H.-J. Deussen et al. / Bioorg. Med. Chem. 8 (2000) 507±513
511
Diethyl 11-[(1,1,1-trimethylsilyl)oxy]undecylphosphonate
(3). A mixture of [(11-bromoundecyl)oxy](trimethyl)-
silane (2) (50.0 g, 154.6 mmol) and triethyl phosphite
(51.4 g, 309.3 mmol) was slowly heated to 156 ꢀC and
this temperature was maintained overnight. The ethyl
bromide evolved was trapped with a condenser and
a receiver cooled in an ice bath. Excess triethyl phos-
phite was removed under reduced pressure (1 mbar) and
the residual oil was fractionally distilled under high
vacuum (bp >157 ꢀC/0.02 mbar) to give 3 as a colorless
pound 5 (4.50 g, 10.0 mmol) was dissolved in a mixture
of dry CH2Cl2 (50 mL). Oxalyl chloride (3.17 g,
25.0 mmol) was added at 0 ꢀC with stirring under an
inert atmosphere. The mixture was slowly allowed to
reach room temperature and stirred for 18 h at this
temperature. The solvents and excess oxalyl chloride
were removed in vacuo. The intermediate mono-
chlorophosphonate (31P NMR (162 MHz, CDCl3)
d 46.03) and 4-nitrophenol (1.39 g, 10.0 mmol) were
dissolved in dry CH2Cl2 (60 mL). Triethylamine
(2.02 g, 20.0 mmol) was added dropwise at room
temperature and the mixture was stirred for 3 h. The
triethylamine hydrochloride formed was ®ltered o
using a glass frit (G5). A yellow oil was obtained
after evaporation of the solvent in vacuo, which was
puri®ed by ¯ash chromatography (silical gel,
CH2Cl2:AcOEt (7:1, v:v)) to give pure 6 as an oil
(2.8 g, 52%). 1H NMR (400 MHz, CDCl3) d 1.2±1.4
(m, ca. 14H), 1.24 (t, 3H), 1.56 (m, 2H), 1.67 (m, 2H),
2.01 (m, 2H), 2.81 (s, 4H), 4.14 (m, 2H), 4.33 (t, 2H),
7.47 (d, 2H), 8.29 (d, 2H); 13C NMR (100 MHz, CDCl3)
d 16.39 (Jc,p=6 Hz), 22.23 (Jc,p= 5 Hz), 25.83, 25.50,
26.09 (Jc,p=137 Hz), 28.38, 28.99, 29.04, 29.26, 29.31,
29.36, 30.42 (Jc,p=17 Hz), 62.91 (Jc,p=7 Hz), 71.64,
120.98 (Jc,p=5 Hz), 125.67, 144.57, 151.65, 155.85
(Jc,p=8 Hz), 168.67. MS (EI) m/z 543 (M+H); 31P
NMR (162 MHz, CDCl3) d 31.17. Anal. calcd for
C24H35N2O10P: C, 53.13; H, 6.50; N, 5.16. Found: C,
52.89; H, 6.61; N, 5.24.
1
liquid (51.2 g, 87%). H NMR (400 MHz, DMSO-d6)
d 0.05 (s, 9H), 1.20±1.35 (m, ca. 14H), 1.21 (t, 6H), 1.42
(m, 4H), 1.65 (m, 2H), 3.50 (t, 2H), 3.95 (dq, 4H). MS
(ESI): m/z 403 (M+Na); 31P NMR (162 MHz, CDCl3)
d 33.27.
Diethyl (11-hydroxyundecyl)phosphonate (4). Compound
3 (15.0 g, 39.4 mmol) was dissolved in acetone (100 mL).
Dilute H2SO4 (5%, 100 mL) was added and the mixture
stirred at room temperature for 30 min. The mixture
was diluted with water (1 L) and diethylether (200 mL)
was added to improve phase separation. The organic
phase was separated and the water phase was extracted
2Â with diethylether. The combined organic phases
were dried over MgSO4 and the solvent was removed in
vacuo after ®ltration to give 4 as a colorless liquid
(11.91 g, 98%), which was used in the next step without
1
puri®cation. H NMR (400 MHz, CDCl3) d 1.25±1.40
(m, ca. 14H), 1.32 (t, 6H), 1.57 (m, 4H), 1.72 (m, 2H),
3.63 (t, 2H), 4.08 (m, 4H). MS (ESI): m/z 309 (M+H);
31P NMR (162 MHz, CDCl3) d 33.34.
