PULKA ET AL.
5f (cis D-Ala/Ala(pep): Yield: 44% (after purification), HPLC
tR = 15.52 min. (Gradient 1), Rf (CHCl3-Acetone 6:4) = 0.50, 1H-
NMR (700 MHz, CDCl3), d 1.39 (d, 3H, J = 5.6 Hz, H-20), 1.47 (d, 3H,
J = 7 Hz, CH3Alapep), 2.77 (ddd, 1H, J = 15.4 Hz, J = 11.9 Hz,
J = 2.1 Hz, H-4), 3.19 (ddd, 1H, J = 14.9 Hz, J = 4.4 Hz, J = 1.6 Hz,
H-4), 3.69 (dd, 1H, J = 11.2 Hz, J = 4.4 Hz, H-3), 3.78 (s, 3H,
OCH3), 4.29 (bs, 1H, H-1), 4.46 (bs, 1H, H-10), 4.67 (p, 1H,
J = 7.0 Hz, CHAlapep), 4.95 (bs, 2H, CH2Z), 5.19 (bs, 1H, NHZ),
7.07 to 7.47 (m, 10H, Ar + NHAlapep), 8.47 (s, 1H, NHind). 13C-NMR
(175 MHz, as HSQC, CDCl3), d 17.7 (C-20), 18.3 (CH3Alapep), 25.7 (C-4),
47.7 (CHAlapep), 47.8 (C-10), 52.5 (OCH3), 57.5 (C-1), 57.8 (C-3), 66.8
(CH2Z), 111.3 to 128.4 (Ar). ROESY (700 MHz) H-1 (H-H-3, H-10), H-3
(H-1, H-4, NHAlapep), H-4 (H-3, Ar), H-20 (H-1, H-10, NHZ), CHAlapep
(CH3Alapep). ESI-MS, m/z: 479 [M+ H]+, 501 [M + Na]+. Exact mass
calculated for C26H30N4O5Na [M + Na]+: 501.2114, found: 501.2119
Compound 7a (cis L-Ala/Leu(pep): Yield: 16% (after purifica-
tion), HPLC tR = 16.98 min. (Gradient 1), Rf (CHCl3-Acetone
6:4) = 0.71, 1H-NMR (700 MHz, CDCl3), d 0.98 (pseudot, 6H,
J = 6.0 Hz, CH3Leupep), 1.12 (d, 3H, J = 6.3 Hz, H-20), 1.61 to 1.75
(m, 3H, CH2Leupep + CHLeupep), 2.71 (ddd, 1H, J = 15.3 Hz,
J = 11.2 Hz, J = 2.5 Hz, H-4), 3.26 (ddd, 1H, J = 15.2 Hz, J = 4.0 Hz,
J = 1.4 Hz, H-4), 3.60 (dd, 1H, J = 11.2 Hz, J = 4.3 Hz, H-3), 3.76 (s,
3H, OCH3), 4.35 (bs, 1H, H-10), 4.38 (bs, 1H, H-1), 4.70 (td, 1H,
J = 8.4 Hz, J = 5.6 Hz, a-CHLeupep), 5.12 to 5.17 (m, 2H, CH2Z), 5.33
(d, 1H, J = 7.4 Hz, NHZ), 7.10 to 7.50 (m, 10H, Ar + NHLeupep), 8.28
(bs, 2H, CH2Z), 517 (d, 1H, J = 7.0 Hz, NHZ), 6.92 (d, 1H, J = 8.5 Hz,
NHLeupep), 7.10 to 7.48 (m, 9H, Ar), 8.44 (s, 1H, NHind). 13C-NMR
(175 MHz, as HSQC, CDCl3), d 17.7 (C-20), 21.9 (CH3Leupep), 22.7
(CH3Leupep), 24.9 (CHLeupep), 25.8 (C-4), 41.5 (CH2Leupep), 47.9 (C-10),
50.3 (a-CHLeupep), 52.3 (OCH3), 57.5 (C-1), 57.9 (C-3), 66.9 (CH2Z),
111.2 to 128.6 (Ar). ROESY (700 MHz, CDCl3) H-1 (H-3, H-20, NHind),
H-3 (H-1, H-4, NHLeupep), H-4 (H-3, NHLeupep, Ar), H-10 (H-1, H-20,
NHind), H-20 (H-1, H-10, NHZ), a-CHLeupep (CH2Leupep + CHLeupep,
CH3Leupep NHLeupep). ESI-MS, m/z: 521 [M + H]+, 543 [M + Na]+ ESI-
MS, m/z: 521 [M+ H]+, 543 [M+ Na]+. Exact mass calculated for
C29H36N4O5Na [M + Na]+: 543.2583, found: 543.2586.
