M. M. Murr et al. / Bioorg. Med. Chem. 9 (2001) 1141±1148
1147
and the solution heated to re¯ux for 18 h. The mixture
was concentrated in vacuo and partitioned between
CH2Cl2 and 0.1 M sodium citrate buer (pH 4.5). The
organiclayer was extracted with buer (2 Â) saturated
NaHCO3 (2Â) and brine and dried over sodium sulfate,
®ltered, and concentrated in vacuo. The resulting solid
was suspended in absolute ethanol and purged with
nitrogen. Pd on C (10%, 5 g) was added and the ¯ask
charged with H2 at atmosphericpressure and stirred 48
h. The solution was concentrated to 100 mL and 10%
triethylamine in CH2Cl2 was added and the solution ®l-
tered through a pad of Celite and washed with 10%
triethyl amine. The ®ltrate was concentrated and puri-
®ed by silica gel chromatography with 0±10% MeOH in
10% Et3N in CH2Cl2. The desired product was con-
centrated, redissolved in 10% MeOH in CH2Cl2 and 20
mL of HOAc was added to precipitate the product and
the suspension was stirred at room temperature over-
night. The precipitate was ®ltered, rinsed with MeOH
and dried in vacuo to yield the product (3.03 g, 28%) as
N,N-diisopropylethylamine (3.57 mL, 20.5 mmol) were
added and the solution stirred at room temperature for
3 h, diluted with CH2Cl2 and washed with 0.1 M citric
acid buer (pH 4) (2Â) and water (2Â). The solution
was dried over sodium sulfate, ®ltered, and con-
centrated in vacuo. The resulting oil was dissolved in
absolute EtOH and purged with Ar. Pd on C (10%) was
added and the ¯ask charged with H2 at atmospheric
pressure and stirred at room temperature for 3 h. Filter
the reaction through Celite and rinse with 10% MeOH
in CH2Cl2. The ®ltrate was concentrated and puri®ed by
silica gel chromatography (2±10% MeOH in CH2Cl2).
The product was concentrated and crystallized with
30% ethyl acetate/CH2Cl2 and pentane, and the white
1
solid ®ltered. H NMR (300 MHz, DMSO-d6) d 8.17 (t,
1H, J=5.4 Hz), 8.09 (t, 1H, J=5.8 Hz) 7.87 (d, 2H,
J=7.4 Hz), 7.72 (dd, 2H, J=7.2, 4.3 Hz), 7.53 (d, 1H,
J=7.8 Hz), 7.41 (t, 2H, J=6.9 Hz), 7.32 (t, 2H, J=7.0
Hz), 6.76 (t, 1H, J=5.3 Hz), 4.32.19 (m, 3H), 3.96 (br q,
1H), 3.74 (t, 4H, J=6.1 Hz), 2.88 (q, 2H, J=4.5 Hz),
1.7.1 (c, 6H), 1.35 (s, 9H); 13C NMR (300 MHz,
DMSO-d6) d 172.3, 171.1, 169.1, 156.1, 155.6, 143.9,
143.8, 140.7, 127.6, 127.1, 125.3, 120.1, 77.3, 65.6, 54.7,
46.7, 40.6, 39.7, 31.4, 29.2, 28.3, 22.8; HRMS (FAB)
m/z 583.2793 (583.2768 calcd for C30H39N4O8, MH).
1
a tan solid. H NMR (DMSO-d6) d 8.58 (s, 4H), 6.9 (br
t, 1H), 4.25 (t, J=7.5 Hz, 2H), 4.12 (br t, 2H), 3.27 (br
q, 2H), 2.62 (t, J=7.4 Hz, 2H), 1.20 (s, 9H); 13C NMR
(DMSO-d6) d 172.4, 162.7, 162.4, 155.8, 130.4, 130.3,
126.4, 126.1, 77.5, 37.6, 36.1, 32.0, 28.1; HRMS (FAB)
m/z 482.1547 (482.1563 calcd for C24H24N3O8).
