Diels–Alder Ligation
FULL PAPER
was purified by flash chromatography, eluting with DCM and DCM/
15.0, 10.4 Hz, CH=CHCH3), 6.28 (dd, 1H, J=15.0, 10.6 Hz, CH2CH=
CH), 7.18 (d, 2H, J=8.4 Hz, Ar Mtt), 7.27–7.33 (m, 6H, Ar Mtt), 7.43–
7.49 ppm (m, 6H, Ar Mtt); FAB-HRMS: m/z: calcd for C34H52N6O6:
909.5602; found 909.5684 [M+H]+.
MeOH 100:1!20:1 to give a colorless solid (70 mg, 0.09 mmol, 37%). Rf
=
0.31 (DCM/MeOH 10:1); MALDI-TOF: m/z: calcd for
C40H51NaN5O8S2: 816.3; found: 816.6 [M+Na]+, 833.5 [M+K]+. Com-
pound 3h was not further characterized and immediately converted to
compound 4h.
H-Lys-Cys
C
=
General procedure for Fmoc-protecting group removal to give hexadien-
yl esters 4: A solution of 5–30 mg Fmoc-protected peptide 3 was treated
with 0.5–2.0 mL 20% piperidine in DMF or DCM for 40 minutes at
room temperature. The reaction mixture was coevaporated with metha-
nol to remove excess of piperidine. The product was purified by re-
versed-phase-HPLC, fractions containing the product (evaluated by
MALDI-TOF) were combined and dried by lyophilization.
ꢁ9.08(c=0.1, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.73 (d, 3H, J=
6.8 Hz, CH3 Val), 0.82(d, 3H, J=6.8 Hz, CH3 Val), 1.34 (s, 9H, tBu),
1.45–1.55 (m, 2H, g-CH2 Lys), 1.63–1.70 (m, 2H, d-CH2 Lys), 1.74 (d,
3H, J=6.6 Hz, CH3 Hxd), 1.84–1.95 (m, 2H, b-CH2 Lys), 2.01–2.10 (m,
1H, b-CH Val), 2.94 (t, 2H, J=7.6 Hz, e-CH2 Lys), 2.98–3.07 (m, 2H, b-
CH2 Phe), 3.20 (dt, 2H, J=13.9, 6.4 Hz, b-CH2 Cys), 3.68 (d, 1H, J=
16.1 Hz, a-CH Val), 3.94 (s, 2H, a-CH2 Gly), 3.98 (t, 1H, J=6.4 Hz, a-
CH Lys), 4.08 (t, 2H, J=8.2Hz, a-CH2 Gly), 4.63 (d, 2H, J=6.6 Hz,
CH2 Hxd), 4.63–4.66 (m, 2H, a-CH Cys, a-CH Phe), 5.58–5.65 (m, 1H,
CH=CHCH3), 5.72–5.82 (m, 1H, CH2CH=CH), 6.03–6.10 (dd, 1H, J=
15.0, 10.4 Hz, CH=CHCH3), 6.25–6.32 (dd, 1H, J=15.1, 10.5 Hz,
CH2CH=CH), 7.18–7.30 ppm (m, 5H, Ar Phe); MALDI-TOF: m/z: calcd
for C37H59N7O7S2: 778.4; found 778.8 [M+H]+, 800.8 [M+Na]+, 816.7
[M+K]+.
H-Lys-Pro-Phe-Leu-Gly-OHxd (4a): Starting from 3a (33 mg,
0.032mmol) a colorless solid (15 mg, 0.023 mmol, 72%) was obtained.
