PAPER
Enkephalin Analogs with Two Catechol Units
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13C NMR (CDCl3, 100 MHz): d = 143.6 (C), 141.1 (C), 131.2 (C),
127.5 2 (CH), 126.9 (2 CH), 125.1 (2 CH), 124.1 (CH), 121.3 (CH),
119.7 (2 CH), 112.0 (CH), 82.2 (C), 67.1 (2 CH), 60.6 (3 CH), 55.7
(CH3), 51.3 (CH), 47.1 (CH), 39.1 (CH2), 38.0 (CH2), 27.8 (3 CH3);
the missing (C) can not be observed.
DMF, and Fmoc-Phe-OH (522 mg, 1.35 mmol), Fmoc-Gly-OH
(401 mg, 1.35 mmol), Fmoc-Asp(CH2Ver)-OH (6; 510 mg, 1.01
mmol), Fmoc-Tyr(Bu-t)-OH (619 mg, 1.35 mmol) and Ac2O (3.5
mL)–DMF (3.5 mL) were attached successively, and if appropriate
deprotected. The peptide was cleaved using Cu(OAc)2 (61 mg, 0.34
mmol) in DMF (6 mL) in the presence of 5 (2.2 mL, 14.9 mmol).
The obtained crude product was washed with Et2O to obtain 419 mg
(69%) of a colorless solid; mp 216 °C.
MS (ESI): m/z = 583.3 [M + Na]+.
Anal. Calcd for C32H36N2O7: C, 68.55; H, 6.47; N, 5.00. Found: C,
68.62; H, 6.62; N, 4.98.
IR (drift, KBr): 3288, 1638, 1544, 1482, 1275, 1233, 1168, 1083
cm–1.
Fmoc-Asp(CH2Ver)-OH (8)
Fmoc-Asp(CH2Ver)-OH (6; 750 mg, 1.34 mmol) was stirred for 30
min with Et2O saturated with gaseous HCl (30 mL). The solvent
was removed in vacuum and the residue was washed with Et2O to
give 576 mg (85%) of a white solid; mp 163 °C.
1H NMR (DMSO-d6, 400 MHz): d = 8.42 (t, J = 5.5 Hz, 1 H, NH),
8.31 (d, J = 7.4 Hz, 1 H, NH), 8.21–8.13 (m, 3 H, NH), 8.07 (d,
J = 8.2 Hz, 1 H, NH), 7.21 (s, 5 H, Phe), 7.11 (d, J = 8.5 Hz, 2 H,
Tyr), 7.01–6.96 (m, 1 H, Cat.), 6.95–6.94 (m, 1 H, Ver), 6.93–6.91
(m, 2 H, Ver), 6.83 (d, J = 8.2 Hz, 2 H, Tyr), 6.82–6.80 (m, 1 H,
Ver), 6.65 (dd, J = 7.0, 1.7 Hz, 1 H, Ver), 4.58–4.43 (m, 3 H, a-H),
4.28 (d, J = 6.3 Hz, 2 H, benzyl CH2), 4.25 (d, J = 6.8 Hz, 2 H, ben-
zyl CH2), 3.78 (s, 3 H, OCH3), 3.77 (s, 3 H, OCH3), 3.70 (s, 3 H,
OCH3), 3.68 (s, 3 H, OCH3), 3.38 (d, J = 7.1 Hz, 2 H, CH2 Gly),
3.07–3.01 (m, 1 H, CH2), 2.95–2.90 (m, 1 H, CH2), 2.90–2.83 (m, 1
H, CH2), 2.71–2.63 (m, 2 H, CH2), 2.58–2.52 (m, 1 H, CH2), 1.74
(s, 3 H, OCOCH3), 1.25 (s, 9 H, t-C4H9).
IR (drift, KBr): 3300, 2940, 1702, 1642, 1542, 1481, 1448, 1430,
1275, 1226, 1172, 1084, 1050, 1004, 740 cm–1.
1H NMR (DMSO-d6, 400 MHz): d = 8.32 (t, J = 5.8 Hz, 1 H, NH),
7.94 (d, J = 7.2 Hz, 2 H, Fmoc), 7.76 (d, J = 7.4 Hz, 2 H, Fmoc),
7.65 (d, J = 8.2 Hz, 1 H, NH), 7.47 (t, J = 7.4 Hz, 2 H, Fmoc), 7.38
(t, J = 7.4 Hz, 2 H, Fmoc), 7.04–6.95 (m, 2 H, Ver), 6.88 (d, J = 7.2
Hz, 1 H, Ver), 4.50–4.42 (m, 1 H, a-H), 4.35–4.31 (m, 3 H, CH2 +
CH Fmoc), 4.30–4.25 (m, 2 H, benzyl CH2), 3.83 (s, 3 H, OCH3),
3.77 (s, 3 H, OCH3), 2.76–2.70 (m, 1 H, CH2 Asp), 2.61 (dd,
J = 10.9, 8.0 Hz, 1 H, CH2 Asp).
13C NMR (DMSO-d6, 100 MHz): d = 173.5 (C), 169.5 (C), 156.2
(C), 152.2 (C), 146.6 (C), 144.2 (2 C), 141.1 (2 C), 132.9 (C), 128.1
(2 CH), 127.5 (2 CH), 125.7 (2 CH), 124.2 (CH), 120.6 (3 CH),
112.1 (CH), 66.3 (CH2), 60.6 (CH3), 45.2 (CH3), 51.2 (CH), 47.2
(CH), 37.6 (CH2), 37.5 (CH2).
MS (ESI): m/z = 919.6 [M + Na]+, 897.3 [M + H]+.