Peptide 9
0.42 mmol (0.2 g) of compound 6a was dissolved in 15 mL CH3OH
and 10% Pd/C was added. The suspension was vigorously stirred
and saturated with hydrogen under atmospheric pressure, until
the starting material was not detected (5 h). The reaction mixture
was filtrated through a Celite pad and the filtrate evaporated to
dryness. 0.38 mmol (0.13 g) of crude product was redissolved
in 10 mL CH2Cl2 and cooled in the ice bath. 0.42 mmol (0.093 g)
Z-Ala-OH, followed by 0.42 mmol (56 mg) HOBt and 0.42 mmol
(0.134 g) TBTU, and 0.76 mmol (0.133 mL) DIPEA were added.
The reaction mixture was stirred for 4 h and then the solvent
was evaporated. The residue was redissolved in 15 mL AcOEt
and extracted with 1 M HCl, sat. NaHCO3 and washed with brine,
dried over Na2SO4. The crude product was purified by flash chro-
matography (CHCl3-Acetone).
13
(s, 1H, NHind). C-NMR (175 MHz, as HSQC, CDCl3), d 15.3 (C-20),
21.8 (CH3Leupep), 22.6 (CH3Leupep), 25.0 (CHLeupep), 25.8 (C-4), 41.4
(CH2Leupep), 49.3 (C-10), 50.4 (a-CHLeupep), 52.3 (OCH3), 56.8 (C-1),
57.3 (C-3), 66.9 (CH2Z), 111.0-128.5 (Ar). ROESY (700 MHz, CDCl3)
H-1 (H-3, H-20, NHZ, NHind), H-3 (H-1, H-4, NHLeupep), H-4 (H-3,
Yield: 62% (after purification), Rf (CHCl3-Acetone 6:4) = 0.38,
1H-NMR (700 MHz, CDCl3), d 1.16 (d, 3H, J = 6.7 Hz, CH3AlaCbz),
1.28 (d, 3H, J = 6.8 Hz, H-20), 1.40 (d, 3H, J = 7.2 Hz, CH3Alapep),
2.91 (ddd, 1H, J = 15.9 Hz, J = 7.5 Hz, J = 1.4 Hz, H-4), 3.16 (dd, 1H,
J = 15.9 Hz, J = 5.2 Hz, H-4), 3.68 (s, 3H, OCH3), 3.87 (bt, J = 6.1 Hz,
H-3), 4.13 (bs, 1H, CHAlaZ), 4.24 (bd, 1H, J = 3.4 Hz H-1), 4.35
(m, 1H, H-10), 4.57 (p, 1H, J = 7.0 Hz, CHAlapep) 5.06 to 5.16
(bs, 2H, CH2Cbz), 5.33 (bs, 1H, NHZ), 6.49 (d, 1H, J = 8.1 Hz, NHHꢀ10),
7.06 to 7.50 (m, 9H, Ar), 7.21 (bs, 1H, NHAlapep), 8.48 (s, 1H, NHind).
13C-NMR (175 MHz, as HSQC, CDCl3), d 17.1 (C-20), 17.8 (CH3Alapep),
17.9 (CH3AlaZ), 23.9 (C-4), 47.8 (CHAlapep), 47.9 (C-10), 51.2 (CHAlaZ),
52.1 (OCH3), 53.8 (C-3), 53.9 (C-1), 66.9 (CH2Z), 94.3-128.6 (Ar).
Ar), H-10 (H-20, NHind), H-20 (H-1, H-10), a-CHLeupep (CH2Leupep
+
CHLeupep, CH3Leupep, NHLeupep). ESI-MS, m/z: 521 [M + H]+, 543
[M + Na]+. Exact mass calculated for C29H36N4O5Na [M + Na]+:
543.2583, found: 543.2591.
Compound 8a (trans L-Ala/Leu(pep): Yield: 41% (after purifi-
cation), HPLC tR = 16.91 min. (Gradient 1), Rf (CHCl3-Acetone
6:4) = 0.63, 1H-NMR (700 MHz, CDCl3), d 0.89 (d, 6H, J = 6.5 Hz,
CH3Leupep), 1.35 (d, 3H, J = 6.5 Hz, H-20), 1.49 to 1.53 (m, 1H,
CH2Leupep), 1.58 (sept, 1H, J = 6.3 Hz, CHLeupep), 1.62 to 1.66
(m, 1H, CH2Leupep), 2.96 (bdd, 1H, J = 15.4 Hz, J = 6.3 Hz, H-4), 3.16
(dd, 1H, J = 15.8 Hz, J = 5.3 Hz, H-4), 3.67 (s, 3H, OCH3), 3.90
(t, 1H, J = 6.2Hz, H-3), 4.12 (d, 1H, J= 5.0Hz H-1), 4.22 (bs, 1H, H-10),
4.60 (td, 1H, J = 8.6Hz, J = 5.4Hz, a-CHLeupep), 5.00 to 5.06 (m, 2H,
CH2Z), 5.10 (d, 1H, J = 7.4Hz, NHZ), 6.97 (bd, 1H, J =5.9 Hz, NHLeupep),
7.10 to 7.51 (m, 9H, Ar), 8.19 (s, 1H, NHind). 13C-NMR (175 MHz, as
HSQC, CDCl3), d 18.2 (C-20), 21.8 (CH3Leupep), 22.7 (CH3Leupep), 23.8
(C-4), 24.8 (CHLeupep), 41.4 (CH2Leupep), 49.3 (C-10), 50.5 (a-CHLeupep),
52.1 (OCH3), 54.0 (C-3), 55.1 (C-1), 66.8 (CH2Z), 111.0-128.5 (Ar).
