Approaches towards enkephalin analogs exhibiting two catechol units

Article abstract of DOI:10.1055/s-2004-834914

Peptide-bridged dicatechols were prepared by a combination of solid- and solution-phase synthesis. The target compounds are potential precursors for constrained metal containing enkephalin analogs. While a simple model compound selectively forms a metallamacrocycle with the cis-molybdenum(VI)dioxo unit, this goal could not be achieved with the corresponding peptide bridged derivatives.

Full text of DOI:10.1055/s-2004-834914

PAPER
211
Approaches Towards Enkephalin Analogs Exhibiting Two Catechol Units
Enkephalin
Analogs
w
a
ith Two Ca
r
techol
U
k
nits us Albrecht,* Patrick Stortz, Miriam Baumert
Institut für Organische Chemie der RWTH-Aachen, Professor-Pirlet-Straße 1, 52074 Aachen, Germany
Fax +49(241)8092385; E-mail: markus.albrecht@oc.rwth-aachen.de
Received 9 August 2004; revised 16 September 2004
HO
Abstract: Peptide-bridged dicatechols were prepared by a combi-
nation of solid- and solution-phase synthesis. The target compounds
are potential precursors for constrained metal containing enkephalin
analogs. While a simple model compound selectively forms a met-
allamacrocycle with the cis-molybdenum(VI)dioxo unit, this goal
could not be achieved with the corresponding peptide bridged deriv-
atives.
O
H
N
H
N
H2N
N
H
O
O
1
HN
O
Key words: peptides, solid-phase synthesis, enkephalin, macrocy-
HO
cles, phenols
O
H
N
H
N
XHN
N
Enkephalins are pentapeptides, which act as neurotrans-
mitters as well as neuromodulators. They are endogenous
ligands for the opiate receptor in the brain. Methionine en-
kephalin as well as leucine enkephalin were first isolated
from porcine brain and have the Tyr-Gly-Gly-Phe-se-
quence in common, followed either by Met or Leu.1
H
O
O
O
HN
O
HN
X = MeCO
X = 4-Br-C6H4CO 2b
2a
HO
OH
OH
Due to the interesting biological activity of the enkepha-
lins, numerous analogs were prepared.2 In this context,
cyclic enkephalin analogs 1 were obtained, e.g. by the
group of Goodman (Figure 1). In vivo, the cyclic com-
pounds are up to 100 times more potent than morphine.3
This high activity is probably due to a higher conforma-
tional preorganization of the macrocycles compared to the
more flexible open chain enkephalins.
OH
Figure 1 General representation of cyclic enkephalin analogs 1 as
described by Goodman and the dicatechol analogs 2 proposed by us.
heptamethylene chain is a very much simplified model for
the peptidic spacer, due to its similar chain length. Here,
we introduced one atom less, than is found in the peptide
spacer, to mimic the constrain of the amide units.
Recently we introduced catechol units at the termini of
short naturally occurring peptide sequences and by metal
coordination fixed the peptides in a macrocyclic confor-
mation.4–6 We were now interested to use this concept for
the formation of metallamacrocycles which contain a con-
strained enkephalin unit, similar as in 1. Therefore, we ex-
pected the ligands 2a,b to be good candidates for the
formation of metallacyclopeptides. In 2a,b one of the gly-
cine units of the enkephalins is substituted by aspartic acid
to enable the side-chain attachment of a catechol moiety
(Figure 1). This work was performed in order to test,
whether short peptides, which bear catechol units at the
side chain are able to specifically form metal complexes.
In future studies the gained knowledge should help to de-
sign metallacyclopeptides with desired conformations.
