Syntheses of azido amino acids
6.80 (1H, t, J = 5.6, –NH–CO–O), 7.52 (1H, br d, J = 8.2, –NH–CO–O),
7.33 (2H, m, Ar–H), 7.41 (2H, m, Ar–H), 7.72 (2H, m, Ar–H), 7.89 (2H,
m, Ar–H), 12.70 (1H, br s, COOH). 13C NMR (125.7 MHz, DMSO):
26.46 (–CH2–), 28.47 ((CH3)3), 28.62 (–CH2–), 40.00 (–CH2–N), 54.13
(>CH–Ν), 65.78 (–CH2–O–CO), 77.58 (O–C(CH3)3), 120.30(2),
125.50, 125.51, 127.28(2) and 127.83(2) (8× Ar=CH–), 140.91,
140.92, 144.02 and 144.08 (4× Ar>C=), 155.79 (N–CO–Ο), 156.24
(N–CO–Ο), 174.30 (–COOH). IR (KBr) νmax (cmꢀ1) 3348 s (NH); 2976
vs, 1393 s, 1366 s (CH3); 1169 vs (C(CH3)3); 1716 vs (C=O) acid;
1698 vs (C=O) carbamates; 1528 s (amide II); 3066 w, 3041 w,
1451 m, 1105 m, 759 m, 740 m (ring). HRMS (ESI) calc for
C25H30O6N2Na [M + Na]+ 477.19961, found: 477.19964.
(cmꢀ1) 3391 s, 3339 s (NH); 2874 m (CH2); 2096 vs (N3); 1711 vs,
(C=O) acid; 1729 vs, 1687 vs (C=O) carbamate; 1534 s (amide II);
3043 m, 1451 s, 1103 m, 1032 m, 759 s, 737 s, 622 s (ring). HRMS
(ESI) calc for C20H20O4N4Na [M + Na]+ 403.13768, found: 403.13774.
6-Azido-2-(S)-(9-fluorenylmethyloxycarbonylamino)hexanoic
Acid 42
With the procedure described for 41, acid 42 was prepared starting
from 40 (14.9 g; 31.8 mmol), NaHCO3 (26.7 g; 318 mmol),
CuSO4·5H2O (79 mg; 0.32 mmol), NaN3 (20.7 g; 318 mmol) and triflic
anhydride (17.9 g; 63.6 mmol). Yield 11.1 g (89%). White solid, 73–
75 °C. Rf
= 0.74 (ethyl acetate–acetone–methanol–water
1
6 : 1 : 1 : 0.5). [α]2D0 = ꢀ15.7 (c = 0.261; DMF). H NMR (500 MHz,
DMSO): 1.38 (2H, m, –CH2–), 1.53 (2H, m, –CH2–), 1.63 and 1.73
(2H, 2× m, –CH2–), 3.32 (2H, t, J = 6.8, –CH2–N3), 3.94 (1H, ddd,
J = 9.5, 8.0 and 4.6, >CH–Ν), 4.22 (1H, br t, J = 7.0, >CH–), 4.28
(2H, m, CO–O–CH2–), 7.33 (2H, m, Ar–H), 7.42 (2H, m, Ar–H), 7.67
(1H, br d, J = 8.0, –NH–CO–O), 7.73 (2H, m, Ar–H), 7.89 (2H, m,
Ar–H), 12.62 (1H, br s, COOH). 13C NMR (125.7 MHz, DMSO): 23.14
(–CH2–), 28.06 (–CH2–), 30.50 (–CH2–), 46.87 (>CH–), 50.73 (–CH2–
N3), 53.88 (>CH–Ν), 65.81 (–CH2–O–CO), 120.34, 120.35, 125.49,
125.52, 127.28(2) and 127.87(2) (8× Ar=CH–), 140.94, 140.95,
144.02 and 144.07 (4× Ar>C=), 156.40 (N–CO–Ο), 174.11 (–COOH).
