Preparation of tripeptide-bridged dicatechol ligands and their macrocyclic molybdenum(VI) complexes: Fixation of the RGD sequence and the WKY sequence of urotensin II in a cyclic conformation

Article abstract of DOI:10.1002/chem.200400144

Dicatechol ligands were prepared with caprylic acid (6-H₄) or the naturally occurring RGD (23-H₄) or WKY sequences (32-H ₄) as spacers. 6-H₄ was prepared by solution-phase amide coupling chemistry, while 16, the

Full text of DOI:10.1002/chem.200400144

FULL PAPER
Preparation of Tripeptide-Bridged Dicatechol Ligands and Their Macrocyclic
Molybdenum(vi) Complexes: Fixation of the RGD Sequence and the WKY
Sequence of Urotensin II in a Cyclic Conformation
Markus Albrecht,*[a] Patrick Stortz,[a] Jan Runsink,[a] and Patrick Weis[b]
Abstract: Dicatechol ligands were pre-
pared with caprylic acid (6-H4) or the
naturally occurring RGD (23-H4) or
WKY sequences (32-H4) as spacers. 6-
H4 was prepared by solution-phase
amide coupling chemistry, while 16, the
precursor of 23-H4, was obtained by
solution-phase and solid-phase prepa-
ration. In the latter case, a polystyrene
resin with a hydrazine benzoate linker
was used as the solid support. The last
coupling step was performed simulta-
neously with cleavage of the peptide
from the resin. The protecting groups
of 16 were all removed in one step to
yield the free ligand 23-H4. The WKY-
bridged derivative 32-H4 was obtained
by a similar solid-phase synthesis fol-
lowed by deprotection. The reaction of
all three ligands with dioxomolybde-
num(vi) bis(acetylacetonate) afforded
19-membered metallamacrocycles in
which the short peptides are conforma-
tionally fixed in a turn-type structure.
Hereby, the side-chain functionalities
of the peptides do not interfere in the
metal complexation.
Keywords:
molybdenum
macrocycles
¥ peptides
¥
¥
solid-phase synthesis
Introduction
ple, the zinc finger protein adopts a random-coil structure
when no metal is present.[5] However, in the presence of
zinc(ii), the metal coordinates to two cysteine and two histi-
dine residues and induces a b-sheet domain as well as an a-
helix domain. The latter is able to bind to DNA and, for ex-
ample, plays a crucial role in transcription factors.[6]
Following nature×s example, we had the idea to attach
metal-binding sites to both termini of short linear tri- or tet-
rapeptides.[7] Upon coordination of the ligand units to ap-
propriate metals a metallamacrocycle should be formed in
which the peptide conformation is fixed in a turn- or loop-
type structure (Scheme 1).[8] For the metal-binding site we
chose catechol units, which are known to show extraordinar-
ily strong binding to metal ions,[9] and for the metal-complex
The a helix, b sheet, and various turns are common structur-
al motifs that are found in proteins. Hereby, the secondary
structures of different protein domains are enforced, either
by intra- or interstrand noncovalent interactions, like hydro-
gen bonding, electrostatic attraction or repulsion, and hy-
drophobic/hydrophilic interactions, or by the formation of
covalent disulfide bridges. The spatial arrangement of the
side-chain functionalities of the amino acids is crucial for bi-
ological activity. Very often this activity depends on the
action of only a short peptide sequence, which is conforma-
tionally fixed by attachment to the protein. Short linear pep-
tides (up to 15 residues) do not usually adopt a well-defined
structure.[1] However, in small cyclopeptides a turn structure
is fixed, which often is responsible for the observed activi-
ty.[2]
Metal coordination is a strong noncovalent interaction
that can also induce a secondary peptide structure. This is
found in nature[3] as well as in artificial systems.[4] For exam-
[a] Prof. Dr. M. Albrecht, P. Stortz, Dr. J. Runsink
Institut f¸r Organische Chemie der RWTH
Prof.-Pirlet-Strasse 1, 52074 Aachen (Germany)
Fax : (+49)241-80-92385
E-mail: markus.albrecht@oc.rwth-aachen.de
[b] Dr. P. Weis
Institut f¸r Physikalische Chemie der Universit‰t Karlsruhe
Fritz-Haber-Weg, 76128 Karlsruhe (Germany)
Figure 1. The arginine glycine aspartic acid (RGD) sequence and the
structure of urotensin II containing the tryptophan lysine tyrosine
(WKY) sequence.
