(138 mg, 0.277 mmol, 52%). [a]2D0 -58.2 (c 1.00, CHCl3). IR (film)
3263, 2959, 1699, 1666, 1641, 1537, 1263, 1238, 1121, 1045, 1028,
961 cm-1. 1H NMR (500 MHz, CD3OD): d ppm 7.35-7.29 (m, 6H),
6.74 (dd, J1 = 16.3, J2 = 5.0 Hz), 5.59-5.55 (m, 1H), 5.13-5.05 (m,
2H), 4.56-4.55 (m, 1H), 4.38-4.35 (m, 1H), 4.12-4.09 (m, 1H), 1.70-
34 (m, 9H), 0.96-0.97 (m, 18H). 13C NMR (125 MHz, CD3OD):
d ppm 175.11, 175.03, 173.93, 173.85, 158.24, 156.50, 137.78,
129.34, 129.27, 128.87, 118.16, 99.83, 67.50, 54.90, 53.13, 50.20,
42.94, 41.95, 41.40, 25.76, 25.60, 25.54, 23.37, 22.26, 22.14, 22.00.
HRMS: calcd. For [C28H43N4O4]+ 499.32788, found 499.32764.
chromatography yielded the title compound (1.21 g, 3.38 mmol,
75%). H NMR (400 MHz, CD3OD): d ppm 4.46-4.39 (m, 1H),
1
4.11 (t, J = 7.45, 7.45 Hz, 1H), 1.76-1.46 (m, 6H), 1.43 (s, 9H),
0.95-0.87 (m, 12H).
General protocol for azide couplings
The Boc-protected warhead was dissolved in TFA : DCM (1 : 1,
v/v) and stirred for 20 min. Coevaporation with toluene (3¥)
afforded the warhead TFA-salt, which was used without further
purification. The appropriate hydrazide (16 or 18) was dissolved in
1 : 1 DMF–DCM (v/v) and cooled to -30 ◦C. tBuONO (1.1 equiv.)
and HCl (4 M sln. in 1,4-dioxane, 2.8 equiv.) were added, and the
mixture was stirred for 3 h. at -30 ◦C after which TLC analysis
(10% MeOH–DCM, v/v) showed complete consumption of the
starting material. The warhead-TFA salt was added to the reaction
mixture as a solution in DMF with 1.1 equivalent of DiPEA. A
further 3.9 equivalents were added to the reaction mixture, and
this mixture was allowed to warm to RT slowly overnight. The
mixture was diluted with EA and extracted with H2O (3¥). The
organic layer was dried over MgSO4 and purified by flash column
chromatography.
(S,E)-4-Azido-Phe-Leu2-amino-6-methylhept-2-enenitrile (Ib)
Following the general procedure for block coupling, the ti-
tle compound was obtained from (S,E)-4-Boc-Leu2-amino-6-
methylhept-2-enenitrile 19 (66 mg, 0.14 mmol, 1 equiv.) and azido-
phenylalanine21 21 (29 mg, 0.154 mmol, 1.1 equiv.). Flash column
chromatography (EA : tol (0-30%)) yielded the title compound
(53 mg, 99 mmol, 70%). [a]2D0 -55.6 (c 0.72, CHCl3). IR (film)
1
3271, 2957, 2110, 1639, 1541, 1456, 1387, 1224, 961 cm-1. H
NMR (400 MHz, CDCl3): d ppm 7.35-7.20 (m, 5H), 7.06-6.98
(m, 1H), 6.96-6.86 (m, 1H), 6.59 (dd, J1 = 16.3 Hz, J2 = 5.5 Hz,
1H), 5.44 (d, J = 16.3 Hz, 1H), 4.57-4.42 (m, 3H), 4.24 (dd, J1 =
7.8 Hz, J2 = 4.1 Hz, 1H), 3.21 (dd, J1 = 14.2 Hz, J2 = 3.6 Hz,
1H), 3.05 (dd, J1 = 14.2 Hz, J2 = 7.9 Hz, 1H), 1.82-1.22 (m, 9H),
1.04-0.73 (m, 18H). 13C NMR (100 MHz, CDCl3): d ppm 171.78,
171.43, 169.05, 154.39, 135.61, 129.37, 128.69, 127.35, 117.05,
99.73, 64.75, 51.95, 51.87, 49.17, 42.42, 41.10, 40.36, 38.00, 24.92,
24.67, 24.59, 22.80, 22.69, 22.61, 22.20, 21.77. HRMS: calcd. for
[C29H44N7O3]+ 538.35001, found 538.34979.
