Homochiral 4-Azalysine Building Blocks
J . Org. Chem., Vol. 67, No. 12, 2002 4027
ether/petroleum ether bp 40-60 °C to give the free acid 29a
(0.42 g, 72%) as an amorphous solid: mp 79-85 °C; TLC
(chloroform/methanol/acetic acid, 2:0.2:0.02) Rf ) 0.68; δH 1.38
(9H, s), 2.01 (3H, s), 3.00-3.90 (6H, m), 4.25-4.65 (1H, m),
4.90-5.15 (2H, m), 5.15-5.40 (1H, br m), 6.00-6.40 (1H, br
d), 7.28 (5H, s), 10.96 (1H, br s); m/z (ES-MS) 423.62 (MH+),
calcd 424.2. Anal. Calcd for C20H29N3O7‚H2O: C, 54.42; H, 7.02;
N, 9.52. Found: C, 54.44; H, 6.94; N, 9.65.
7.45 (4H, m), 7.45-7.65 (2H, m), 7.65-7.85 (2H, m), 8.39 (1H,
br s); δC 28.21, 28.30, 42.07, 46.98, 47.91, 49.01, 54.22, 67.28,
80.29, 81.25, 119.86, 125.14, 127.01, 127.62, 127.93, 141.17,
143.59, 143.82, 156.11, 156.53, 172.98; m/z (ES-MS) 571 (MH+),
calcd 570.3. Anal. Calcd for C30H39N3O8: C, 63.25; H, 6.90; N,
7.37. Found: C, 62.70; H, 6.84; N, 7.36.
(S)-N2-9-F lu or en ylm et h oxyca r b on yl-N6-ter t-b u t oxy-
ca r bon yl-N4-cycloh exylca r ba m oyl-4-a za lysin e (31d ). Us-
ing 30d (0.32 g, 0.86 mmol), NaHCO3 (0.216 g, 2.57 mmol),
and FmocOSu (0.32 g, 0.94 mmol), in the procedure described
for 31a , on recrystallization from ethyl acetate/hexane gave
the title compound 31d (0.38 g, 75%): mp 110-112 °C; TLC
N2-Ben zyloxyca r bon yl-N4,N6-bis-ter t-bu toxyca r bon yl-
4-a za lysin e (29c). Treatment of 28c (1.301 g, 2.63 mmol) with
aqueous LiOH (0.122 g, 2.9 mmol), as described in the
procedure for 29a , gave 29c as colorless plates (1.0 g, 79%):
mp 45-46 °C; TLC (chloroform/methanol/acetic acid, 2.0:0.2:
(chloroform/methanol/acetic acid, 3.5:0.2:0.1) Rf ) 0.38; [R]27
D
0.02) Rf ) 0.4; [R]26 -12.44 (c 2.09, MeOH); δH 1.42 (18H, s),
-3.48 (c 1.15, MeOH); δH 0.90-2.00 (11H, m), 1.36 (9H, s),
2.98-3.81 (6 H, m), 4.12-4.68 (4H, m), 5.42 (1H, br s), 6.39
(1H, br s), 6.52 (1H, d, J 6.62), 7.12-7.46 (4H, m), 7.46-7.69
(2H, m), 7.69-7.86 (2H, m); δC 25.04, 25.59, 33.25, 28.29, 39.20,
47.00, 47.79, 50.25, 51.61, 56.50, 67.29, 80.30, 119.94, 125.19,
127.71, 141.22, 143.55, 143.81, 156.40, 157.00, 158.86, 172.34;
m/z (ES-MS) 595.3 (MH+), calcd 595.31. Anal. Calcd for
D
3.00-3.90 (6H, m), 4.20-4.80 (1H, m), 5.09 (2H, br s), 5.20-
5.60 (1H, m), 5.90-6.50 (1H, m), 7.30 (5H, s), 9.75 (1H, s); δC
27.94, 28.09, 38.90, 47.60, 48.58, 53.41, 66.71, 79.30, 80.75,
127.75, 128.16, 135.94, 155.95, 156.28, 172.78; m/z (FAB) 482
(MH+), calcd 482.2; HRMS (FAB) calcd for C23H36N3O8 (MH+)
482.2503, found 482.2507.
(S)-N6-ter t-Bu toxyca r bon yl-N4-a cetyl-4-a za lysin e (30a ).