[a]2D0 =ꢁ27.98 (c=0.3, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.91
(d, 3H, J=6.1 Hz, CH3 Leu), 0.94 (d, 3H, J=6.2Hz, CH 3 Leu), 1.46–
1.82(m, 7H, d-CH2 Lys, g-CH2 Lys, b-CH2 Leu, g-CH Leu), 1.74 (d, 3H,
J=6.7 Hz, CH3 Hxd), 1.84–2.23 (m, 6H, b-CH2 Pro, g-CH2 Pro, b-CH2
Lys), 2.90–3.00 (m, 2H, e-CH2 Lys), 3.02(dd, 1H, J=13.9, 7.7 Hz, b-CH2
Phe), 3.13 (dd, 1H, J=13.9, 6.5 Hz, b-CH2 Phe), 3.56–3.63 (m, 1H, e-CH2
Pro), 3.67–3.73 (m, 1H, e-CH2 Pro), 3.81–3.96 (m, 2H, a-CH2 Gly), 4.25
(t, 1H, J=6.1 Hz, a-CH), 4.42(dd, 1H, J=9.5, 5.5 Hz, a-CH), 4.50 (t,
1H, J=5.2Hz, a-CH), 4.57 (t, 1H, J=6.6 Hz, a-CH), 4.61 (d, 2H, J=
6.6 Hz, CH2 Hxd), 5.61 (ddd, 1H, J=13.5, 6.5, 6.5 Hz, CH=CHCH3),
5.72–5.81 (m, 1H, CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.4 Hz, CH=
CHCH3), 6.28 (dd, 1H, J=15.0, 10.4 Hz, CH2CH=CH), 7.17–7.30 ppm
(m, 5H, Ar Phe); ESI-MS: m/z: calcd for C34H52N6O6: 641.4; found:
641.4 [M+H]+; FAB-HRMS: m/z: 640.3945 [M]+.
H-Lys-Phe-Pro-Ile-Gly-Leu-Phe-Gly-OHxd (4 f): Starting from 3 f
(14.5 mg, 10.3 mmol) a colorless solid (9.0 mg, 9.4 mmol, 91%) was ob-
1
tained. [a]2D0 =ꢁ28.68 (c=0.3, MeOH); H NMR (CD3OD, 400 MHz): d=
0.82(dd, 3H, J=6.3, 4.3 Hz, CH3 Ile), 0.87 (d, 3H, J=6.3 Hz, CH3 Ile),
0.91–1.00 (m, 6H, 6CH3 Leu), 1.21–1.71 (m, 10H, CH2 Ile, d-CH2 Lys,
g-CH2 Lys, b-CH2 Leu, b-CH2 Lys), 1.74 (d, 3H, J=6.6 Hz, CH3 Hxd),
1.83–2.14 (m, 6H, b-CH2 Pro, g-CH2 Pro, g-CH Leu, b-CH Ile), 2.90–3.04
(m, 4H, 21Hb-CH2 Phe, e-CH2 Lys), 3.17–3.26 (m, 2H, 21H b-CH2
Phe), 3.32–3.52 (m, 1H, e-CH2 Pro), 3.76–3.95 (m, 6H, e-CH2 Pro, 2 a-
CH2 Gly), 4.08 (t, 1H, J=7.0 Hz, a-CH Pro), 4.22–4.28 (m, 2H, 2a-
CH), 4.53–4.67 (m, 2H, 2 a-CH), 4.62(d, 2H, J=6.5 Hz, CH2 Hxd),
5.61 (ddd, 1H, J=13.6, 6.4, 6.4 Hz, CH=CHCH3), 5.72–5.81 (m, 1H,
CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.5 Hz, CH=CHCH3), 6.28 (dd,
1H, J=15.2, 10.5 Hz, CH2CH=CH), 7.17–7.34 ppm (m, 10H, Ar Phe);
ESI-MS: m/z: calcd for C51H75N9O9: 958.6; found: 958.7 [M+H]+;
MALDI-TOF: m/z: 958.9 [M+H]+, 980.9 [M+Na]+, 996.8 [M+K]+;
FAB-LRMS: m/z: 958.75 [M+H]+.
