5284 J. Am. Chem. Soc., Vol. 119, No. 23, 1997
Fox and Galoppini
Vacuo overnight (T(bath) ) 40 °C) to afford a pale yellow solid (690
mg, 91%) which was used in the next step without further purification.
Boc-Ala-Pro-Ala-dmaPhe-Ala-Pro-Ala-OEt was prepared follow-
ing the general coupling procedure using Boc-Ala-Pro-Ala-OH (2
mmol), H-dmaPhe-Ala-Pro-Ala-OEt (1.6 mmol), HOBT (270 mg, 2
mmol), EDCI (385 mg, 2 mmol), and NMM (0.5 mL) in DMF (30
mL). Purification by column chromatography (90% ethyl acetate/10%
MeOH, v/v) and trituration with ether afforded the product as an off-
white solid (780 mg, 86%), which was further purified with a second
column chromatography using the same solvent mixture to afford a
white solid: the 1H NMR spectrum shows about 10% of another isomer
white solid obtained was dried in Vacuo overnight, in the dark, to afford
the acid as a white solid, which was used in the next step without further
purification.
Boc-Ala-Pro-Ala-pyrAla-Ala-Pro-Ala-OEt was prepared following
the general coupling procedure from H-Ala-Pro-Ala-OEt (1 mmol),
Boc-Ala-Pro-Ala-pyrAla-OH (0.68 mmol), HOBT (115 mg, 1 mmol),
EDCI (160 mg, 1 mmol), NMM (30 µL), and DMF (10 mL).
Purification by two consecutive column chromatography runs (ethyl
acetate, followed by 80% ethyl acetate/20% MeOH, v/v) and trituration
with ether afforded the peptide as a white solid (410 mg, 67%): 1H
NMR δ 8.27 (d, 1H, J ) 9.0, pyrene), 8.15 (d, 1H, J ) 7.5, pyrene),
8.12 (d, 1H, J ) 7.5, pyrene), 7.95-8.05 (m, 5 H, pyrene), 7.8 (d, 1H,
J ) 7.7, pyrene), 7.14 (d, 1H, J ) 6, NH), 6.54 (br s, 1H, NH), 5.25
(d, 1H, J ) 9.0, NH), 4.40-4.30 (m, 4H), 4.14 (q, 2H, J ) 7.5, OCH2-
CH3), 3.9 (m, 1 H), 3.6 (m, 2H), 3.47 (dd, 2H, J1 ) J2 ) 7), 3.40 (m,
1 H), 3.30 (m, 1H), 2.0-1.85 (br m, 9H), 1.43 (s, 9H, C(CH3)2), 1.42
(br s, 3H, CH3), 1.3-1.2 (m, 16H, CHCH3 and -CH2-) ppm; 13C
NMR δ 173.031, 172.72, 171.82, 171.25, 170.84, 170.62, 169.96,
155.31 (-C(dO)sOEt), 131.31,130.97, 130.73, 130.44, 129.43, 128.03,
127.95, 127.64, 127.48, 126.92, 125.94, 125.11, 124.96, 124.81, 124.77,
123.24, 79.95 (OsC(CH3)3), 65.85, 61.25, 60.32, 59.46, 54.69, 49.44,
48.63, 47.99, 47.24, 46.93, 35.84, 28.58 (3C, OsC(CH3)3), 27.55, 25.19,
24.78, 18.04, 17.99 (2C), 17.67, 15.27, 14.11 ppm; CI-MS m/z 896
(100), 796 (18), 670(1), 626 (7); HRMS calcd for C48H62N7O10
896.4558, found 896.4558.
1
(not reported); H NMR δ 7.6 (br s, 1H, NH), 7.34 (d, 1H, J ) 7.1,
NH), 7.19 (d, 1H, J ) 7.1, NH), 6.95 (d, 2H, phenyl, J ) 8.5, and 1H,
NH), 6.58 (d, 2H, J ) 8.7, phenyl), 5.45 (br s, 1H, NH), 4.8 (m, 3H),
4.3-4.6 (m, 3 H), 4.16 (q, 2H, J ) 6.0, OCH2CH3), 3.5-3.8 (m, 1H),
2.7-3.0 (m, 3 H), 2.8-3.0 (m, 2H), 2.95 (s, 6H, -N(CH3)2), 1.9-2.2
(series of br m, 10 H), 1.44 (s, 9H, C(CH3)2), 1.4-1.2 (m, 15 H, CHCH3
and OCH2CH3) ppm; 13C NMR δ 173.11, 172.78, 172.81, 171.87,
171.78, 171.26, 171.05, 170.68, 155.43 (-C(dO)sOEt), 149.59
(phenyl), 130.28 (2C, phenyl), 124.54 (phenyl), 112.92 (2C, phenyl),
79.05 (OsC(CH3)3), 61.219, 60.35, 59.92, 54.7, 49.35, 48.50, 48.07,
47.40, 47.24, 46.74, 40.73 (2C, -N(CH3)2), 37.68, 28.38 (3C, C(CH3)3),
25.21, 25.06, 18.51, 18.36, 18.11, 17.96, 17.90, 14.14 ppm; CI-MS
m/z 576 (100), 562 (4), 520 (8), 503 (3), 330 (11), 286 (6); HRMS
calcd for C40H63N8O10 815.4667, found 815.4665.