Anal. Calcd for C48H60N2O11·2H2O: C, 61.79; H, 6.91; N, 9.01.
Found: C, 61.38; H, 6.68; N, 8.94.
Ac-Tyr-Asp(CH2Cat)-Gly-Phe-CH2Cat (2a)
At 0 °C, AlCl3 (186 mg, 1.39 mmol, 25 equiv) was dissolved in
ethanethiol (5 mL) and 11 (50 mg) was added and the mixture was
stirred overnight. After hydrolysis with MeOH (15 mL), the volatile
components were removed in vacuum the residue was suspended in
H2O and then filtered to obtain 8 mg (18%) of a grey solid; mp
180 °C (dec.).
MS (ESI): m/z = 504.2 [M – H]–.
Anal. Calcd for C28H28N2O7·0.75H2O: C, 64.92; H, 5.74; N, 5.41.
Found: C, 64.98; H, 5.82; N, 5.26.
IR (drift, KBr): 3331, 2930, 1653, 1518, 1478, 1343, 1259, 742
cm–1.
Solid-Phase Synthesis of the Peptide Sequences; General Proce-
dure
1H NMR (CD3OD + DMSO-d6, 300 MHz): d = 7.20 (br s, 5 H,
Phe), 7.00 (d, J = 8.7 Hz, 2 H, Tyr), 6.72–6.67 (br m, 3 H, Tyr +
Cat), 6.67–6.56 (m, 5 H, Cat), 4.60–4.56 (br m, 1 H, a-H), 4.51–
4.42 (br m, 2 H, a-H), 4.35–4.25 (br m, 4 H, benzyl CH2), 3.70 (br
s, 2 H, CH2 Gly), 3.22–3.17 (br m, 1 H, CH2), 3.05–2.90 (br m, 2 H,
CH2), 2.81–2.70 (br m, 2 H, CH2), 2.67–2.65 (br m, 1 H, CH2), 1.88
(s, 3 H, OCOCH3).
The synthesis of dicatechol peptide precursors was performed on
the 4-Fmoc-hydrazinobenzoyl resin 9 (4-Fmoc-hydrazinobenzoyl
AM resin, Novabiochem)11 by using N-Fmoc-protected amino ac-
ids. Prior to use, the resin was swollen in CH2Cl2 and after washing
with DMF, the Fmoc-group was removed with a 20% solution of pi-
peridine in DMF to obtain 10.
For the preparation of the peptide sequences, the following protocol
was used: The C-terminal-unprotected N-Fmoc amino acid (2
equiv) was activated with DIPEA (4 equiv) and HBTU (2 equiv) in
DMF. After 10 min, this solution was added to 10 and the mixture
was shaken for 1 h. Before attaching the next residue, the Fmoc
group had to be removed by treating the resin with a 20% solution
of piperidine in DMF for 15 min. The N-terminus of the peptide was
either capped by reaction with Ac2O in DMF or by attachment of 4-
bromobenzoic acid (12) which was activated with DIPEA/HBTU.
Finally, the resin was successively washed with DMF, CH2Cl2, and
MeOH. After drying under vacuum, the peptide was cleaved from
the resin with simultaneous attachment of 5 to the C-terminus. This
was achieved by treatment with Cu(OAc)2 (1 equiv) in DMF and
bubbling air through the mixture for 4 h in the presence of 5. The
resin was removed by filtration and washed with CH2Cl2 (in the
case of 2a with DMF), and then the combined organic layers were
washed with aq 1 M KHSO4, H2O, and brine. After drying
(Na2SO4), the organic solvents were removed by distillation in vac-
uum.
Positive ESI-MS: m/z = 807.6 [M + Na]+, 785.4 [M + H]+.
Negative ESI-MS: m/z = 783.5 [M – H]–.
(4-Bromobenzoyl)-Tyr(Bu-t)-Asp-Gly-Phe-CH2Ver (13)
Following the general procedure for the solid-phase peptide cou-
pling, the compound 9 (1.33 g, 1.06 mmol) was treated with piperi-
dine in DMF, and Fmoc-Phe-OH (821 mg, 2.12 mmol), Fmoc-Gly-
OH (630 mg, 2.12 mmol), Fmoc-Asp(Bu-t)-OH (974 mg, 2.12
mmol), Fmoc-Tyr(Bu-t)-OH (974 mg, 2.12 mmol), and 4-bro-
mobenzoic acid (12; 426 mg, 2.12 mmol) were succesively at-
tached, and if appropriate were deprotected. Prior to cleavage, the
resin was treated with 20% TFA in CH2Cl2 (10 mL) for 20 min and
then was washed with DIPEA in DMF. The peptide was cleaved us-
ing Cu(OAc)2 (96 mg, 0.53 mmol) in DMF (12 mL) in the presence
of 5 (1 mL, 10.8 mmol). The obtained crude product was washed
with Et2O to obtain 508 mg (54%) of a yellow solid; mp 210 °C
(dec.).
IR (drift, KBr): 3287, 1636, 1518, 1482, 1273, 1227, 1157, 750
cm–1.
1H NMR (CD3OD, 300 MHz): d = 7.66 (d, J = 8.7 Hz, 2 H, ben-
zoyl), 7.56 (d, J = 8.7 Hz, 2 H, benzoyl), 7.25 (s, 5 H, Phe), 7.11 (d,
Ac-Tyr(tBu)-Asp(CH2Ver)-Gly-Phe-CH2Ver (11)
Following the general procedure for solid-phase peptide coupling,
compound 9 (843 mg (0.67 mmol) was treated with piperidine in
Synthesis 2005, No. 2, 211–216 © Thieme Stuttgart · New York