Xu et al.
butoxy-5-oxopentanoic acid 1 (4.5 g, 10.6 mmol) in DMF (100
mL) were added DIC (0.5 M in DMSO, 70 mL, 35 mmol) and
HOBt (1.7 g, 35 mmol). The mixture was stirred at room
temperature for 30 min and then N1-(2,4-dinitrophenyl)ethane-1,2-
diamine (2) (2.0 g, 8.8 mmol) was added to the mixture. The
reaction was left at room temperature for 10 h and concentrated in
vacuo to remove DMF. The mixture was then diluted with 50%
EtOAc/hexane (500 mL) and washed 3 times with water (500 mL).
The organic layer was dried over Mg SO4 and concentrated. The
crude product was purified by flash column chromatography on
silica gel (50% EtOAc/Hex) to give tert-butyl 2-(((9H-fluoren-9-
yl)methoxy)carbonylamino)-5-(2-(2,4-dinitrophenylamino)ethy-
lamino)-5-oxopentanoate (5.4 g, 8.5 mmol, 96% yield) as a yellow
solid.
from 10 volumes of Et2O (prechilled at -20 °C) and isolated by
centrifugation. After likewise washing the pellet three times further
with Et2O, the peptide was extracted into 50% MeCN/water and
lyophilyzed over 2 days. The dried peptide was then redissolved
in 50% AcOH and purified by RP-HPLC. Isolated yield after
lyophilization is 115 mg (2.5%).
ESI-MS theoretical, MW ) 4497.6; M2+ ) 2249.8, M3+
)
1500.2; observed M2+ ) 2250.8, M3+ ) 1501.2.
Synthesis of Peptides N36 and N36MCA. Peptide N36 was
prepared on a 1.0 mmol scale with use of custom-written DIC/
HOBt protocols for Fmoc SPPS on an automated peptide synthe-
sizer. After removal of the N-terminal Fmoc group, the resin was
divided equally into two potions. To one portion acetic anhydride
(5 mmol) and DIEA (5 mmol) were added to produce the terminal
amide of N36 peptide. To the other portion 7-methoxycoumarinyl-
4-acetic acid was coupled according to standard DIC/HOBt
protocols to give N36MCA peptide. Global deprotection and
cleavage of the peptide-resin anchorage was accomplished by
batchwise treatment with 95:2.5:2.5 TFA:water:triisopropylsilane
(TIPS) for 2 h, followed by filtration and removal of TFA by rotary
evaporation at room temperature. Both peptides were then triturated
from 10 volumes of Et2O (prechilled at -20 °C) and isolated by
centrifugation. After likewise washing the pellet three times further
with Et2O, the peptides were extracted into 50% MeCN/water and
lyophilized over 2 days. The dried peptides was then redissolved
in 50% AcOH and purified by RP-HPLC. Isolated yield of N36
after lyophilization was 79 mg (3.9%); isolated yield of N36MCA
was 76 mg (3.5%)
1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1Η), 8.81 (s, 1H), 8.31
(d, J ) 9.45 Hz, 1H), 7.84 (d, J ) 7.5 Hz, 2H), 7.67 (t, J ) 7.72
Hz, 2H), 7.49 (t, J ) 7.12 Hz, 2H), 7.40 (t, J ) 7.45 Hz, 2H), 7.34
(d, J ) 0.98 Hz, 1H), 7.09 (d, J ) 7.48 Hz, 1H), 6.86 (s, 1H), 5.66
(d, J ) 7.60, 1H), 4.50 (m, 2H), 4.28 (m, 2H), 3.69 (m, 4H), 2.42
(m, 2H), 1.99 (m, 1H), 1.76 (m, 1H), 1.54 (s, 9H). 13C NMR (125
MHz, CDCl3) δ 173.1, 170.7, 156.7, 148.3, 143.7, 141.2, 141.2,
136.22, 130.6, 130.3, 127.7, 127.0, 125.0, 124.9, 124.1, 120.0,
113.9, 83.0, 77.2, 77.0, 76.7, 67.1, 60.3, 53.6, 47.0, 43.0, 38.3,
32.49, 29.6, 27.9, 21.0, 14.1. ESI-TOF calcd for C32H35N5O9 [M
+ H+] 634.2507, found 634.2500.
2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-(2-(2,4-dini-
trophenylamino)ethylamino)-5-oxopentanoic Acid (3). To a
solution of tert-butyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
5-(2-(2,4-dinitrophenylamino)ethylamino)-5-oxopentanoate 3 (5.4
g, 8.5 mmol) in DCM (100 mL) was added TFA (100 mL). The
reaction was left at room temperature for 5 h then concentrated in
vacuo to remove TFA. The crude product was purified by flash
column chromatography on silica gel (10% H2O/ MeCN) to give
2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-(2-(2,4-dinitrophe-
nylamino)ethylamino)-5-oxopentanoic acid (3) (4.3 g, 7.4 mmol,
87% yield) as a yellow solid.
N36 ESI-MS theory, MW ) 4163.9; M2+ ) 2083.0, M3+
1389.0; observed, M2+ ) 2083.5, M3+ ) 1389.5.
