8
R.A.G. Harmsen et al. / Tetrahedron xxx (2014) 1e9
9:1 v/v): 0.58; tR 17.6 min; 1H NMR (500 MHz, CDCl3/CD3OH 14.5:1
v/v) H), 5.73 (br s, 1H, NH), 5.13e5.10 (m,
-Alg1:
¼5.78 (m, 1H, C
2H, C H), 4.13 (m, 1H, C H), 2.57e2.48 (m, 2H, C H), 1.44 (s, 9H,
Boc); Ile2: 7.76 (d, 1H, NH), 4.45 (m, 1H, C
H), 1.87 (m, 1H, C H), 1.65
(m, 2H, C H), 0.84 (m, 6H, C
0H/C H); Ala3: 7.42 (br s, 1H, NH), 4.23
(m,1H, C H),1.38 (br s, 3H, C
H); Leu4: 7.60 (d,1H, NH), 4.45 (m,1H,
H), 1.74 (m, 1H, C H), 1.65 (m, 2H, C H), 0.84 (m, 6H, C H/C
0H);
Alg5: 7.55 (br s, 1H, NH), 5.78 (m, 1H, C
H), 5.13e5.09 (m, 2H, C H),
4.13 (m, 1H, C H), 3.74 (s, 3H, OCH3), 2.57e2.43 (m, 2H, C
H); 13C
NMR (CDCl3, 75.5 MHz):
¼172.7 (two lines), 172.1 (two lines),
the general procedure. The obtained TFA-salt was dissolved in DCM
(10 mL) and Fmoc-Glu(OtBu)-OH (510 mg, 1.20 mmol) followed by
BOP (530 mg, 1.20 mmol) and DIPEA (636 mL, 3.6 mmol) were
added. After stirring for 16 h, the workup of the reaction mixture
was as described in the general procedure and the obtained residue
was purified by column chromatography on silica gel (eluens: DCM/
MeOH 97.5:2.5 v/v). The protected tripeptide was obtained in 93%
yield (870 mg). Rf (DCM/MeOH 9:1 v/v): 0.65; 1H NMR (300 MHz,
D
d
g
d
a
b
a
b
g
a
g
b
d
C
a
g
b
g
d
d
d
a
b
CDCl3) Glu:
2H, arom H Fmoc), 7.40e7.25 (m, 4H, arom H Fmoc), 6.01 (d, 1H,
NH), 4.19e4.15 (m, 4H, C H/CH2 Fmoc/CH Fmoc), 2.32 (m, 2H, C H),
1.90e1.85 (m, 2H, C H), 1.44 (m, 9H, Boc); Alg: 6.99 (d, 1H, NH),
5.74e5.65 (m, 1H, C H), 5.19e5.03 (m, 2H, C H), 4.55 (m, 1H, C H),
2.55e2.44 (m, 2H, C H); Lys: 7.40e7.25 (m, 10H, arom Cbz (5H)/
arom Bzl (5H)), 6.94 (d, 1H, NH), 5.19e5.03 (m, 4H, CH2 Bzl
(2ꢁ2H)), 5.33 (m, 1H, NH), 4.50e4.46 (m, 1H, C H), 3.18e3.08 (m,
2H, C H), 2.01e1.99 (m, 1H, C H), 1.75e1.55 (m, 1H, C H), 1.44e1.21
(m, 4H, C H/C
H); 13C NMR (CDCl3, 75.5 MHz):
¼173.1, 171.7, 171.5,
d
¼7.75 (d (J 4.8 Hz), 2H, arom H Fmoc), 7.55 (d (J 4.8 Hz),
d
171.6,156.0,133.0 (two lines),119.2,118.8, 80.6, 56.3, 54.3, 52.4, 51.7,
50.0, 49.7, 40.6, 36.2 (two lines), 28.3, 27.1, 23.0, 21.8, 17.5, 15.6,
11.4; ES-MS: calcd for C31H53N5O8: 623.8, found: m/z: [MþH]þ
624.4, [MþNa]þ 646.7, [(MꢀC4H8)þH]þ 568.6, [(MꢀC5H8O2)þH]þ
524.7.
a
b
g
g
d
a
b
a
3
a
4.5.9. Cbz-Lys(Boc)1-Ala2-cyclo[
(6). Boc-removal of the cyclic peptide Boc-cyclo[
D
-Alg3-Ile4-Ala5-Leu6-Alg7]-OMe
-Alg-Ile-Ala-Leu-
3
b
b
D
g
d
d
Alg]-OMe (120 mg, 0.20 mmol) was performed as described. The
obtained TFA-salt was dissolved in DMF (15 mL) and Cbz-Lys(Boc)-
Ala-OH (120 mg, 0.27 mmol) was added followed by HOBt.H2O
170.4,156.6,143.7,141.3,136.6,135.3,132.7,128.4,127.7,127.0,125.0,
120.0,119.2, 81.2, 67.1, 66.5, 54.7, 52.6, 52.1, 47.1, 40.9, 36.1, 31.9, 31.6,
29.1, 28.1, 27.6, 22.1,; ES-MS: calcd for C50H59N4O10: 875.4, found:
m/z: [MþH]þ 875.7, [MþNa]þ 897.8, [(MꢀC4H8)þH]þ 821.7. Then,
Fmoc-Glu(OtBu)-Alg-Lys(Cbz)-OBzl (750 mg, 0.86 mmol) was dis-
solved in THF (2 mL) and Et2NH (2 mL) was added. The obtained
reaction mixture was stirred for 1 h. Then, the solvents were re-
moved in vacuo and the residue was coevaporated with toluene
(2ꢁ5 mL) and CHCl3 (2ꢁ5 mL) to remove any residual diethylamine.