Chem. Eur. J. 2004, 10, 3657 3666
DOI: 10.1002/chem.200400144
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FULL PAPER
fragment
the
cis-MoVIO2
moiety, which is able to bind
two catechols, was chosen.[10]
Following this concept, we
have already described metal-
lacyclopeptides[11] that possess
the WAGV or WAG sequence
of the naturally occurring sege-
talins A and B.[12] Other metal-
lacyclopeptides were presented
by Imperiali,[13] Fairlie and
Kelso,[14] and Constable,[15]
with their respective co-work-
ers.
Scheme 2. Solution-phase synthesis of 6-H4 and K2[(6)MoO2]. HBTU=O-(benzotriazol-1-yl)-N,N,N’,N’-tetra-
methyluronium hexafluorophosphate, acac=acetylacetone.
The segetalins do not pos-
sess high functionalization in
their amino acid side chains.
For this reason, they were our first targets. In the present
study we have investigated whether it is possible to obtain
metallacyclopeptides with a high degree of functionality
without the functional groups interfering in the metal coor-
dination.
As nature×s model for our studies we chose the universal
cell recognition sequence arginine glycine aspartic acid
(RGD), which was shown to possess a high affinity for cell
surfaces by binding to the integrins (Figure 1). A cyclic con-
formation even favors a strong interaction.[16]
preparation of 6-H4, we started with 2,3-dimethoxybenzoic
acid (1), which was activated by reaction with HBTU in the
presence of ethyldiisopropylamine (H¸nig×s base). Addition
of 8-aminocaprylic acid (2) resulted in the formation of
compound 3. Again the carboxylic acid had to be activated
before 2,3-dimethoxybenzylamine (4) was attached to the C-
terminus and derivative 5 was obtained.[19,20] The methyl
ethers were cleaved by addition of BBr3, to yield the unpro-
tected ligand 6-H4.[21]
Reaction of ligand 6-H4 with [MoO2(acac)2] and potassi-
um carbonate in methanol resulted in the formation of the
macrocyclic molybdenum(vi)dioxo complex K2[(6)MoO2].[22]
IR spectroscopy of the complex showed the typical frequen-
cies of
a cis-dioxomolybdenum unit at n˜ =896 and
863 cmÀ1.[23] For the free ligand 6-H4, separated signals were
1
observed by H NMR spectroscopy in [D4]-methanol for the
aromatic protons at d=7.24, 7.00, 6.81, 6.75, 6.72, and
6.65 ppm. Upon complex formation the signals converged
and appeared at d=7.20, 6.70, and 6.46 ppm (4H). The reso-
nance of the benzylic methylene group acted as an NMR
spectroscopy probe. In the free ligand 6-H4 it appeared as a
singlet at d=4.52 ppm and upon complex formation it split
into two signals at d=4.44 and 4.31 ppm, due to the pres-
ence of the chiral biscatecholate molybdenum(vi)dioxo
moiety.
Negative ESI-MS showed that the mononuclear 19-mem-
bered metallamacrocycle K2[(6)MoO2] was formed. Corre-
sponding peaks were observed at m/z 543 {H[(6)MoO2]}À
and 581 {K[(6)MoO2]}À.
Scheme 1. Schematic representation of the formation of conformationally
fixed metallacyclopeptides from random-coil peptides.
Just recently urotensin II was thoroughly studied due to
its vasoconstrictor activity.[17] The tryptophan lysine tyro-
sine (WKY) sequence of the cyclopeptide part was shown to
be the active part of this molecule (Figure 1).[18]
The high degree of functionality of the arginine and as-
partic acid residues made the RGD sequence an appropriate
target for our studies, while basic lysine and phenolic tyro-
sine are present in the WKY sequence. However, prior to
coordination studies, appropriate RGD- or WKY-bridged
dicatechols had to be prepared.
