tR = 28.67 min). The desired product was collected and lyophilized
to yield a white fluffy powder (17.4 mg, 15% over 5 steps). H
J = 5.8, 2H), 2.25 (s, 1H), 2.13 (s, 3H), 1.97–1.81 (m, 3H), 0.90 (s,
3H), 0.86 (s, 3H); 13C NMR (D2O, 126 MHz) d 207.77, 174.10,
173.89, 173.16, 170.70, 141.30, 128.72, 128.51, 126.27, 96.70,
78.85, 76.06, 74.94, 72.41, 71.38, 70.31, 68.61, 53.75, 52.11, 49.91,
49.60, 48.46, 39.99, 39.47, 38.18, 38.13, 35.39, 35.26, 34.62, 31.78,
31.24, 29.81, 29.25, 28.22, 22.38, 20.61; HRMS for C38H64N7O12S
[M + H]+ calcd. 842.4328, found 842.4306. Purity, 90% (method
D, tR = 20.17 min; method E, tR = 13.85 min).
1
NMR (D2O, 400 MHz) d 7.23–7.12 (m, 3H), 7.05 (d, J = 6.8, 2H),
5.55 (d, J = 4.0, 1H), 3.75–3.68 (m, 4H), 3.51–3.12 (m, 19H), 2.73
(t, J = 6.4, 2H), 2.54 (t, J = 6.4, 2H), 2.37–2.27 (m, 3H), 2.07
(s, 1H), 1.96 (s, 3H), 1.70 (q, J = 12.4, 1H), 0.80 (s, 3H), 0.74 (s,
3H); 13C NMR (D2O, 126 MHz) d 207.67, 174.12, 173.84, 173.12,
171.20, 138.06, 129.15, 128.84, 126.89, 96.64, 78.76, 75.98, 74.89,
72.36, 71.32, 70.24, 68.55, 53.69, 51.78, 51.62, 49.54, 48.39, 40.10,
39.39, 38.58, 38.11, 35.37, 35.21, 34.54, 33.61, 31.18, 29.82, 28.17,
22.41, 20.62; HRMS for C37H62N7O12S [M + H]+ calcd. 828.4172,
found 828.4169. Purity, 96% (method F, tR = 7.01 min; method G,
tR = 12.61 min).
4¢-Aminopantetheine (3). To Fmoc protected amine 11 (33 mg,
0.03 mmol) dissolved in DMF (5 mL) was added piperidine
(0.25 mL) followed by evaporation of the liquids under high
reduced pressure. The process was repeated once more and the
residue dissolved in H2O (5 mL). The solution was washed with
diethyl ether (5 mL ¥ 3), concentrated and purified by reversed
phase HPLC (method A, tR = 35.87 min). The desired product (as
the disulfide dimer) was collected and lyophilized to yield a white
powder (14 mg, 77%). The compound was fully characterized as
the more stable dimer, but the free thiol was easily accessible by
Neamine
6¢-N-(R)-(3-((2-((2-amino-2-oxoethyl)thio)ethyl)-
amino)-3-oxopropyl)-2-hydroxy-3,3-dimethyl-4-(3-oxo-N-(3-phe-
nylpropyl)butanamido)butanamide (2d). To aldehyde 8 (80 mg,
0.10 mmol) in 1,2-dichloroethane (3 mL) was added anhydrous
sodium sulfate (0.20 g) and 3-phenylpropan-1-amine (0.059 mL,
0.40 mmol). The reaction mixture was stirred at RT for 1 h then
filtered. The filtrate was diluted with toluene (2 ¥ 20 mL) and
evaporated to dryness under reduced pressure to get the imine
intermediate as a clear oil. This crude product was dissolved in
anhydrous MeOH (3 mL). Sodium borohydride (39 mg, 1.0 mmol)
was added. The reaction mixture was stirred overnight at RT,
quenched with saturated aqueous sodium bicarbonate (5 mL),
concentrated under reduced pressure, and extracted with CH2Cl2
(4 ¥ 20 mL). The combined organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to get the amine as a colorless oil. ESI-MS
for C52H92N6O6S2Si2Na [M+Na]+ calcd. 1039.6, found 1039.7.
The amine in THF (3 mL) was treated with 2-hydroxypyridine
(20 mg, 0.20 mmol) and diketene (0.080 mL, 1.0 mmol). The
reaction mixture was warmed up to 50 ◦C and stirred overnight.
The reaction was monitored by ESI-MS for the disappearance of
the amine and the emergence of the amide product; ESI-MS for
the amide C60H100N6O10S2Si2Na [M + Na]+ calcd. 1207.6, found
1207.6. The reaction mixture was concentrated under reduced
pressure, then redissolved in THF (3 mL). TBAF (1 M in THF,
1 mL, 1 mmol) was added and the resulting solution was allowed
to stir at RT overnight. The reaction was quenched with saturated
aqueous sodium bicarbonate (5 mL). Water (3 mL) was added,
and the mixture was extracted with ethyl acetate (3 ¥ 10 mL).