ROESY (700MHz, CDCl3) H-1 (H-20, NHZ), H-3 (H-4, H-10, H-20,
NHLeupep), H-4 (H-3, Ar), H-10 (H-3, H-20), H-20 (H-1, H-3, H-10, NHZ),
a-CHLeupep (CH2Leupep, CHLeupep, CH3Leupep, NHLeupep). ESI-MS, m/z:
521 [M + H]+, 543 [M + Na]+. Exact mass calculated for
C29H36N4O5Na [M + Na]+: 543.2583, found: 543.2575.
Compound 7f (cis D-Ala/Leu(pep): Yield: 53% (after purifica-
tion), HPLC tR = 17.14 min. (Gradient 1), Rf (CHCl3-Acetone
6:4) = 0.54, 1H-NMR (700 MHz, CDCl3), d 0.96 (d, 3H, J = 6.4 Hz,
CH3Leupep), 0.98 (d, 3H, J = 6.3 Hz, CH3Leupep), 1.39 (d, 3H,
J = 6.0 Hz, H-20), 1.60 to 1.73 (m, 3H, CH2Leupep + CHLeupep), 2.77
(ddd, 1H, J = 15.3 Hz, J = 11.2 Hz, J = 2.1 Hz, H-4), 3.20 (ddd, 1H,
J = 15.4 Hz, J = 4.3 Hz, J = 1.4 Hz, H-4), 3.71 (dd, 1H, J = 11.1 Hz,
J = 4.3 Hz, H-3), 3.76 (s, 3H, OCH3), 4.30 (bs, 1H, H-1), 4.45
(bs, 1H, H-10), 4.73 (td, 1H, J = 8.4 Hz, J = 5.6 Hz, a-CHLeupep), 4.96
ROESY (700 MHz, CDCl3, t = 245.2 K) H-1 + CHAlaZ (H-20 + CH3AlaZ
,
NHZ, NHH-10), H-3 (H-20 + CH3AlaZ, H-4, H-10, NHAlapep), H-4 (H-3),
H-10 (H-20 + CH3AlaZ, H-3, NHind), H-20 + CH3AlaZ (H-1 + CHAlaZ, H-3,
H-10, NHZ,), CHAlapep (CH3Alapep, NHAlapep .
)
Compound 11a (cis L-Ala-L-Ala): Yield: 37% (on the basis of
1H-NMR, with reference to the integration of OCH3 signals of
1
products and H-Trp-OCH3 ), HPLC tR = 13.5 min. (Gradient 2), H-
NMR (500 MHz, CDCl3), d 1.07 (d, 3H, J = 5.8 Hz, H-20), 1.39 (d, 3H,
J = 7.1 Hz, CH3AlaZ), 2.81 (bt, 1H, J = 13.0 Hz, H-4), 3.13 (dd, 1H,
J = 14.8 Hz, J = 3.0 Hz, H-4), 3.73 (bd, 1H, J = 9.5 Hz, H-3), 3.81
(s, 3H, OCH3), 4.29 (m, 1H, a-CHAlaZ), 4.43 (bs, 1H, H-1), 4.63
(m, 1H, H-10), 5.12 (q, 2H, J = 12.2 Hz, CH2Z), 5.64 (bd, 1H,
J = 5.5 Hz, NHZ), 7.17 (d, 1H, J = 7.3 Hz, NHAla), 7.07 to 7.46
(m, 9H, Ar), 9.05 (s, 1H, NHind). 13C-NMR (125 MHz, as HSQC,
CDCl3), d 14.6 (C-20), 18.8 (CH3AlaZ), 25.8 (C-4), 47.8 (C-10), 51.1
(a-CHAlaZ), 52.3 (OCH3), 55.8 (C-1), 56.0 (C-3), 67.5 (CH2Z), 111.5
to 128.4 (Ar). ROESY (500 MHz, CDCl3) H-1 (H-3, H-10, H-20), H-3
(H-1, H-4), H-4 (H-3, Ar), H-10 (H-1, H-20, NHind), H-20 (H-1, H-10,
Ar), a-CHAlaZ (CH3AlaZ, NHZ, NHAla). ESI-MS, m/z: 479 [M + H]+, 501
[M + Na]+. Exact mass calculated for C26H30N4O5Na [M + Na]+:
501.2114, found: 501.2111.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 433–440