)
N36MCA ESI-MS theory, MW ) 4337.9; M2+ ) 2170.0, M3+
) 1446.9; observed, M2+ ) 2170.6, M3+ ) 1447.8.
CD Measurements. Far-UV circular dichroism spectra were
obtained with a 1 cm path length fused silica cell at 4 °C. Purified
peptides, C34, C34Dnp, N36, and N36MCA, were reconstituted at
100 µM. Both of the peptides C34Dnp and N36MCA were diluted
5-fold into a solution in phosphate-buffered saline, pH 7.4,
containing 5 vol % EtOH. Data were corrected for blank absorbance
of each compound, and are reported as the average of three scans.
The CD spectrum of a C34/N36 complex was measured in the same
manner. Thermal stability was determined by monitoring the change
in [θ]222 as a function of temperature. Thermal melts were performed
in 2 deg increments with an equilibration time of 120 s at the desired
temperature and an integration time of 30 s. The apparent Tm was
estimated from the thermal dependence of the CD signal at 222
nm.
Characterization of the FRET System by Using a Series of
Fluorescence-Based Assays. All fluorescence measurements were
performed with excitation at 325 nm. The fluorescence emission
(Eem ) 445 nm) of donor peptide was measured at a range of
peptide concentrations in PBS buffer (10 mM, 100 mM NaCl, pH
7.4), containing 5 vol % EtOH, at room temperature in a BSA-
precoated 96-well EIA/RIA plate (1/2 area, flat bottom). In a typical
titration assay, quencher was added to the N36MCA solution (5
µM) in PBS in a sequential manner. The fluorescence emission
was then measured at 445 nm and data were corrected for the actual
volume of the peptide solution.
Displacement of the 2 µM N36MCA/C34Dnp Hexameric
Bundle by Using Unlabled Peptide C34 or N36. A solution of 2
µM N36MCA/C34Dnp hexameric bundle was prepared by mixing
N36MCA (6 µM) and C34Dnp (6 µM) in PBS, containing 5 vol
% EtOH, at room temperature in a BSA-precoated 96-well EIA/
RIA plate (1/2 area, flat bottom,). The solution was allowed to stand
at room temperature for 30 min. Following the addition of unlabeled
peptide C34 (final concentration 16 µM) and a final volume of
200 µL, the fluorescence emission at 445 nm was measured every
10 min over a period of 2 h. The assay was repeated at three other
C34 concentrations, 32, 64, and 100 µM. The same displacement
assays were performed with N36.
1H NMR (400 MHz, MeOD) 8.97 (s, 1H), 8.81 (m, 1H), 8.22
(d, J ) 9.60 Hz, 1H), 7.76 (t, J ) 7.54 Hz, 2H), 7.65 (t, J ) 8.10
Hz, 2H), 7.37 (t, J ) 7.39 Hz, 2H), 7.30 (t, J ) 7.43, 2H), 7.18 (d,
J ) 9.57, 1H), 4.35 (m, 2H), 4.18 (m, 2H), 3.57 (m, 4H), 2.32 (t,
J ) 6.80 Hz, 2H), 2.19 (m, 1H), 1.95 (m, 1H). 13C NMR (100
MHz, MeOD) 188.8, 175.7, 158.5, 149.7, 145.2, 142.4, 130.9,
128.7, 128.1, 127.2, 126.2, 126.2 120.9, 115.6, 110.6, 67.9, 48.3,
43.9, 42.7, 39.1,33.2, 28.7, 23.5. ESI-TOF calcd for C28H27N5O9
[M + H+] 578.1881, found 578.1873.
Synthesis of Peptide C34. Peptide C34 was prepared on a 1.0
mmol scale with use of custom-written DIC/HOBt protocols for
Fmoc SPPS on an automated peptide synthesizer. Global depro-
tection and cleavage of the peptide-resin anchorage was ac-
complished by batchwise treatment with 95:2.5:2.5 TFA:water:
triisopropylsilane (TIPS) for 2 h, followed by filtration and removal
of TFA by rotary evaporation at room temperature. The peptide
was then triturated from 10 volumes of Et2O (prechilled at -20
°C) and isolated by centrifugation. After likewise washing the pellet
three times further with Et2O, the peptide was extracted into 50%
MeCN/water and lyophilyzed over 2 days. The dried peptide was
then redissolved in 50% AcOH and purified by RP-HPLC. Isolated
yield after lyophilization is 130 mg (3.0%).
ESI-MS theoretical, MW ) 4289.6; M2+ ) 2145.8, M3+
)
1430.8; observed M2+ ) 2146.5, M3+ ) 1431.8.
Synthesis of Peptide C34Dnp. Peptide C34Dnp was prepared
on a 1.0 mmol scale with use of custom-written DIC/HOBt
protocols for Fmoc SPPS on an automated peptide synthesizer and
is depicted in Scheme 2. Dnp labeled Glutamic acid 4 was
incorporated into this stepwise synthetic process. Global deprotec-
tion and cleavage of the peptide-resin anchorage was accomplished
by batchwise treatment with 95:2.5:2.5 TFA:water:triisopropylsilane
(TIPS) for 2 h, followed by filtration and removal of TFA by rotary
evaporation at room temperature. The peptide was then triturated
6706 J. Org. Chem., Vol. 72, No. 18, 2007