The dicatechol derivative 3 was prepared starting from
azelic acid (4), which was activated by DIPEA (N,N-di-
isopropylethylamine) and HBTU [O-(benzotriazol-1-yl)-
N,N,N¢,N¢-tetramethyluronium hexafluorophosphate] in
acetonitrile and then was coupled with two equivalents of
2,3-dimethoxybenzylamine (5) (Scheme 1).7 The protect-
ed ligand precursor was isolated in 77% and was then
demethylated in 90% yield by reaction with BBr3 to ob-
tain 3.8 The dicatechol 3 formed a metal complex
K2[3MoO2] by reaction with MoO2(acac)2 in methanol in
the presence of K2CO3. Ligand 3 as well as the molybde-
num(VI) complex could be characterized by spectroscop-
ic methods.9 Hereby negative ESI-MS of K2[3MoO2]
showed, that it has the 1:1 stoichiometry. In the 1H NMR
spectra, the benzylic protons can be considered as a spec-
troscopic probe. In 3, they appear as a singlet at d = 4.48
(in CD3OD) while for K2[3MoO2] a broad multiplet is ob-
served at d = 4.19–4.10 (D2O). This is due to the diaste-
reotopicity of the protons close to the newly introduced
stereocenter at the metal complex unit.
Initially, we prepared a very simple model system 3,
which possesses two 2,3-dihydroxybenzamide units
bound to azelic acid (nonanedicarboxylic acid). The ben-
zyl amides resemble the ligand units of 2a,b while the
SYNTHESIS 2005, No. 2, pp 0211–0216
x
x
.
x
x
.2
0
0
4
Advanced online publication: 24.11.2004
DOI: 10.1055/s-2004-834914; Art ID: T09304SS
© Georg Thieme Verlag Stuttgart · New York

212
M. Albrecht et al.
PAPER
1) DIPEA, HBTU,
MeCN
2) 2,3-dimethoxy-
benzylamine (5)
(77%)
3) BBr3, CH2Cl2
(90%)
then coupled to the hydrazine units of the resin. The
Fmoc-groups were removed by reaction with piperidine in
DMF. The same protocol of activation, coupling and
deprotection was performed successively with Fmoc-Gly-
OH, with Fmoc-Asp(CH2Ver)-OH (6) (Ver = 3-
veratryl = 2,3-dimethoxybenzene), and with Fmoc-
Tyr(Bu-t)-OH. After removal of the Fmoc-protecting
group at the N-terminus, the amino group was N-protect-
ed by reaction with acetic anhydride (Scheme 3).
O
HN
O
HO
HO
HN
HOOC(CH2)7COOH
4
OH
3
OH
An advantage of the hydrazinobenzoyl resin was that
amide coupling at the C-terminus of the peptide could be
performed simultaneously with the cleavage from the res-
in. Therefore, the hydrazine unit was oxidized in the pres-
ence of copper(II) acetate and air and the activated C-
terminus was trapped with an appropriate amine.11 In this
study 2,3-dimethoxybenzyl amine (5) was used, to obtain
the protected ligand precursor 11 over 11 steps in 69%
yield.
O
HN
O
O
O
MoO2(acac)2, K2CO3
methanol
HN
K2
76%
O
Mo
K2[3MoO2]
O
O
O
Finally, the tert-butyl group at tyrosine and the methyl
ethers of the veratryl unit were cleaved in one step using
the AlCl3/ethanethiol reaction conditions.12 The reaction
mixture was worked up by quenching with methanol, re-
moval of volatile components in vacuum and extraction of
the residue with water. The remaining solid material was
isolated by filtration and was dried in vacuum.
Scheme 1 Preparation of a simple model ligand 3 and its molybde-
num(VI)dioxo complex.
For the synthesis of the peptide ligand 2a, the 9-fluorenyl-
methyloxycarbonyl-(Fmoc-)-protected aspartic acid de-
rivative 6 had to be prepared first. As shown in Scheme 2,
the side chain carboxylic acid of Fmoc-Asp-OBu-t (7)
was activated with DIPEA/HBTU and was then reacted
with 2,3-dimethoxybenzylamine (5), to obtain the fully
protected amino acid derivative 8 in 72% yield.7 The tert-
butyl group of 8 could be selectively removed by reaction
with HCl in diethyl ether10 and the Fmoc-protected amino
acid derivative 6 was obtained in 85% yield.