A solution of 29a (0.384 g, 0.90 mmol) in methanol (10 mL)
was hydrogenated at room temperature with stirring in the
presence of 10% Pd-C (0.038 g). On completion, the reaction
mixture was filtered through a Celite pad and the filtrate
evaporated to dryness to give the product 30a as a glassy solid,
which was recrystallized from methanol/diethyl ether (0.262
g, 84%): mp 135-140 °C; TLC (methanol/acetic acid, 5:0.5) Rf
) 0.56; δH 1.35 (9H, s), 2.14 (3H, s), 3.00-4.25 (6H, m), 4.30-
4.75 (1H, br m), 8.12 (3H, br s); m/z (ES-MS) 290.18 (MH+),
calcd 290.17. Anal. Calcd for C12H23N3O5‚1/2H2O: C, 48.31; N,
8.10; N, 14.08. Found: C, 48.05; H, 7.95; N, 13.75.
C32H42N4O7‚H2O: C, 62.71; H, 7.18; N 9.14. Found: C, 62.95;
H, 6.89; N, 8.91.
H -Ar g-Ar g-Tr p -Tr p -Azl(N4-Ac)-P h e-NH 2 (32a ). Rink
amide aminomethyl polystyrene (0.050 g, 0.033 mmol; 0.66
mmol g-1, 200-400 mesh) was swelled in DMF (2 mL) for 1 h
and then Fmoc-deprotected using 20% piperidine in DMF (2.5
mL min-1, 7 min). Using an LKB 4175 Biolynx manual peptide
synthesizer, the peptide sequence Arg(Pmc)-Arg(Pmc)-Trp-
(Boc)-Trp(Boc)-Azl(N4-Ac)-Phe was assembled. Sequential acy-
lation reactions were carried out at ambient temperature for
3 h, except for the coupling of FmocAzl(N4-Ac)OH, which was
left for 18 h. Appropriate Fmoc-amino acids [FmocPheOH,
0.051 g, 0.132 mmol; FmocTrp(Boc)OH, 0.069 g, 0.132 mmol;
FmocArg(Pmc)OH, 0.087 g, 0.132 mmol; FmocAzl(N4-Ac,N6-
Boc)OH 31a (0.051 g, 0.099 mmol), HOBt (0.015 g, 0.099
mmol), TBTU (0.032 g, 0.099 mmol) and DIEA (0.034 mL,
0.198 mmol)] were used, and carboxyl-activated using TBTU
(0.043 g, 0.132 mmol), HOBt (0.020 g, 0.132 mmol), and DIEA
(0.046 mL, 0.264 mmol) in DMF (1 mL). Repetitive N-Fmoc-
deprotection was achieved using 20% v/v piperidine in DMF
(2.5 mL min-1, 7 min). Following assembly, the resin product
was filtered, washed with DMF (20 mL), CH2Cl2 (50 mL), and
hexane (50 mL), and dried in vacuo to afford the required
peptidyl-resin (98 mg, 98%). The resin (98 mg) was suspended
in a mixture of TFA/H2O/1,2-ethanedithiol/i-Pr3SiH (9:0.7:0.1:
0.2, 10 mL) for 3 h with occasional stirring. The mixture was
filtered and the filtrate evaporated to dryness in vacuo. The
residue was triturated several times with ether (3 × 10 mL),
dissolved in water, and lyophilized to yield the title hexapep-
tide sequence as an amorphous powder (0.012 g). RP-HPLC
conditions: solvent A, 0.06% aqueous TFA; solvent B, 0.06%
TFA in acetonitrile/water, 90:10 v/v, gradient elution 20%-60%
B in 25 min; tR 9.38 min; m/z (ES-MS) 1018.8 (MH+), calcd
1020.56.
N4,N6-Bis-ter t-bu toxyca r bon yl-4-a za lysin e (30c). Hy-
drogenation of 29c (1.0 g, 2.07 mmol) using 10% Pd-C (0.10
g) in methanol (10 mL) in the procedure for 30a gave the title
compound 30c (0.70 g, 97%) as a glassy solid that was
recrystallized from CHCl3/hexane: mp 170-172 °C; [R]27
D
-8.46 (c 1.04, MeOH); δH 1.38, 1.42 (18H, 2 × s), 3.10-4.30
(7H, m), 5.63 (1H, br s), 8.26 (3H, br s); δC 28.20, 28.33, 39.00,
48.74, 49.92, 54.84, 78.85, 81.08, 156.10, 157.33, 171.69; m/z
(FAB) 348 (MH+), calcd 348.2; HRMS (FAB) calcd for
C
15H30N3O6 (MH+) 348.2134, found 348.2132.