Ethyl (4-nitrophenyl)(11-[(2-[(2-[6-(5-[(3aR,4R,6aS)-2-
oxoperhydrothieno[3,4-d]imidazol-4-yl]pentanoylamino)-
hexanoyl]aminoethyl)disulfanyl]ethylamino)carbonyl]oxy-
Diethyl [11-([(2,5-dioxotetrahydro-1H-1-pyrrolyl)oxy]-
carbonyloxy)undecyl]phosphonate (5). Compound
(11.91 g, 38.6 mmol) was dissolved in CH2Cl2 (200 mL).
Phosgene (1.93 solution in toluene) (100 mL,
4
undecyl) phosphonate (8). Compound 6 (264 mg,
0.49mmol) and biotinyl-N-e-aminocaproyl-N-cystamine-
N0-cystaminammonium tri¯uoroacetate7 (297 mg,
0.49 mmol) were dissolved in dry DMF (30 mL) under
an inert atmosphere. Dry triethylamine (149 mg,
1.47 mmol) was added and the mixture was stirred for
4 h. The mixture was poured into water (500 mL) and
the water phase was extracted with AcOEt (3Â150 mL).
The combined organic phases were dried over MgSO4
and the solvent was removed in vacuo after ®ltration to
give an oil, which was puri®ed by ¯ash chmromato-
graphy (silical gel, CH2Cl2:MeOH (8:1, v:v)) to give
pure 8 as a white solid (220 mg, 49%). 1H and 13C NMR
see Table 1, mp 127±132 ꢀC. MS (FAB+) m/z 919
(M+H), 31P NMR (162 MHz, CDCl3) d 31.71. Anal.
calcd for C40H67N6O10PS3: C, 52.27; H, 7.35; N, 9.14.
Found: C, 52.10; H, 7.31; N, 8.93.
M
193.0 mmol) was added to the cooled mixture (ice bath)
with stirring. The mixture was stirred overnight slowly
allowing it to reach room temperature. The solvents and
excess phosgene were removed in vacuo. The inter-
mediate [11-(chlorocarbonyloxy)undecyl](diethoxy) was
redissolved in dry CH2Cl2 (200 mL) followed by addi-
tion of N-hydroxy succinimide (4.54 g, 39.4 mmol). Dry
triethylamine (6.76 g, 66.8 mmol) was added dropwise
to the solution with stirring at room temperature. The
solvents were removed in vacuo after stirring overnight.
The remainder was redissolved in a small volume of
diethylether, insoluble triethylamine hydrochloride
was ®ltered o using a glass frit (5). Evaporation of
the solvents in vacuo yielded an oil, which crystallized
on standing (17.17 g, 99%): mp 58±59 ꢀC (ether); 1H
NMR (400 MHz, CDCl3) d 1.25±1.43 (m, ca. 14H), 1.32
(t, 6H), 1.60 (m, 2H), 1.73 (m, 4H), 2.84 (s, 4H), 4.08
(m, 4H), 4.31 (t, 2H); 13C NMR (100 MHz, CDCl3)
d 16.45 (Jc,p=5 Hz), 22.35 (Jc,p=5 Hz), 25.35, 25.41,
25.62 (Jc,p=140 Hz), 28.29, 28.99, 29.17, 29.25, 29.27,
29.34, 30.53 (Jc,p=17 Hz), 61.23 (Jc,p=6 Hz), 71.50,
151.24, 168.30; m/z 450 (M+H); 31P NMR (162 MHz,
CDCl3) d 33.38. Anal. calcd for C20H36NO8P: C, 53.44;
H, 8.07; N, 3.12. Found: C, 53.22; H, 8.08; N, 3.36.
Enzyme activity and inhibition
Puri®ed recombinant Lipolase1 (100 mg/mL in 100 mM
Tris±HCl, 150 mM NaCl, 0.3 mM CaCl2, 0.1 mM
MgCl2, pH 7.5) was mixed with suicide inhibitor 8
(50 mM in DMSO) to a ®nal inhibitor concentration of
25 mM). At t=0, 5, 10 and 20 min 10 mL samples were
withdrawn and added to 990 mL lipase assay buer
(80 mM PNP-butyrate in 50 mM Tris±HCl, 0.1% Triton
X-100, 10 mM CaCl2, pH 7.5). Release of 4-nitrophenol
was recorded continuously at OD405 on a Milton Roy
1201 spectraphotometer.
Ethyl (4-nitrophenyl)[11-([(2,5-dioxotetrahydro-1H-1-pyr-
rolyl)oxy]carbonyloxy)undecyl]phosphonate (6). Com-