The desired dicatechol functionalized peptide Ac-Tyr-
Asp(CH2Cat)-Gly-Phe-CH2Cat (2a) (Cat = 3-catechyl =
2,3-dihydroxyphenyl) was obtained in 18% yield. Com-
pound 2a was characterized by ESI-MS, showing molar
peaks in the positive mode at m/z = 807.6 [M + Na]+ and
785.4 [M + H]+. In the negative mode the corresponding
peak of [M – H]– was found at m/z = 783.5. Due to the low
solubility of 2a, the 1H NMR spectrum was recorded in a
mixture of CD3OD and DMSO-d6. The spectrum was
characteristic for 2a (see Experimental Section).
O
1) DIPEA, HBTU,
MeCN
O
2) 2,3-dimethoxy-
benzylamine (5)
However, the low yield of 2a was not satisfying. We sup-
posed that it was due to the low solubility of 2a in com-
mon solvents but a reasonable solubility in water, which
results in the loss of product during work up. To overcome
this problem, we synthesized the dicatechol 2b in a some-
what modified procedure (Scheme 4).
HN
O
O
O
72%
OH
7
Following the above described solid-phase protocol, we
deprotected the resin 9 and successively activated, cou-
pled and deprotected Fmoc-Phe-OH, Fmoc-Gly-OH,
Fmoc-Asp(Bu-t)-OH, and Fmoc-Tyr(Bu-t)-OH. Finally,
4-bromoacetic acid (12) was attached to the N-terminus.
O HO
O
O
O
O
HCl
Et2O
HN
O
FmocHN
H3CO
O
85%
NH
NH
8
6
Prior to cleavage of the peptide from the resin, the tert-bu-
tyl protecting group at the aspartic acid residue was selec-
tively (vide infra) removed by reaction with 20%
trifluoroacetic acid (TFA) in dichloromethane for 20 min-
utes.10 Finally the peptide was cleaved with simultaneous
attachment of 2,3-dimethoxybenzylamine (5) as was de-
scribed before, to yield 13 in 54% over 12 steps.
OCH3
H3CO
OCH3
Scheme 2 Synthesis of the aspartic acid derivative 6.
The enkephalin analogs were assembled by solid-phase
synthesis. Therefore, the 4-Fmoc-hydrazinobenzoyl resin
9 was deprotected with piperidine in DMF.5,11 The resin
10 was the starting point for the successive introduction of
the amino acid residues. In a first reaction sequence
Fmoc-Phe-OH was activated with DIPEA/HBTU and was
The obtained peptide 13 was characterized by negative
and positive ESI-MS, 1H NMR spectroscopy, and elemen-
tal analysis. The data showed that, as desired, one tert-bu-
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PAPER
Enkephalin Analogs with Two Catechol Units
213
Scheme 3 Solid-phase synthesis of the enkephalin analogue dicatechol derivative 2a (Ver = 3-veratryl = 2,3-dimethoxybenzene).
Scheme 4 Solid-phase synthesis of the enkephalin analogue dicatechol derivative 2b.
tyl group was still attached. However, spectroscopy did yield as a colorless solid. The protecting groups were
not allow to distinguish, whether the remaining group is cleaved in one reaction step with AlCl3/ethanethiol,12 to
bound to tyrosine or to aspartic acid.
obtain the dicatechol functionalized enkephalin analog 2b
in 94% yield as crude product in good purity. However, it
could further be purified by recrystallization from metha-
nol–water to obtain a yield of 64% of very pure 2b. Com-
pound 2b was characterized by positive ESI-MS, 1H and
13C NMR, IR spectra, and elemental analysis (see experi-
mental section).