Boc-Ala-Pro-Ala-dmaPhe-Ala-Pro-Ala-OH was prepared follow-
ing the general procedure for ester hydrolysis from Boc-Ala-Pro-Ala-
dmaPhe-Ala-Pro-Ala-OEt (123 mg, 0.15 mmol) and NaOH (8 mg in 5
mL of H2O) in ethanol (40 mL). The peptide solution was cooled with
an ice bath before the addition of NaOH, allowed to warm to room
temperature over 5 h, and then stirred for 2 h at room temperature and
neutralized to pH 6-7 with 5% NaHSO4(aq). The white solid obtained
was used in the next step without further purification.
Boc-Ala-Pro-Ala-pyrAla-Ala-Pro-Ala-OH. The acid was prepared
following the general procedure for ester hydrolysis from Boc-Ala-
Pro-Ala-pyrAla-Ala-Pro-Ala-OMe (140 mg, 0.15 mmol) and NaOH
(8 mg in 10 mL of H2O) in ethanol (20 mL). The peptide solution
was cooled with an ice bath before adding the NaOH solution. The
solution was allowed to warm to room temperature over 4 h, stirred
overnight at room temperature, and then acidified to pH 5 with 5%
NaHSO4(aq). The solvent was evaporated, and the white solid obtained
was dried in Vacuo overnight to afford the acid as a white solid, which
was used in the next step without further purification.
H-Ala-Pro-Ala-pyrAla-Ala-Pro-Ala-OEt. The deprotected peptide
was prepared following the procedure used for Boc-dmaPhe-Ala-Pro-
Ala-OEt from Boc-Ala-Pro-Ala-pyrAla-Ala-Pro-Ala-OEt (145 mg, 0.15
mmol) and Me3SiI (40 µL, 0.28 mmol) in CH3CN (10 mL) followed
by MeOH (0.5 mL).
Boc-Ala-Pro-Ala-pyrAla-(Ala-Pro-Ala)2-Ala-dmaPhe-Ala-Pro-
Ala-OEt (3). The peptide 3 was prepared following the general
coupling procedure from H-Ala-Pro-Ala-dmaPhe-Ala-Pro-Ala-OEt
(0.15 mmol), Boc-Ala-Pro-Ala-pyrAla-Ala-Pro-Ala-OH (0.15 mmol),
HOBT (30 mg, 0.2 mmol), EDCI (40 mg, 0.2 mmol), and NMM (110
µL) in DMF (10 mL). Purification by a short silica gel column (ethyl
acetate followed by increasing amounts of EtOH and then pure EtOH)
and trituration with ether afforded 3 as a white solid (30 mg, 12%):
1H NMR δ 8.36 (d, 1H, J ) 9.8, pyrene), 8.15 (d, 1H, J ) 8.4, pyrene),
8.12 (d, 1H, J ) 8.4, pyrene), 7.9-8.1 (m, 6 H, pyrene), 7.8 (br s, 1H,
NH), 7.4-7.6 (series of br m, 6 H, NH), 7.17 (d, 2H, J ) 8.7, phenyl),
6.54 (d, 2H, J ) 8.7, phenyl), 5.29 (br s, 1H, NH), 4.90 (m, 1H), 4.0-
4.55 (m, 13 H and q, 2H, J ) 7.0, OCH2CH3), 3.5-3.9 (m, 10 H), 3.2
(br m, 1H), 2.7-3.0 (br m, 2H), 2.69 (s, 6H, -N(CH3)2), 1.6-2.4 (br
m, 16 H), 1.57 (d, 3H, J ) 8, CHCH3), 1.52 (d, 3H, J ) 7, CHCH3),
1.49 (s, 9H, tBu, C(CH3)2), 1.44 (d, 3H, J ) 3, CHCH3), 1.40 (d, 3H,
J ) 7, CHCH3), 1.38 (d, 3H, J ) 7, CHCH3), 1.34 (d, 3H, J ) 7,
CHCH3), 1.29 (d, 3H, J ) 6, CHCH3), 1.24 (t, 3H, J ) 7, OCH2CH3),
1.16 (d, 3H, J ) 7, CHCH3) ppm; 13C NMR δ 173.39, 173.43, 173.28,
173.03, 172.83, 172.74, 172.62, 172.57 (2C), 172.77 (2C), 171.67,
171.63 (2C), 156.08 (-C(dO)sOEt), 149.34 (phenyl), 131.47, 131.24,
130.71, 130.40, 130.26 (2C, phenyl), 129.91, 129.01, 127.78, 127.35,
127.07, 126.03, 125.24, 125.08, 124.82, 124.32 (phenyl), 122.82, 112.73
(2C, phenyl), 80.96 (OsC(CH3)2), 65.85, 63.15, 62.77, 62.65, 61.11,
60.17, 58.44, 55.32, 54.51, 53.68, 52.44, 51.27, 50.71, 49.06, 48.24,
48.01, 47.79, 47.74, 47.26, 40.69 (2C, -N(CH3)2), 28.76, 28.38 (3C,
C(CH3)2), 27.76, 26.15, 26.10, 25.97, 25.21, 18.45, 17.95, 17.75, 17.47,
16.95, 16.57, 16.46, 16.19, 15.27, 15.14, 14.15 ppm; FAB-MS m/z 1586
(88), 1565 (90), 1564 (100), 1563 (75), 1562 (70), 1487 (35), 1487
(50), 1250 (40); HRMS calcd for C81H110N15O17 1564.8204, found
1564.8209.