The obtained free amine was dissolved in CH2Cl2 (15 mL) and Fmoc-
(40 mg, 0.26 mmol), BOP (115 mg, 0.26 mmol) and DIPEA (104 mL,
0.6 mmol). After stirring for 16 h, the solvent was evaporated in
vacuo and the residue was triturated with EtOAc to obtain com-
pound 6 in 85% yield (158 mg). tR 17.5 min; Rf (CH2Cl2/MeOH 9:1 v/
v): 0.55 and 0.60; 1H NMR (500 MHz, CDCl3) Lys1:
NH), 7.33 (m, 5H, arom Cbz), 6.13 (m, 1H, NH), 5.10 (s, 2H, CH2 Bzl),
d
¼7.58 (m, 1H,
4.53 (m, 1H, C
a
H), 3.07 (m, 2H, C H), 1.78e1.25 (m, 6H, CbH/CgH/
3
C
C
d
a
H (3ꢁ2H)), 1.42 (s, 9H, Boc); Ala2: 7.36 (m, 1H, NH), 4.35 (m, 1H,
D-Alg-Pro-OH (375 mg, 0.86 mmol) followed by BOP (380 mg,
H), 1.78e1.25 (m, 3H, C
b
H);
D
-Alg3: 7.71 (m, 1H, NH), 5.63/5.51
0.86 mmol) and DIPEA (318 L, 1.80 mmol) were added and the
m
(m, 1H, C
7.33 (m, 1H, NH), 4.17 (m,1H, C
g
H), 5.06/4.83 (m, 1H, C
a
H), 2.21e1.99 (m, 2H, C
b
H); Ile4:
H (1H)/C
reaction mixture was stirred for 16 h. Then, the solvent was re-
moved in vacuo and the workup of the residue was as described in
the general procedure. After purification by column chromatogra-
phy on silica gel (eluent: CH2Cl2/MeOH 97.5:2.5 v/v) pentapeptide 8
was obtained in 82% yield (754 mg). Rf (CH2Cl2/MeOH 9:1 v/v): 0.55
a
H), 1.78e1.25 (m, 3H, C
b
gH
(2H)), 0.89 (m, 6H, C
(m, 1H, C
(m, 1H, C
H/C
g
0H/C
d
H (2ꢁ3H)); Ala5: 8.26 (m, 1H, NH), 4.17
a
H), 1.78e1.25 (m, 3H, Cb
H); Leu6: 7.30 (m, 1H, NH), 4.17
a
H), 1.78e1.25 (m, 3H, CbH (2H)/CgH (1H)), 0.89 (m, 6H,
C
d
d
0H (2ꢁ3H)); Alg7: 7.92 (m, 1H, NH), 5.63/5.51 (m, 1H, C
g
H),
and 0.59; tR 16.9 min; 1H NMR (300 MHz, CDCl3)
D
-Alg1:
d
¼7.73 (d,
2H, arom H Fmoc), 7.55 (d (J 4.8 Hz), 2H, arom H Fmoc), 7.40e7.26
(m, 4H, arom H Fmoc), 5.85e5.67 (m, 2H, C H/NH), 5.15e5.05 (m,
2H, C H), 4.68e4.58 (m, 1H, C H), 4.47e4.19 (m, 3H, CH2/CH Fmoc),
2.62e2.33 (m, 2H, C H), 3.70e2.89
H); Pro2: 4.47e4.19 (m, 1H, C
(m, 2H, C H), 2.18e1.74 (m, 4H, C H/C
H); Glu3: 7.98 (d, 1H, NH),
4.47e4.19 (m, 1H, C H), 2.18e1.74 (m, 4H, C H/C H), 1.47 (m, 9H,
Boc); Alg4: 7.40e7.26 (m, 1H, NH), 5.85e5.67 (m, 1H, C
H),
5.15e5.05 (m, 2H, C H), 4.68e4.58 (m, 1H, C H), 2.62e2.33 (m, 2H,
H); Lys5: 7.43 (d, 1H,
NH), 7.40e7.26 (m, 10H, arom Cbz (5H)/
arom Bzl (5H)), 5.52 (m, 1H,
5.06/4.83 (m, 1H, CaH), 3.79 (s, 3H. OCH3), 2.21e1.99 (m, 2H, Cb
H);
ES-MS: calcd for C46H73N8O12: 929.5 found: m/z: [MþH]þ 930.3,
[MþNa]þ 952.3, [(MꢀC5H8O2)þH]þ 829.5; MALDI-TOF: [MþNa]þ
952.4.