Activation of the free carboxylic acid function of the as-
partic acid residue with DIPEA/HBTU and amide forma-
tion with 2,3-dimethoxybenzylamine (5) finally showed
that the t-butyl group was bound to the phenol of tyrosine.
The protected ligand precursor 14 was obtained in 74%
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214
M. Albrecht et al.
PAPER
13C NMR (CD3OD, 100 MHz): d = 172.5 (2 C), 152.4 (2 C), 131.8
(2 C), 124.1 (2 CH), 121.4 (2 CH), 111.7 (2 CH), 60.6 (2 CH3), 55.7
(2 CH3), 38.9 (2 CH2), 36.7 (2 CH2), 29.0 (2 CH2), 28.8 (CH2), 25.5
(2 CH2).
The synthesis of 2b described herein has major advantag-
es over the one described for the preparation of 2a: Selec-
tive cleavage of the aspartic acid protecting group and
attachment of 2,3-dimethoxybenzylamine (5) to this posi-
tion avoids the preparation of the building block 6.
MS (EI-DIP,70 eV): m/z (%) = 466.2 (6.35 [M]+), 166.0 (100).
Anal. Calcd for C27H38N2O6: C, 66.64; H, 7.87; N, 5.76. Found: C,
66.21; H, 7.76; N, 5.72.
The 4-bromobenzoyl group induces solubility properties,
which enable a simple and effective removal of the pro-
tecting group in the final step.
Azelic Acid Bis(2,3-dihydroxybenzylamide) (3)
At –15 °C, the protected ligand precursor azelic acid bis(2,3-
dimethoxybenzylamide) (298 mg, 0.61 mmol, 1 equiv) was dis-
solved in CH2Cl2 (30 mL) and BBr3 (590 mL, 6.12 mmol, 10 equiv)
was added. After stirring overnight, MeOH (20 mL) was added and
the volatile components were removed in vacuum. The residue was
washed with H2O to afford 236 mg (90%) of a slightly brown solid;
Finally a series of coordination studies of 2b with
MoO2(acac)2 were performed in methanol in the presence
of potassium or sodium carbonate. However, in contrast to
the simple model compound 3, compounds 2a and 2b did
not yield defined mononuclear macrocyclic complexes.
Probably mixtures of oligomeric coordination compounds mp 143 °C.
were formed.
IR (drift, KBr): 3395, 2933, 2859, 1623, 1552, 1480, 1376, 1259,
1223, 1077, 1043, 741 cm–1.
Our described results showed, that the chain length of the
dicatechol compounds 2a and 2b (as well as 3) should be
appropriate for coordination of both catechols to one met-
al center. However, in the peptidic systems, the preferred
conformation of the peptide chain seems to disfavor a cy-
clic arrangement, which would be present in a 1:1 com-
plex.
1H NMR (CD3OD, 400 MHz): d = 6.97 (dd, J = 9.4, 1.7 Hz, 2 H),
6.71 (dd, J = 7.7, 1.9 Hz, 2 H), 6.68–6.64 (m, 4 H), 4.48 (s, 4 H, ben-
zyl CH2), 2.47 (t, J = 7.7 Hz, 4 H), 1.68–1.59 (br m, 4 H), 1.31 (br
s, 6 H).
MS (EI-DIP, 70 eV): m/z (%) = 430.1 (10.73 [M]+), 128.0 (100).
Anal. Calcd for C23H30N2O6·2.5H2O: C, 58.04; H, 6.57; N, 5.89.
Found: C, 58.48; H, 6.81; N, 5.58.
In conclusion, we have presented a synthetic entry to di-
catechol functionalized enkephalin analogues, which
have the potential to form conformationally constrained
metallacyclopeptides. Although no defined coordination
compounds are observed with the ligand 2b, we have es-
tablished the preparative methods, which we need to sys-
tematically screen this system. The L-aspartic acid residue
can be substituted by D-aspartic acid or by glutaric acid.
Side-chain catechol-functionalized lysine or ornithine res-
idues could be introduced in this position. The respective
studies will be performed in our laboratories and finally
should result in the desired metallamacrocycles.