H-Pro-Cys
C
ꢁ17.18 (c=0.1, MeOH); 1H NMR (CD3OD, 400 MHz): d=1.35 (s, 9H,
tBu), 1.75 (d, 3H, J=6.6 Hz, CH3 Hxd), 1.90–2.21 (m, 6H, g-CH2 Pro, b-
CH2 Pro, b-CH2 Met), 2.09 (s, 3H, CH3 Met), 2.38–2.64 (m, 2H, g-CH2
Met), 3.02(dd, 1H, J=13.7, 9.1 Hz, b-CH2 Cys), 3.23 (dd, 1H, J=13.6,
5.0 Hz, b-CH2 Cys), 3.30–3.45 (m, 2H, e-CH2 Pro), 3.75 (dd, 1H, J=10.9,
5.9 Hz, b-CH2 Ser), 3.85 (dd, 1H, J=10.9, 5.4 Hz, b-CH2 Ser), 3.94 (m,
2H, a-CH2 Gly), 4.31 (dd, 1H, J=8.4, 6.3 Hz, a-CH Pro), 4.40 (t, 1H,
J=5.7 Hz, a-CH Met), 4.56 (dd, 1H, J=9.0, 4.9 Hz, a-CH Ser), 4.62(d,
2H, J=6.7 Hz, CH2 Hxd), 4.69 (dd, 1H, J=9.0, 5.1 Hz, a-CH Cys), 5.61
(ddd, 1H, J=13.2, 6.5, 6.5 Hz, CH=CHCH3), 5.73–5.81 (m, 1H, CH2CH=
CH), 6.06 (dd, 1H, J=15.0, 10.4 Hz, CH=CHCH3), 6.27 ppm (dd, 1H,
J=15.1, 10.3 Hz, CH2CH=CH); ESI-MS: m/z: calcd for C28H47N5O7S3:
662.3; found: 662.2 [M+H]+, 684.3 [M+Na]+; MALDI-TOF: m/z: 662.8
[M+H]+, 684.8 [M+Na]+, 700.8 [M+K]+.
H-Lys-Phe-Pro-Ile-Gly-Leu-Gly-Phe-Gly-OHxd (4g): Starting from 3g
(5.4 mg, 3.7 mmol) a colorless solid (3.5 mg, 3.4 mmol, 92%) was obtained.
[a]2D0 =ꢁ25.78 (c=0.2, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.82
(dd, 3H, J=6.3, 3.1 Hz, CH3 Ile), 0.87 (d, 3H, J=6.5 Hz, CH3 Ile), 0.90–
1.00 (m, 6H, 2CH3 Leu), 1.17–1.72(m, 10H, CH Ile, d-CH2 Lys, g-CH2
2
Lys, b-CH2 Leu, b-CH2 Lys), 1.73 (d, 3H, J=6.6 Hz, CH3 Hxd), 1.83–2.04
(m, 5H, b-CH2 Pro, g-CH2 Pro, g-CH Leu), 2.04–2.13 (m, 1H, b-CH Ile),
2.89–3.04 (m, 4H, 21H b-CH2 Phe, e-CH2 Lys), 3.17–3.26 (m, 2H, 2
1H b-CH2 Phe), 3.32–3.52 (m, 1H, e-CH2 Pro), 3.72–4.02 (m, 8H, e-CH2
Pro, 3a-CH2 Gly), 4.10 (m, 1H, a-CH), 4.21–4.28 (m, 2H, 2a-CH),
H-Lys-Leu-Gly-Phe-Ala-Gly-OHxd (4c): Starting from 3c (7 mg,
0.0063 mmol) a colorless solid (3.5 mg, 0.0052mmol, 83%) was obtained.
[a]2D0 =ꢁ7.28 (c=0.3, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.95 (d,
3H, J=6.5 Hz, CH3 Leu), 0.97 (d, 3H, J=6.5 Hz, CH3 Leu), 1.38 (d, 3H,
J=7.2Hz, CH 3 Ala), 1.45–1.91 (m, 9H, 3CH2 Lys, b-CH2 Leu, g-CH
Leu), 1.74 (d, 3H, J=6.4 Hz, CH3 Hxd), 2.90–3.20 (m, 4H, b-CH2 Phe,
4.49–4.56 (m, 2H, 2 a-CH), 4.62(d, 2H,
J=6.5 Hz, CH2 Hxd), 5.62
(ddd, 1H, J=13.2, 6.7, 6.7 Hz, CH=CHCH3), 5.71–5.82(m, 1H, CH CH=
2
CH), 6.06 (dd, 1H, J=14.9, 10.5 Hz, CH=CHCH3), 6.27 (dd, 1H, J=
14.6, 10.0 Hz, CH2CH=CH), 7.19–7.35 ppm (m, 10H, Ar Phe); ESI-MS:
m/z: calcd for C53H78N10O10: 1015.6; found: 1015.7 [M+H]+; MALDI-
TOF: m/z: 1015.9 [M+H]+, 1037.8 [M+Na]+, 1053.8 [M+K]+.