IR (KBr) νmax (cmꢀ1) 3378 m (NH); 2937 m, 2886 m, 1477 m (CH2);
2097 vs (N3); 1717 vs, (C=O) acid; 1746 vs, 1698 vs (C=O) carbamate;
1525 s (amide II); 3065 m, 3043 m, 1451 s, 1102 m, 758 s, 739 s (ring).
HRMS (ESI) calc for C21H22O4N4Na [M + Na]+ 417.15333, found:
417.15346.
6-(tert-Butoxycarbonylamino)-2-(S)-(9-
fluorenylmethyloxycarbonylamino)hexanoic Acid 40
Acid 40 was prepared by the reaction of 38 (9.3 g; 35.2 mmol),
NaHCO3 (5.9 g; 70.4 mmol) and Fmoc-OSu (11.1 g; 35.2 mmol),
using the protocol described for 39. Yield 15.6 g (95%). White solid,
m.p. 125–127 °C. Rf = 0.75 (ethyl acetate–acetone–methanol–water
6 : 1 : 1 : 0.5). [α]2D0= ꢀ8.5 (c = 0.272; DMF). 1H NMR (500 MHz, DMSO):
1.30 (2H, m, –CH2–), 1.36 (9H, s, (CH3)3), 1.36 (2H, m, –CH2–), 1.59 and
1.69 (2H, 2× m, –CH2–), 2.90 (2H, m, –CH2–N), 3.89 (1H, ddd, J = 9.2,
8.2 and 4.5, >CH–Ν), 4.22 (1H, m, >CH–), 4.27 (2H, m, CO–O–CH2–),
6.79 (1H, br t, J = 5.6, –NH–CO–O), 7.54 (1H, br d, J = 8.2, –NH–CO–
O), 7.33 (2H, m, Ar–H), 7.41 (2H, m, Ar–H), 7.73 (2H, m, Ar–H), 7.89
(2H, m, Ar–H). 13C NMR (125.7 MHz, DMSO): 23.17 (–CH2–), 28.49
((CH3)3), 29.34 (–CH2–), 30.83 (–CH2–), 39.82 (–CH2–N), 46.88
(>CH–), 54.21 (>CH–Ν), 65.78 (–CH2–O–CO), 77.56 (O–C(CH3)3),
120.33, 120.34, 125.50, 125.52, 127.29(2) and 127.85(2) (8× Ar=CH–),
140.93, 140.94, 144.03 and 144.09 (4× Ar>C=), 155.80 (N–CO–Ο),
156.32 (N–CO–Ο), 174.40 (–COOH). IR (KBr) νmax (cmꢀ1) 3391 s,
3369 s (NH); 2978 vs, 1393 s, 1367 s (CH3); 2936 m, 1478 m (CH2);
1173 vs (C(CH3)3); 1711 vs (C=O) acid; 1693 vs + br (C=O) carba-
mates; 1525 s (amide II); 3067 w, 3041 w, 1451 m, 1105 m, 760 m,
740 m (ring). HRMS (ESI) calc for C26H32O6N2Na [M + Na]+
491.21526, found: 491.21527.
General Protocol for the Manual Synthesis of Tripeptides 43–47
(i) Rink amide resin (400 μmol, loading 0.68 mmol/g) was placed
in a 20 ml polypropylene syringe equipped with a polypropyl-
ene frit and swelled in 10 ml of DMF for 1 h.
(ii) Fmoc group was cleaved by treatment with 20%
piperidine/DMF (5 ml for 5 and 20 min), followed by five wash-
ings with 5 ml of DMF.
(iii) Fmoc-Phe (619 mg; 1.6 mmol), HBTU (607 mg; 1.6 mmol) and
DIPEA (418 μmol; 2.4 mmol) were added in 5 ml of DMF. The
resin was stirred for 2 h and then washed five times with
5 ml of DMF. Step (iii) was repeated, and the resin was washed
successively with DMF, MeOH, DCM and DMF (each solvent
five times with 5 ml). Step (ii) was repeated. Step (iii) was re-
peated twice (Fmoc-Val was used in the case of tripeptide
43; 543 mg; 1.6 mmol). Step (ii) was repeated.