Metal Complex K2[3MoO2]
Ligand 3 (28 mg, 0.07 mmol, 1 equiv), K2CO3 (35 mg, 0.25 mmol,
4 equiv) and MoO2(acac)2 (25 mg, 0.08 mmol, 1.2 equiv) were dis-
solved in MeOH (10 mL). The mixture was stirred overnight, the
solvent was removed in vacuum and the residue was filtered over
Sephadex LH to give 20.3 mg (76%) of a red solid.
IR (drift, KBr): 3395, 2928, 1632, 1535, 1457, 1278, 1252, 1211,
890 cm–1.
1H NMR (D2O, 400 MHz): d = 6.52–6.40 (br m, 6 H), 4.19–4.10 (br
m, 4 H, benzyl CH2), 2.20–1.62 (m, 4 H), 1.50–1.18 (m, 4 H), 1.14–
0.98 (br, 6 H).
Negative ESI-MS: m/z = 557.3 [{3MoO2} + H]–.
1H and 13C NMR spectra were recorded on a Varian Inova 400 spec-
trometer. FT-IR spectra were recorded on a Bruker IFS spectrome-
ter (drift = diffuse reflection FT-IR spectroscopy). Mass spectra
(EI, 70 eV; FAB) were taken on a Finnigan MAT 95 or 212 mass
spectrometer. Elemental analyses were obtained with a Heraeus
CHN-O-Rapid analyzer. Melting points: Büchi B-540 (uncorrect-
ed). The 4-Fmoc-hydrazinobenzoyl AM resin 9 and the protected
amino acid building blocks were purchased from Novabiochem.
Anal. Calcd for C23H26K2MoN2O8·3H2O: C, 40.23; H, 4.70; N,
4.08. Found: C, 40.23; H, 4.68; N, 3.92.
Fmoc-Asp(CH2Ver)-OBu-t (8)
Fmoc-Asp-OBu-t (7; 850 mg, 2.07 mmol, 1 equiv) was dissolved in
MeCN (15 mL) and DIPEA (0.39 mL, 2.27 mmol, 1.2 equiv) as
well as HBTU (940 mg, 2.48 mmol, 1.2 equiv) were added. The
mixture was activated for 30 min and then benzylamine 5 (0.34 mL,
2.27 mmol, 1.1 equiv) was added and the mixture was stirred for 3
days. The precipitate was collected by filtration and dried in vacu-
um to yield a colorless solid (837 mg, 72%); mp 157 °C.
Azelic Acid Bis(2,3-dimethoxybenzylamide)
Azelic acid (4; 1.00 g, 5.31 mmol, 1 equiv) was dissolved in MeCN
(60 mL), and DIPEA (2.00 mL, 11.7 mmol, 2.2 equiv) and HBTU
(4.84 g, 12.8 mmol, 2.4 equiv) were added. After 15 min, 2,3-
dimethoxybenzylamine (5; 1.95 mL, 11.7 mmol, 2.2 equiv) was
added. The mixture was stirred for 2 h and the precipitate was re-
moved by filtration and washed with MeCN to give 1.98 g (77%) of
a colorless solid; mp 128 °C.
IR (drift, KBr): 3345, 3259, 2977, 2938, 1728, 1699, 1649, 1546,
1481, 1451, 1369, 1347, 1296, 1273, 1231, 1157, 1079, 1036, 1007,
743 cm–1.