H-Ala-Pro-Ala-dmaPhe-Ala-Pro-Ala-OEt. The deprotected tet-
rapeptide was prepared following the procedure used for H-dmaPhe-
Ala-Pro-Ala-OEt from Boc-Ala-Pro-Ala-dmaPhe-Ala-Pro-Ala-OEt (125
mg, 0.15 mmol), and Me3SiI (34 µL, 0.2 mmol) in CH3CN (10 mL)
followed by MeOH (0.5 mL). The reaction flask was protected from
light. The off-white solid obtained was dried for 6 h in high vacuum
and immediately used in the next step without further purification.
Boc-Ala-Pro-Ala-pyrAla-OMe was prepared following the general
coupling procedure from Boc-Ala-Pro-Ala-OH (570 mg, 1.6 mmol),
H-pyrAla-OMe (490 mg, mmol),4a,26 HOBT (230 mg, 1.7 mmol), EDCI
(326 mg, 1.7 mmol), and NMM (0.3 mL) in DMF (20 mL). Purification
by column chromatography (CH2Cl2 followed by 95% ethyl acetate/
5% MeOH, v/v) and trituration with ether afforded the peptide as an
1
off-white solid (560 mg, 53%): the H NMR spectrum shows about
1
20% of another isomer (not reported); H NMR δ 8.36 (d, 1H, J )
9.3, pyrene), 8.2-8.0 (m, 7 H, pyrene), 7.85 (d, 1H, J ) 7.8), 7.03 (d,
1H, J ) 7.3, NH), 6.85 (d, 1H, J ) 7.6, NH), 5.26 (d, 1H, J ) 7.6,
NH), 5.08 (dd, 1H, J1 ) 7.6, J2 ) 6.5), 4.5-4.2 (series of m, 2H),
3.9-3.8 (m, 3 H), 3.86 (s, 3 H, OCH3), 3.7-3.5 (series of m, 3 H),
1.95 (m, 2 H), 1.85 (m, 2H), 1.43 (s, 9H, C(CH3)2 ), 1.27 (d, 3H, J )
7.0, CHCH3), 1.24 (d, 3H, J ) 6.8, CHCH3) ppm; 13C NMR δ 173.11,
171.96, 171.74, 170.93, 155.20 (-C(dO)sOEt), 131.32, 130.79,
130.61, 130.06, 129.58, 127.91 (2C), 127.42, 127.21, 126.00, 125.92,
125.10 (2C), 124.70, 123.09, 79.77 (OsC(CH3)3), 65.84, 63.20, 59.77,
53.72, 52.35, 48.91, 48.00, 47.11, 42.58, 37.51, 35.31, 28.36 (3C,
C(CH3)3), 27.07, 25.06, 18.33, 17.50, 15.27 ppm; CI-MS m/z 644 (28),
643 (10), 543 (8), 414 (8), 386 (45), 372(19), 332 (100), 286 (12);
HRMS calcd for C36H43N4O7 643.3131, found 643.3120.
Boc-Ala-Pro-Ala-pyrAla-OH. The acid was prepared following
the general procedure for ester hydrolysis from Boc-Ala-Pro-Ala-
pyrAla-OMe (0.44 g, 0.68 mmol) and NaOH (30 mg in 5 mL of H2O)
in ethanol (20 mL), but by adding the NaOH to the solution cooled
with an ice bath. The solution was allowed to warm to room
temperature, stirred overnight at room temperature, and then acidified
to pH 5 with 5% NaHSO4(aq). The solvent was evaporated, and the
(26) The synthesis of H-pyrAla-OMe was adapted from (a) Schmidt,
U.; Lieberknecht, A.; Wild, J. Synthesis 1988, 159. (b) Schmidt, U.;
Lieberknecht, A.; Wild, J. Synthesis 1984, 53. (c) Burk, M. J; Feaster, J.
E.; Nugent, W. A.; Harlow, R. L. J. Am. Chem. Soc. 1993, 115, 10125.
Boc-Ala-Pro-Ala-dmaPhe-(Ala-Pro-Ala)2-pyrAla-Ala-Pro-Ala-