(iv) 14 (563 mg; 1.6 mmol) or 29 (586 mg; 1.6 mmol) or 41
(608 mg; 1.6 mmol) or 42 (630 mg; 1.6 mmol) with HBTU
(607 mg; 1.6 mmol) and DIPEA (418 μmol; 2.4 mmol) in 5 ml
of DMF was added, stirred for 2 h and then washed five times
with 5 ml of DMF. Only in the case of the synthesis of 44 did
couplings with 14 proceed for 5 and 18 h. Step (iv) was re-
peated and the resin successively washed with DMF, MeOH,
DCM and DMF (each solvent five times with 5 ml). Step (ii)
was repeated.
(v) Thereafter, 300 μl of Ac2O and 300 μl of DIPEA, each in 1 ml of
DMF, were added; the resin was stirred for 15 min and washed
five times with 5 ml of DMF. Step (v) was repeated. Thereafter,
the resin was transported to a small glass reactor equipped
with a frit, rinsed with 50 ml of dichloromethane and dried
overnight under deep vacuum. The resin was cleaved for
60 min with 5 ml of a cocktail of TFA/water/triisopropylsilane
5-Azido-2-(S)-(9-fluorenylmethyloxycarbonylamino)pentanoic
Acid 41
Compound 39 (10.4 g; 22.9 mmol) was treated with 20 ml of DCM,
20 ml of TFA and 2 ml of water. After 2 h stirring, volatile materials
were evaporated to give a yellow oil, which was then suspended
with NaHCO3 (19.2 g; 229 mmol) and CuSO4·5H2O (57 mg;
0.229 mmol) in 100 ml of water and 150 ml of methanol. TfN3, pre-
pared by the reaction of NaN3 (14.9 g; 229 mmol) and triflic anhy-
dride (12.9 g; 45.8 mmol), was added dropwise to the slurry in
100 ml of DCM, followed by the work-up, which was the same as
in the case of the diazotransfer reaction leading to the product
14. Yield 8 g (92%). White solid, 127–128 °C. Rf = 0.71 (ethyl
acetate–acetone–methanol–water 6 : 1 : 1 : 0.5). [α] 20 = ꢀ6.7
D
1
(c = 0.254; DMF). H NMR (500 MHz, DMSO): 1.59 (2H, m, –CH2–),
1.66 and 1.78 (2H, 2× m, –CH2–), 3.34 (2H, m, –CH2–N3), 3.98 (1H,
ddd, J = 9.2, 8.2 and 5.0, >CH–Ν), 4.23 (1H, br t, J = 7.0, >CH–),
4.30 (2H, m, CO–O–CH2–), 7.33 (2H, m, Ar–H), 7.42 (2H, m, Ar–H),
7.70 (1H, br d, J = 8.2, –NH–CO–O), 7.73 (2H, m, Ar–H), 7.89 (2H,
m, Ar–H), 12.66 (1H, br s, COOH). 13C NMR (125.7 MHz, DMSO):
25.31 (–CH2–), 28.21 (–CH2–), 46.89 (>CH–), 50.45 (–CH2–N3), 53.57
(>CH–Ν), 65.81 (–CH2–O–CO), 120.33, 120.35, 125.48, 125.50,
127.28(2) and 127.86(2) (8× Ar=CH–), 140.94, 140.96, 144.00 and
144.07 (4× Ar>C=), 156.38 (N–CO–Ο), 173.88 (–COOH). IR (KBr) νmax
J. Pept. Sci. 2017
© 2017 The Authors Journal of Peptide Science
published by European Peptide Society and John Wiley & Sons Ltd
wileyonlinelibrary.com/journal/jpepsci