General protocol for block couplings
The Boc-protected tripeptide was dissolved in TFA : DCM (1 : 1,
v/v) and stirred for 20 min. Coevaporation with toluene (3¥)
afforded the tripeptide TFA-salt, which was used without further
purification. The carboxylic acid (21 or 22, 1 equiv.) was dissolved
in DCM–DMF (1/1, v/v). HBTU (1.1 equiv.), DiPEA (3.5 equiv.)
were added and the mixture was stirred for 5 min. A solution of
the tripeptide TFA salt in DMF was added and the mixture was
stirred for 2 h. before being concentrated. The residue was taken
up in DCM, washed with 1 M HCl (2¥), sat. aq. NaHCO3 (4¥),
brine, and dried with Na2SO4. The residue was purified by flash
column chromatography.
(S,E)-4-Ada-Ahx3-Leu2-amino-6-methylhept-2-enenitrile (Ic)
Following the general procedure for block coupling, the title com-
pound was obtained from (S,E)-4-Boc-Leu2-amino-6-methylhept-
2-enenitrile 19 (64 mg, 138 mmol) and Ada-Ahx3-OH 22 (81 mg,
152 mmol, 1.1 equiv.). Flash column chromatography (MeOH–
DCM (2-8%)) yielded Ic (108 mg, 123 mmol, 80%). [a]2D0 -38.2 (c
1.00, MeOH). IR (film) 3277, 2928, 2905, 2849, 1636, 1541, 1456,
1368, 1244, 1171, 962 cm-1. 1H NMR (400 MHz, CD3OD): d ppm
8.12-8.04 (m, 2H), 8.00 (d, J = 8.24 Hz, 1H), 7.95 (t, J = 5.06,
5.06 Hz, 2H), 7.87-7.82 (m, 1H), 6.73 (dd, J = 16.36, 5.04 Hz,
1H), 5.55 (d, J = 16.35 Hz, 1H), 4.60-4.49 (m, 1H), 4.39-4.25
(m, 2H), 3.13 (dd, J = 12.14, 6.00 Hz, 6H), 2.23 (t, J = 7.27,
7.27 Hz, 2H), 2.15 (t, J = 7.39, 7.39 Hz, 4H), 1.95-1.87 (m, 5H),
1.76-1.24 (m, 38H), 0.96-0.84 (m, 18H). 13C NMR (100 MHz,
CD3OD): d ppm 176.45, 175.95, 175.17, 174.37, 173.73, 156.76,
118.25, 99.86, 53.71, 53.45, 51.93, 50.40, 43.74, 43.12, 41.67, 41.51,
40.31, 40.26, 40.18, 37.90, 37.04, 36.66, 33.77, 30.22, 30.15, 30.12,
27.65, 27.59, 27.56, 26.73, 26.56, 25.94, 25.89, 25.78, 23.47, 23.42,
22.12, 21.93, 21.88. HRMS calcd. for [C50H86N7O6]+ 880.66341,
found 880.66397.
(S,E)-4-Boc-Leu2-amino-6-methylhept-2-enenitrile (19)
Following the general procedure for azide coupling the title
compound was obtained from (S,E)-4-Boc-amino-6-methylhept-
2-enenitrile 13 (49 mg, 0.21 mmol, 1.1 equiv.) and Boc-Leu2-
NHNH2 (18, 67 mg, 0.187 mmol, 1 equiv.). Purification by
flash chromatography (EA : PE (0-30%)) gave the title compound
1
(66 mg, 0.14 mmol, 76%). H NMR (400 MHz, CDCl3): d ppm
7.00-6.90 (m, 1H), 6.62 (dd, J1 = 16.4 Hz, J2 = 4.9 Hz, 2H), 5.56
(d, J = 16.4 Hz, 1H), 5.12-5.00 (m, 1H), 4.67-4.58 (m, 1H), 4.41-
4.35 (m, 1H), 4.08-4.00 (m, 1H), 1.82-1.25 (m, 18H), 1.10-0.85
(m, 18H). 13C NMR (100 MHz, CDCl3): d ppm 172.89, 171.45,
156.17, 154.54, 117.31, 99.64, 80.81, 53.92, 52.23, 48.92, 42.69,
40.57, 40.09, 28.23, 24.88, 24.82, 24.69, 22.96, 22.89, 22.61, 21.86,
21.75, 21.67.
(S,E)-4-Cbz-Leu2-amino-6-methylhept-2-enenitrile (Ia)
Competition experiments in vitro
Following the general procedure for azide coupling the title
compound was obtained from (S,E)-4-Boc-amino-6-methylhept-
2-enenitrile 13 (141 mg, 0.59 mmol, 1.1 equiv.) and Cbz-Leu2-
NHNH2 (16, 210 mg, 0.534 mmol, 1 equiv.). Purification by
flash chromatography (EA : PE (0-40%)) gave the title compound
HEK293T cells were cultured on DMEM supplemented with 10%
Fetal Calf Serum (FCS), 10 units/ml penicillin and 10 mg ml-1
streptomycin in a 7% CO2 humidified incubator at 37 ◦C. Cells
were harvested, washed with PBS (2¥) and permeated in digitonin
lysis buffer (4¥ pellet volume, 50 mM Tris pH 7.5, 250 mM
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1885–1893 | 1891
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