The combined organic layer was dried over anhydrous sodium
sulfate, concentrated under reduced pressure, and redissolved in
acetone/H2O (2 mL, 1/1 v/v). Dithiothreitol (15 mg, 0.10 mmol)
and DIPEA (1 mL, 5.7 mmol) were added and the resulting
mixture was sonicated for 2 min. The mixture was transferred into
a solution of 1311 (0.20 mmol) in acetone/H2O (4 mL, 1/1 v/v).
The resulting mixture was sonicated for 2 min and then stirred for
16 h at RT. The reaction mixture was then evaporated in vacuo to
~1 mL, diluted with H2O (10 mL), and acidified to pH 4 using
TFA. The solution was washed with ethyl acetate (3 ¥ 5 mL),
concentrated and purified by reversed phase HPLC (method C,
tR = 29.13 min). The desired product was collected and lyophilized
to yield a white fluffy powder (19 mg, 16% over 5 steps). 1H NMR
(D2O, 400 MHz) d 7.38–7.33 (m, 2H), 7.29–7.24 (m, 3H), 5.70
(d, J = 4.0, 1H), 3.90–3.76 (m, 4H), 3.66–3.24 (m, 19H), 2.69 (t,
J = 6.4, 2H), 2.62 (t, J = 6.8, 2H), 2.51 (t, J = 4.0, 1H), 2.47 (t,
1
treatment with dithiothreitol (DTT, ~1 mM). H NMR (D2O,
500 MHz) d 4.08 (s, 1H), 3.61–3.58 (m, 4H), 3.10 (s, 2H), 2.92
(t, J = 6.5, 2H), 2.59 (t, J = 6.5, 2H), 1.16 (s, 3H), 1.09 (s, 3H);
13C NMR (D2O, 125 MHz) d 173.75, 173.74, 77.38, 47.70, 37.83,
36.24, 35.67, 35.40, 34.99, 21.77, 19.76; HRMS for C22H45N6O6S2
[M + H]+ calcd. 553.2837, found 553.2848. Purity, 98% (method
D, tR = 10.15 min; method E, tR = 7.41 min).
D-Pantetheine (4)45. D-Pantethine (5) (1.0 g, 1.8 mmol) was
dissolved in degassed H2O (10 mL) and MeOH (10 mL). The
mixture was cooled to 4 ◦C and D/L-dithiothreitol (DTT, 0.44 g,
2.8 mmol) was added. The reaction mixture was stirred at 4 ◦C
for 16 h under N2. After evaporation of the solvent, the residue
was purified by silica gel column chromatography with a mixture
of CHCl3–MeOH (10 : 1 → 9 : 1) as the eluent to give compound
4 as a white solid (0.99 g, 99%). Rf 0.31 (CH3Cl–MeOH 7 : 1); 1H
NMR (D2O, 500 MHz) d 3.97 (s, 1H), 3.55–3.46 (m, 3H), 3.38–
3.34 (m, 3H), 2.63 (t, J = 6.5, 2H), 2.50 (t, J = 6.5, 2H), 0.90 (s, 3H),
0.87 (s, 3H); 13C NMR (D2O, 126 MHz) d 175.26, 174.18, 75.87,
68.47, 42.41, 38.72, 35.60, 35.41, 23.25, 20.61, 19.20; ESI-MS for
C11H22N2O4SNa [M + Na]+ calcd. 301.1, found 301.2.
(R)-S-2-(3-(2,4-tert-butyldimethylsilyloxy-3,3-dimethylbutan-
amido)propanamido)ethyl disulfide (6). D-Pantethine 5 (1.05 g,
1.89 mmol) in anhydrous DMF (10 mL) was treated with imidazole
(1.54 g, 22.6 mmol) and tert-butyldimethylsilyl chloride (3.42 g,
22.6 mmol) at RT. The reaction mixture was stirred overnight, and
then quenched with water (60 mL). The mixture was extracted with
EtOAc (3 ¥ 100 mL). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The resulting colorless oil was purified by
flash column chromatography on silica gel (EtOAc–hexane 1 : 1
→ EtOAc) to get the title compound as a clear oil (1.86 g, 98%).
Rf 0.40 (EtOAc); 1H NMR (CDCl3, 300 MHz) d 6.91 (t, J = 6.0,
1H), 6.84 (t, J = 6.0, 1H), 3.98 (s, 1H), 3.58–3.46 (m, 4H), 3.40
(d, J = 6.3), 3.29 (d, J = 6.3), 2.77 (t, J = 6.6, 2H), 2.44 (t, J = 6.0,
2H), 0.91 (s, 9H), 0.87 (s, 9H), 0.85 (s, 3H), 0.81 (s, 3H), 0.06 (s,
3H), 0.02 (s, 3H), 0.01 (s, 3H), -0.02 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 172.95, 171.34, 76.49, 68.76, 40.07, 38.33, 37.52, 35.88,
34.60, 25.96, 25.79, 20.97, 19.85, 18.33, 17.96, -5.08, -5.28, -5.36,
1544 | Org. Biomol. Chem., 2011, 9, 1538–1546
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The Royal Society of Chemistry 2011
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