g
d
a
b
a
d
b
g
4.5.10. Boc-Alg-Lys(Cbz)-OBzl (7). Boc-Lys(Cbz)-OBzl was synthe-
sized according to the method of Shin and co-workers13 and was
obtained with 80% yield. Boc-Lys(Cbz)-OBzl (611 mg, 1.3 mmol) was
treated with TFA to remove the Boc-functionality as described in
the general procedure (vide supra). The corresponding amine was
dissolved in CH2Cl2 (15 mL) and coupled to Boc-Alg-OH (258 mg,
1.2 mmol) in the presence of BOP (530 mg, 1.2 mmol) and DIPEA
a
b
g
g
d
a
Cb
a
NH), 5.15e4.05 (m, 4H, CH2 Bzl
(2ꢁ2H)), 4.68e4.58 (m, 1H, C
a
H), 3.18e3.08 (m, 2H, C H), 2.18e1.74
3
(m, 2H, C
b
H), 1.47e1.32 (m, 4H, C
gH/Cd
H); 13C NMR (CDCl3,
(636
m
L, 3.6 mmol). The coupling conditions and the subsequent
125 MHz): d
¼174.5, 172.9 (two lines), 171.9, 171.3, 170.3, 156.8 (two
workup of the reaction mixture were as described in the general
procedure. Protected dipeptide 7 was obtained as a white solid in
84% (573 mg) yield. Rf (CH2Cl2/MeOH 9:1 v/v): 0.65; 1H NMR
lines), 143.9 (two lines), 141.4 (two lines), 136.9 (two lines), 134.9,
134.4, 129.2, 128.6, 128.3, 128.1, 127.2, 124.4, 119.2, 117.7 (two lines),
81.7, 67.8, 66.9, 66.5, 61.7, 55.1, 54.5, 52.4, 50.4, 47.5, 46.1, 40.5, 35.0
(two lines), 31.8, 31.3, 29.2, 28.5, 28.1, 24.9, 22.7, 21.1; ES-MS: calcd
for C60H72N6O12: 1068.3 found: m/z: [MþH]þ 1069.8, [MþNa]þ
1091.7, [(MꢀC4H8)þH]þ 1013.8.
(CDCl3, 300 MHz) Alg:
urethane NH), 5.18e5.02 (m, 2H, C
(m, 2H, C H), 1.49 (m, 9H, Boc); Lys: 7.19e7.33 (m, 11H, arom Cbz
(5H)/arom Bzl (5H)/ NH),
NH), 5.18e5.02 (m, 5H, CH2 Bzl (2ꢁ2H)/
4.57 (m, 1H, C H), 3.11e3.04 (m, 2H, C H), 1.79e1.69(m, 2H, C H),
1.49e1.21 (m, 4H, C H/C
H); 13C NMR (CDCl3, 75.5 MHz):
¼171.5,
d
¼5.69e5.63 (m, 1H, C
g
H), 5.36 (d, 1H,
d
H), 4.30 (m, 1H, C
aH), 2.35e2.47
b
a
3
a
3
b
4.5.12. H-cyclo[D
-Alg1-Pro2-Glu(OtBu)3-Alg4]-Lys(Cbz)5-OBzl
(9). Linear pentapeptide 8 (500 mg, 0.47 mmol) was dissolved in
CH2Cl2 (250 mL) and the solution was flushed with N2 for 30 min.
The solution was heated to reflux and a solution of second-gener-
g
d
d
170.3, 155.9, 155.7, 136.1, 132.7, 128.9, 127.6, 127.1, 116.4, 79.8, 66.5,
66.4, 56.8, 52.8, 40.3, 36.7, 30.6, 29.9, 28.4, 22.7; ES-MS: calcd for
C
30H39N3O8: 569.6, found: m/z: [MþH]þ 570.7, [MþNa]þ 593.3,
ation Grubbs’ catalyst (40 mg, 47 mmol) in CH2Cl2 (1 mL) was added.
[(MꢀC5H8O2)þH]þ 469.1.
The obtained reaction mixture was refluxed for 4.5 h under a ni-
trogen atmosphere. After evaporation of the solvent, the residue
was redissolved in THF/Et2NH (4 mL, 1:1 v/v) and stirred for 1 h to
remove the Fmoc-functionality. Then, the solvents were removed
in vacuo and the residue was coevaporated with toluene (2ꢁ5 mL)
4.5.11. Fmoc-
-Alg1-Pro2-Glu(OtBu)3-Alg4-Lys(Cbz)5-OBzl (8). Fmoc-
D
Glu(OtBu)-Alg-Lys(Cbz)-OBzl: Dipeptide 7 (539 mg, 0.95 mmol) was
treated with TFA to remove the Boc-functionality as described in