1H NMR (CDCl3, 400 MHz): d = 7.74 (d, J = 7.4 Hz, 2 H, Fmoc),
7.59 (d, J = 7.4 Hz, 2 H, Fmoc), 7.39 (t, J = 7.4 Hz, 2 H, Fmoc),
7.33–7.28 (m, 2 H, Fmoc), 6.97 (t, J = 8.0 Hz, 1 H, Ver), 6.87-6.81
(m, 2 H, Ver), 6.12–6.03 (m, 2 H, NH), 4.50–4.46 (m, 1 H, a-H),
4.44 (d, J = 5.8 Hz, 2 H, CH2 Fmoc), 4.37 (dd, J = 10.2, 7.4 Hz, 1
H, benzyl CH2), 4.31–4.25 (m, 1 H, benzyl CH2), 4.20 (t, J = 7.1 Hz,
1 H, CH Fmoc), 3.85 (s, 3 H, OCH3), 3.83 (s, 3 H, OCH3), 2.91–2.84
(m, 1 H, CH2 Asp), 2.74–2.67 (m, 1 H, CH2 Asp), 1.43 (s, 9 H, t-
C4H9).
IR (drift, KBr): 3432, 3293, 2928, 1631, 1541, 1482, 1276, 1086,
1007, 751 cm–1.
1H NMR (CDCl3, 400 MHz): d = 7.04 (t, J = 8.0 Hz, 2 H), 6.90–
6.86 (m, 4 H), 4.45 (d, J = 5.8 Hz, 4 H, benzyl CH2), 3.87 (s, 6 H,
OCH3), 3.86 (s, 6 H, OCH3), 2.15 (t, J = 7.6 Hz, 4 H), 1.64–1.56 (m,
4 H), 1.27 (br s, 6 H).
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PAPER
Enkephalin Analogs with Two Catechol Units
215
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

216
M. Albrecht et al.
PAPER
J = 8.4 Hz, 2 H, Tyr), 6.99–6.90 (m, 2 H, Ver), 6.71 (d, J = 8.4 Hz,
2 H, Tyr), 6.70–6.66 (m, 1 H, Ver), 4.74 (t, J = 7.4 Hz, 1 H, a-H),
4.69–4.61 (m, 2 H, a-H), 4.33 (s, 2 H, benzyl CH2), 3.93 (d, J = 16.8
Hz, 1 H, CH2 Gly), 3.86 (s, 3 H, OCH3), 3.79 (s, 3 H, OCH3), 3.71
(d, J = 16.8 Hz, 1 H, CH2 Gly), 3.24–3.13 (m, 2 H, CH2 Tyr/Phe),
3.07–2.96 (m, 2 H, CH2 Tyr/Phe), 2.92–2.83 (m, 1 H, CH2 Asp),
2.64 (dd, J = 16.4, 7.0 Hz, 1 H, CH2 Asp), 1.45 (s, 9 H, t-C4H9).
Cat), 7.09 (d, J = 8.5 Hz, 2 H, Tyr), 6.73–6.68 (m, 4 H, Tyr + Cat),
6.60 (t, J = 7.7 Hz, 2 H, Cat), 6.52 (dd, J = 7.7, 1.7 Hz, 1 H, Cat),
4.74–4.69 (m, 1 H, a-H), 4.65 (t, J = 6.3 Hz, 1 H, a-H), 4.58 (dd,
J = 8.5, 6.0 Hz, 1 H, a-H), 4.26 (br s, 4 H, benzyl CH2), 3.88 (d,
J = 17.0 Hz, 1 H, CH2 Gly), 3.70 (d, J = 17.0 Hz, 1 H, CH2 Gly),
3.19–3.15 (m, 1 H, CH2), 3.14–3.11 (m, 1 H, CH2), 3.02 (d, J = 8.0
Hz, 1 H, CH2), 2.97–2.92 (m, 1 H, CH2), 2.91–2.88 (m, 1 H, CH2),
2.81–2.74 (m, 1 H, CH2).
