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was washed successively with 95% EtOH and anhydrous ethyl
ether to yield 7‚Et3N (0.1538 g, 60.8%): mp 203 °C dec; 1H
NMR (DMSO-d6) δ 1.62 (br s, 4H, CH2CH2), 5.79 (m, 1H, CH);
MS (FAB) m/z 852.3 (MH+). Anal. (C44H39N5O7SCl2‚(C2H5)3N)
C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1R,S)-1-[N-(N-
flu or escein yl-5-th iou r eid oglycylglycin a m id o)-4-a m in o-
p h en yl]-2-(1-p yr r olid in yl)eth yl}a ceta m id e (8). Interme-
diate 16‚HCl (0.1195 g, 0.1729 mmol) and 24 mg of 10% Pd/C
were mixed in MeOH (6 mL) and hydrogenated at 25 °C under
10 psi for 138 h before the mixture was filtered through Celite,
and the Pd/C was washed thoroughly with hot MeOH. After
evaporation of the filtrate, the residue was partitioned between
1 N NaOH (150 mL) and EtOAc (300 mL), and the organic
fraction was dried (Na2SO4), filtered through Celite, and
evaporated. To a solution of the deprotected intermediate
(0.0615 g, 0.1182 mmol) in absolute EtOH (3 mL) and Et3N (2
mL) were added FITC-I (0.0479 g, 0.1229 mmol) and freshly
distilled THF (1 mL) with stirring at 25 °C under N2. After 7
days in the dark, the mixture was evaporated, and Et3N was
removed azeotropically with dry CH3CN (16 mL) to yield a
solid which was suspended in absolute EtOH (8 mL) and
collected by filtration. Washing with anhydrous ethyl ether
and drying yielded 8‚Et3N (0.0887 g, 74%): mp 208 °C dec;
1H NMR (DMSO-d6) δ 1.61 (m, 4H, CH2CH2), 5.80 (m, 1H, CH);
MS (FAB) m/z 909.3 (MH+). Anal. (C46H42N6O8SCl2‚(C2H5)3N)
C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1R,S)-1-[N-(N-
flu or escein yl-5-th iou r eidoglycylglycylglycylglycin am ido)-
4-a m in op h en yl]-2-(1-p yr r olid in yl)et h yl}a cet a m id e (9).
Intermediate 17‚HCl (0.0729 g, 0.0905 mmol) and 16 mg of
10% Pd/C were mixed in MeOH (20 mL) and hydrogenated at
25 °C under 10 psi. After 72 h, the mixture was filtered
through Celite. The Pd/C was washed thoroughly with hot
MeOH (100 mL), and the combined filtrate was evaporated.
To a solution of the 1 HCl salt of the deprotected intermediate
(0.0500 g, 0.0745 mmol) in absolute EtOH (5 mL) and Et3N (4
mL) was added FITC-I (0.0309 g, 0.0794 mmol) in dry THF (2
mL) with stirring at 25 °C under N2 in the dark. After 3 days,
more fluorescein isothiocyanate isomer I (0.0298 g, 0.0765
mmol) and 100% EtOH (4 mL) were added, and the stirring
continued for 2 days. The mixture was then evaporated, and
Et3N was azeotropically removed with dry CH3CN (8 mL). The
solid was purified on 0.25 mm reverse-phase-18 TLC plates
eluting with 100% MeOH. The residue solidified upon addition
of dry CH3CN, and the solid was collected by filtration to yield
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1
9‚Et3N (0.0343 g, 41%): mp 198 °C dec; H NMR (DMSO-d6)
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Ack n ow led gm en t. We thank Veronika Phillips,
J oan Naeseth, and Michael Powers for in vitro testing
of the compounds. We also acknowledge funding from
U.S. Public Health Service Grants DA-08131, DA-04381,
and DA-09924.
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