(S)-N2-9-F lu or en ylm et h oxyca r b on yl-N6-ter t-b u t oxy-
ca r bon yl-N4-a cetyl-4-a za lysin e (31a ). To a stirred solution
of 30a (0.187 g, 0.65 mmol) in NaHCO3 (0.163 g, 1.95 mmol)
solution in water (10 mL) at 5 °C was added a solution of
FmocOSu (0.240 g, 0.715 mmol) in dioxane (10 mL) over a
period of 15 min. The reaction mixture was stirred for 5 h at
room temperature, after which time it was evaporated to
dryness and the residue redissolved with water (30 mL). The
aqueous solution was washed with diethyl ether (2 × 30 mL),
acidified with 2 M aqueous KHSO4, and extracted with ethyl
acetate (3 × 50 mL). The pooled extracts were dried and
concentrated under reduced pressure to give the title com-
pound as a white solid. Recrystallization of the crude product
from ethyl acetate/hexane gave the title product 31a (0.268 g,
81%): mp 145-149 °C; TLC (chloroform/methanol/acetic acid,
2:0.2:0.02) Rf ) 0.38; [R]27D -11.9 (c 1.35, MeOH); δH 1.27 (9H,
s), 2.16 (3H, s), 3.00-3.95 (6H, m), 4.10-4.30 (1H, m), 4.30-
4.60 (3H, m), 5.00-5.30 (1H, m), 6.25 (1H, d, J 6.8), 7.20-
7.50 (4H, m), 7.50-7.77 (2H, m), 7.70-7.90 (2 H, m); δC 21.10,
28.28, 39.06, 47.00, 47.09, 49.86, 54.08, 67.94, 81.04, 119.98,
125.06, 127.08, 127.74, 141.25, 143.76, 155.86, 156.09, 172.48,
174.90; m/z (ES-MS) 512.19 (MH+), calcd 512.24. Anal. Calcd
for C27H33N3O7: C, 63.46; H, 6.50; N, 8.22. Found: C, 63.11;
H, 6.40; N, 8.00.
H-Ar g-Ar g-Tr p -Tr p -Azl-P h e-NH2 (32c). Use of FmocAzl-
(N4-Boc,N6-Boc)OH 31c afforded the title peptide as white
powder (0.032 g): RP-HPLC analysis, tR 8.30 min; m/z (ES-
MS) 978.4 (MH+), calcd 978.55.
H-Ar g-Ar g-Tr p-Tr p-Azl(N4-C6H11NHCO)-P h e-NH2 (32d).
Use of FmocAzl(N4-C6H11NHCO,N6-Boc)OH 31d afforded the
title peptide as a white amorphous powder (0.031 g): RP-
HPLC analysis, tR 12.56 min; m/z (ES-MS) 1104.0 (MH+), calcd
1103.64.
N2,N4,N6-Tr is-ter t-bu toxyca r bon yl-4-a za lysin e (2). Di-
tert-butyl dicarbonate (0.627 g, 2.88 mmol) was added to a
stirred solution of 30c (1.00 g, 2.88 mmol) in dry CH2Cl2
followed by Et3N (0.4 mL, 2.88 mmol) and the stirring was
continued at room temperature for 6 h. Following evaporation
in vacuo to dryness, the residue was redissolved in ethyl
acetate (100 mL), washed with 1 M aqueous KHSO4 (2 × 30
mL) and brine (1 × 30 mL). The organic phase was dried and
concentrated in vacuo to give the crude product as a sticky
solid. The crude compound was dissolved in aqueous NaHCO3
(50 mL) and washed with ethyl acetate (1 × 30 mL), acidified
with 1 M aqueous KHSO4, followed by extraction with ethyl
(S)-N2-9-F lu or en ylm eth oxyca r bon yl-N4,N6-bis-ter t-bu -
toxyca r bon yl-4-a za lysin e (31c). Using 30c (0.60 g, 1.72
mmol), NaHCO3 (0.433 g, 5.16 mmol), and FmocOSu (0.637 g,
1.89 mmol), in the procedure described for 31a , and on
recrystallization from ethyl acetate/hexane gave the title
compound 31c (0.5 g, 51%): mp 104-108 °C; TLC (chloroform/
methanol/acetic acid, 2:0.2:0.02) Rf ) 0.4; [R]28D -11.0 (c 1.09,
MeOH); δH 1.41, 1.44 (18H, 2 × s), 3.00-3.90 (6H, m), 4.10-
4.70 (4H, m), 4.90-5.40 (1H, m), 5.80-6.50 (1H, m), 7.10-