13C NMR (CD3OD, 100 MHz): d = 173.0 (C), 172.4 (C), 172.3 (C),
171.9 (C), 169.9 (C), 167.9 (C), 163.4 (C), 155.9 (C), 136.9 (C),
132.8 (C), 131.3 (2 CH), 130.1 (2 CH), 129.0 (4 CH), 128.9 (2 CH),
128.1 (2 CH), 127.4 (C), 126.3 (CH), 125.9 (C), 124.6 (C), 119.8
(CH), 119.2 (CH), 114.9 (CH3), 114.2 (CH), 55.9 (CH), 55.2 (CH),
50.2 (CH), 42.6 (CH2), 38.6 (CH2), 37.4 (CH2), 36.4 (CH2), 35.7
(CH2), 35.1 (CH2); four carbon atoms cannot be observed.
MS (FAB, 3-NBA/DMSO); Positive Mode: m/z = 913.1 [M + Na]+,
891.0 [M + H]+.
MS (FAB, 3-NBA/DMSO); Negative Mode: m/z = 888.8 [M – H]–.
Anal. Calcd for C44H50BrN5O10·H2O): C, 58.28; H, 5.78; N, 7.72.
Found: C, 58.33; H, 5.86; N, 7.86.
(4-Bromobenzoyl)-Tyr(Bu-t)-Asp(CH2Ver)-Gly-Phe-CH2Ver
(14)
MS (ESI): m/z = 844.1 [M – Br]+.
A solution of 13 (505 mg, 0.57 mmol, 1 equiv) and DIPEA (107 mL,
0.63 mmol, 1.1 equiv) in DMF (5 mL) and CH2Cl2 (25 mL) was
combined with a solution of HBTU (259 mg, 0.68 mmol, 1.2 equiv)
in DMF (3 mL). After 20 min of activation, 5 (190 mL, 1.14 mmol,
2 equiv) was added and the mixture was stirred overnight. EtOAc
was added and the mixture was washed with sat. aq NH4Cl,
NaHCO3, H2O, and brine. The organic phase was dried (Na2SO4)
and the solvent was removed under reduced pressure. The crude
product can be recrystallized from hexane–i-PrOH; yield: 431 mg
(74%); colorless solid; mp 208 °C.
Anal. Calcd for C45H45BrN6O11·6H2O: C, 52.28; H, 5.56; N, 8.13.
Found: C, 52.45; H, 5.09; N, 7.67.
References
(1) Sewald, N.; Jakubke, H.-D. Peptides: Chemistry and
Biology; VCH: Weinheim, 2002.
(2) Evans, S. M.; Lenz, G. R. Chemtracts: Org. Chem. 1995, 8,
122.
IR (drift, KBr): 3419, 3298, 2936, 1633, 1519, 1481, 1275, 1227,
1159, 1079, 1009, 846 cm–1.
(3) (a) Baker, T. J.; Rew, Y.; Goodman, M. Pure. Appl. Chem.
2000, 72, 347. (b) Rew, Y.; Malkmus, S.; Svensson, C.;
Yaksh, T. L.; Chung, N. N.; Schiller, P. W.; Cassel, J. A.;
DeHaven, R. N.; Goodman, M. J. Med. Chem. 2002, 45,
3746. (c) Rew, Y.; Goodman, M. J. Org. Chem. 2002, 67,
8820.
(4) (a) Albrecht, M.; Stortz, P.; Nolting, R. Synthesis 2003,
1307. (b) Albrecht, M.; Stortz, P.; Weis, P. Supramol. Chem.
2003, 15, 477.
(5) (a) Albrecht, M.; Stortz, P.; Weis, P. Synlett 2003, 867.
(b) Albrecht, M.; Stortz, P.; Runsink, J.; Weis, P. Chem. Eur.
J. 2004, 10, 3657.
(6) For other metal stabilized peptide microstructures, see:
(a) Torrado, A.; Imperiali, B. J. Org. Chem. 1996, 61, 8940.
(b) Kelso, M. J.; Hoang, H. N.; Appleton, T. G.; Fairlie, D.
P. J. Am. Chem. Soc. 2000, 122, 10488. (c) Constable, E.
C.; Housecroft, C. E.; Mundwiler, S. J. Chem. Soc., Dalton
Trans. 2003, 2112.
