-exchange resin
and polystyrene cross-linked by divinylbenzene with
sulfone groups as the active exchange sites for physio-
logical amino acid analysis. We measured the absorp-
tion of the solutions at 570 nm by filter photometer
using a reference wavelength of 700 nm. The mobile
20
223.0753). ½aꢀD + 23 (c = 1, 1 M HCl). mp: 283–
286 °C (decomp.).
4-[(2R)-3-tert-Butoxy-2-(tert-butoxycarbonyl)amino-
3-oxopropyl]thio-[(3S)-3-(tert-butoxycarbonyl)amino]
butanoic acid tert-butyl ester (14). CsCO3 (629 mg,
1.93 mmol) was added to a solution of 4 (540 mg,
1.95 mmol) and 13 (754 mg, 1.95 mmol) in anhydrous