CH2 Lys), 3.88–3.97 (m, 4H, 2 a-CH2 Gly), 4.34–4.42(m, 2H, 2
a-
CH), 4.58–4.61 (m, 2H, 2 a-CH), 4.62(d, 2H, J=6.2Hz, CH 2 Hxd),
5.62(ddd, 1H, J=13.6, 6.6, 6.6 Hz, CH=CHCH3), 5.72–5.81 (m, 1H,
CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.5 Hz, CH=CHCH3), 6.28 (dd,
1H, J=15.1, 10.3 Hz, CH2CH=CH), 7.18–7.29 ppm (m, 5H, Ar Phe);
ESI-MS: m/z: calcd for C34H53N7O7: 671.4; found 671.4 [M]+; MALDI-
TOF: m/z: 672.9 [M+H]+, 694.9 [M+Na]+, 710.9 [M+K]+.
H-Cys(StBu)-Gly-Pro-Ala-Gly-OHxd (4h): Starting from 3h (70 mg,
E
0.09 mmol) a colorless solid (18 mg, 0.05 mmol, 35%) was obtained.
1
[a]2D0 =ꢁ18.58 (c=0.2, MeOH); H NMR (CD3OD, 400 MHz): d=1.37 (s,
9H, tBu), 1.38 (d, 3H, J=7.3 Hz, CH3 Ala), 1.75 (d, 2H, J=6.7 Hz, CH3
Hxd), 1.86–2.38 (m, 4H, b-CH2, g-CH2 Pro), 3.07 (dd, 1H, J=8.5,
14.3 Hz, b-CH2 Cys), 3.26 (dd, 1H, J=5.2, 14.1 Hz, b-CH2 Cys), 3.52–3.74
(m, 2H, d-CH2 Pro), 3.89–4.01 (m, 2H, a-CH2 Gly), 4.05–4.17 (m, 3H, a-
H-Lys-Leu-Gly-Lys(Mtt)-Ala-Gly-OHxd (4d): Starting from 3d (21 mg,
A
15.5 mmol) a colorless solid (10.2mg, 11.2 mmol, 73%) was obtained.
1H NMR (CD3OD, 400 MHz): d=0.94 (d, 3H, J=6.4 Hz, CH3 Leu), 0.96
(d, 3H, J=6.4 Hz, CH3 Leu), 1.29–1.40 (m, 2H, g-CH2 Lys), 1.39 (d, 3H,
J=7.2Hz, CH 3 Ala), 1.44–1.55 (m, 2H, g-CH2 Lys), 1.58–1.93 (m, 11H,
2d-CH2 Lys, b-CH2 Leuk, 2 b-CH2 Lys, g-CH Leu), 1.74 (d, 3H, J=
6.6 Hz, CH3 Hxd), 2.38 (s, 3H, CH3 Mtt), 2.85–2.99 (m, 4H, 2e-CH2
Lys), 3.77–3.96 (m, 5H, 2 a-CH2 Gly, a-CH), 4.26–4.43 (m, 3H, 3a-
CH), 4.58 (d, 2H, J=6.6 Hz, CH2 Hxd), 5.59 (ddd, 1H, J=13.3, 6.6,
6.6 Hz, CH=CHCH3), 5.71–5.80 (m, 1H, CH2CH=CH), 6.05 (dd, 1H, J=
CH2 Gly, a-CH Cys), 4.36–4.46 (m, 2H, 2 a-CH), 4.62(d, 2H,
J=
6.6 Hz, CH2 Hxd), 5.61 (ddd, 1H, J=13.8, 7.1, 7.1 Hz, CH=CHCH3),
5.72–5.81 (m, 1H, CH2CH=CH), 6.06 (dd, 1H, J=15.1, 10.5 Hz, CH=
CHCH3), 6.28 (dd, 1H, J=15.2, 10.4 Hz, CH2CH=CH); ESI-MS: m/z:
calcd for C25H41N5O6S2: 572.3; found: 572.3 [M+H]+, 594.3 [M+Na]+;
FAB-HRMS: m/z: 571.2521 [M]+.
H-Lys-Cys-Gly-Val-Phe-Gly-OHxd (5): Peptide 4d (4.2mg, 5.4 mmol)
was dissolved in a degassed solution (800 mL) of 0.1m ammonium bicar-
Chem. Eur. J. 2006, 12, 6095 – 6109
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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