Solid-phase peptide synthesis. Fmoc-Lys(BOC) function-
alized Tenta-Gel Resin (NovaBiochem, 0.75 g, 0.13
mmol/g loading capacity) was added to 10 mL fritted
®lter ¯ask with a screw cap and Te¯on stopcock. The
resin was washed with DMF (3Â5 mL), iPOH (3Â3
mL), and DMF (3Â5 mL), and then deprotected with 5
mL of 20% piperidine in DMF for 20 min. The resin
was washed with DMF/iPOH/DMF. A 0.195 mM stock
solution of PyBOP was made in DMF and a 0.195 mM
solution of N-methyl morpholine in DMF. Compound
A (128.8 mg, 0.195 mmol) was dissolved in 1 mL of the
PyBOP stock solution and added to the resin followed
by 2 mL of the NMM stock solution. The coupling
reacted for 45 min, was ®ltered, and the resin washed
with DMF/iPOH/DMF. This coupling was repeated,
and then the resin was capped with a solution of 20%
acetic anhydride with 1 mL NMM stock solution for 20
min and the resin rinsed. The resin was deprotected,
washed and then the coupling was repeated with com-
pound B (113.6 mg, 0.195 mmol). This process was
repeated with alternating couplings until the octamer
had been synthesized. After the ®nal piperidine depro-
tection the resin was rinsed with MeOH and CH2Cl2
and dried in vacuum dessicator for 2 h. The compound
was then cleaved o the resin with 95% TFA/5% water
for 12 h and the resin beads ®ltered o. The ®ltrate was
concentrated in vacuo and ether was added to pre-
cipitate the product. The resulting reddish brown solid
was puri®ed by reverse-phase C18 column (PrepRPC
15m, Pharmacia) with a 2-h gradient (0±100% 0.07%
TFA in ACN in 0.07% TFA in water). The product
fractions were combined and lyopholized to yield the
octamer (15 mg, 2.6%) as a light-yellow solid.
N-2-(Nꢀ-9-Fluorenylmethoxycarbonylglycyl)aminoethyl-
N0 - (2 - carboxyethyl) - 1,4,5,8 - napthalenetetracarboxylic
diimide1 (compound A). The above product (2 g, 4.16
mmol) was suspended in 15 mL CH2Cl2 and 15 mL
TFA was added slowly. After standing for 10 min, the
solution was evaporated and the residual TFA removed
by azeotropicevaporation (2 Â) from heptane. The
resulting solid was triturated with ether, ®ltered, and
dried in vacuo. The solid was suspended in DMF (15
mL) and N-9-¯uorenylmethoxycarbonylglycine penta-
¯uorophenyl ester (1.92 g, 4.16 mmol) was added, fol-
lowed by 1-hydroxybenztriazole (HOBT, 561 mg, 4.16
mmol) and 2,6-lutidine (890 mg, 8.32 mmol). After stir-
ring for 4 h, the mixture was poured slowly into rapidly
stirred H2O (150 mL), and the resulting suspension was
allowed to stand for several hours. The mixture was ®l-
tered and the resulting yellow solid was rinsed with H2O
and dried in the presence of P2O5 in a vacuum desicca-
tor overnight. The crude product was triturated with
Et2O several times (to remove the residual 1-hydroxy-
benztriazole and penta¯uorophenol), ®ltered, and dried
1
in vacuo to the product (77%) as a yellow powder. H
NMR (DMSO-d6) d 8.58 (s, 4H), 6.90 (br t, 1H), 4.25 (t,
J=7.5 Hz, 2H), 4.12 (br t, 2H), 3.27 (br q, 2H), 2.62 (t,
J=7.4 Hz, 2H), 1.20 (s, 9H); 13C NMR (DMSO-d6)
172.4, 162.7, 162.4, 155.8, 130.4, 130.3, 126.4, 126.1,
77.5, 37.6, 36.1, 32.0, 28.1; HRMS (FAB) m/z 482.1547
(482.1563 calcd for C24H24N3O8).
Nꢀ-9-Fluorenylmethyoxycarbonyl-(N"-tert-butoxycarbo-
nyl)lysylglycylglycine (compound B). Na-9-Fluorenyl-
methyoxycarbonyl-(Ne-tert-butoxycarbonyl)lysine (3.0
g, 6.4 mmol) and glycylglycine benzyl ester p-toluene-
sulfonate salt (1.42 g, 6.4 mmol) were dissolved in
CH2Cl2. (Benzotriazole-1-yloxy)tripyrrolidinophospho-
nium hexa¯uorophospate (PyBOP, 5.0 g, 9.6 mmol) and
1
Octamer 4. H NMR (500 MHz, D2O) d 8.24.14 (m,
32H), 4.30.50 (m, 100H), 3.02.64 (m, 32H), 1.83.44 (m,
48H) MS (ESI) m/z 744.9 (744.5 calcd for (M+7H)7+),