(7) (a) Li, P.; Xu, J.-C. Tetrahedron 2000, 56, 9949.
(b) Speicher, A.; Eicher, T. J. Prakt. Chem. 1998, 340, 581.
(8) McOmie, J. F. W.; West, D. E. Org. Synth. Collect. Vol. V.;
Wiley: New York, 1973, 412.
(9) Duhme, A.-K. J. Chem. Soc., Dalton Trans. 1997, 773.
(10) Jones, J. Synthese von Aminosäuren und Peptiden; VCH:
Weinheim, 1995.
(11) (a) Millington, C. R.; Quarrell, R.; Lowe, G. Tetrahedron
Lett. 1998, 39, 7201. (b) Rosenbaum, C.; Waldmann, H.
Tetrahedron Lett. 2001, 42, 3677. (c) Peters, C.;
Waldmann, H. J. Org. Chem. 2003, 68, 6053.
(12) Node, M.; Nishide, K.; Fuji, K.; Fujita, E. J. Org. Chem.
1980, 45, 4275.
1H NMR (DMSO-d6, 400 MHz): d = 8.63 (d, J = 8.5 Hz, 1 H, NH),
8.45 (d, J = 7.7 Hz, 1 H, NH), 8.36 (t, J = 5.9 Hz, 1 H, NH), 8.10 (d,
J = 8.2 Hz, 1 H, NH), 7.92 (m, 2 H, NH), 7.72 (d, J = 8.7 Hz, 2 H,
benzoyl), 7.64 (d, J = 8.7 Hz, 2 H, benzoyl), 7.22 (s, 5 H, Phe),
7.12–7.05 (m, 4 H), 6.95–6.91 (m, 3 H), 6.63–6.59 (m, 3 H), 4.67–
4.50 (br m, 3 H), 4.28 (d, J = 5.8 Hz, 2 H, benzyl CH2), 4.25–4.20
(m, 2 H, benzyl CH2), 3.81 (s, 3 H, OCH3), 3.78 (s, 3 H, OCH3), 3.75
(s, 3 H, OCH3), 3.69 (s, 3 H, OCH3), 3.71–3.60 (m, 2 H, CH2 Gly),
3.04–2.96 (m, 2 H, CH2 Tyr/Phe), 2.85–2.80 (m, 2 H, CH2 Tyr/Phe),
2.74–2.66 (m, 2 H, CH2 Asp), 1.34 (s, 9 H, t-C4H9).
MS (ESI): m/z = 968.4 [M – (OBu-t)]+, 912.4 [M – Ver + Na]+.
Anal. Calcd for C53H61BrN5O11·4H2O: C, 57.35; H, 6.27; N, 7.57.
Found: C, 57.54; H, 6.01; N, 7.98.
(4-Bromobenzoyl)-Tyr-Asp(CH2Cat)-Gly-Phe-CH2Cat (2b)
At 0 °C, AlCl3 (481 mg, 3.61 mmol, 25 equiv) was dissolved in
ethanethiol (12 mL) and 14 (150 mg, 0.14 mmol) was added and the
mixture was stirred overnight. The mixture was diluted with CH2Cl2
(9 mL) and under cooling was quenched with MeOH (30 mL). The
volatile components were removed in vacuum and the residue was
suspended in H2O and then filtered to obtain 125 mg (94%) of crude
product, which can be recrystallized from MeOH–H2O; yield: 78
mg (64%); mp 185 °C (dec.).
IR (drift, KBr): 3307, 3089, 2931, 1652, 1594, 1518, 1480, 1229,
745 cm–1.
1H NMR (CD3OD, 400 MHz): d = 7.64 (d, J = 8.5 Hz, 2 H, ben-
zoyl), 7.55 (d, J = 8.8 Hz, 2 H, benzoyl), 7.22–7.15 (m, 6 H, Phe +
Synthesis 2005, No. 2, 211–216 © Thieme Stuttgart · New York