Our investigations with the simple ligand 6 showed that
the chain length of 8-aminocaprylic acid, which corresponds
to that of a tripeptide, is appropriate to form a macrocycle
by coordination of the two terminal catechol units to a mo-
lybdenum(vi)dioxo moiety.[8]
Results and Discussion
The RGD-bridged dicatechol ligand 23-H4 and its molybde-
num(vi)dioxo complex K2[(23)MoO2]: The precursor for the
RGD-bridged dicatechol ligand, 16, was prepared by two
different approaches. The possibilities were to follow a 9-flu-
orenylmethoxycarbonyl (Fmoc) protection/deprotection
strategy in solution or to prepare the compound by solid-
phase synthesis.[20]
Investigation of the simple model system 6-H4 and
[(6)MoO2]2À: Initially we synthesized the simple ligand 6-H4
(Scheme 2). The spacer, 8-aminocaprylic acid (2), possesses
the same number of backbone atoms as is found in a tripep-
tide. However, 2 shows a higher degree of flexibility. For the
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Molybdenum Complexation of Tripeptides
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Solution-phase synthesis of 16:
In the solution-phase synthesis
of 16 we started with the N-ter-
minus- and side-chain-protect-
ed aspartic acid derivative 7
and applied a repeating se-
quence of amide coupling and
Fmoc cleavage (Scheme 3).[20]
Fmoc-Asp(OtBu)-OH
(7)
was activated with HBTU and
H¸nig×s base in DMF and the
benzyl amine 4 was added to
obtain amide 8 in 90% yield
after work up. The Fmoc
group of 8 was quantitatively
removed by treatment with pi-
peridine to yield the corre-
sponding amine 9. Fmoc-Gly-
OH (10) was first activated
(H¸nig×s base, HBTU) and
then amine 9 was added to
produce 11 in 79%. This was
also deprotected with piperi-
dine to yield the free amine 12
(quantitative yield). The same
sequence was used to prepare
amide 14 (79%) from amine
12 and activated Fmoc-
Arg(Mtr)-OH (13). Fmoc re-
moval with piperidine afforded
amine 15 (quantitative yield).
Finally, 2,3-dimethoxybenzoic
acid (1) was activated with
HBTU and H¸nig×s base and
amine 15 was added. The pro-
tected RGD-bridged ligand
precursor 16 was obtained in
86% yield. Thus, by starting
from 7, the ligand precursor 16
was prepared in seven steps in
48% overall yield.
Scheme 3. Solution-phase synthesis of precursor 16. Mtr=4-methoxy-2,3,6-trimethylbenzoylsulfonyl.
Synthesis of 16 on solid sup-
port: In a second approach, 16
was prepared by solid-phase
Scheme 4. Solid-phase synthesis of precursor 16.
synthesis on
a
polystyrene
resin bearing the 4-Fmoc-hy-
drazinobenzoyl linker, 17 (Scheme 4).[24,25]
of 22 was oxidized by copper(ii) acetate and air and the labi-
lized C terminus was attacked by 2,3-dimethoxybenzylamine
(4) as a nucleophile. The protected ligand 16 was purified by
column chromatography and was finally obtained in 64%
overall yield.
With the higher overall yield and the more simple reac-
tion procedures, the solid-phase synthesis of 16 was clearly
superior to the synthesis in solution.
First, the Fmoc protecting group of 17 was removed by
treatment with piperidine to yield the free hydrazine 18.
Next, the amino acids were successively attached by treat-
ment with Fmoc-protected acids 7, 10, and 13 that had been
activated with HBTU and H¸nig×s base. Prior to amide cou-
pling the Fmoc groups were cleaved. Thus, successively, the
derivatives 19, 20, and 21 were obtained. The N-terminal
ligand unit was introduced by addition of activated 2,3-di-
methoxybenzoic acid (1). Attachment of the C-terminal
ligand moiety was performed with concomitant cleavage of
the peptide from the solid support. Thus, the hydrazyl amide
Deprotection of 16 to obtain the RGD-bridged ligand 23-
H4: The ligand precursor 16 possessed an Mtr group at the
arginine residue, a tert-butyl group at the aspartic acid resi-
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M. Albrecht et al.
FULL PAPER
due, and four methyl groups
protecting the catechol moiet-
ies. Our intention was to
remove all of those groups in
only one step (Scheme 5). Usu-
ally, the Mtr group is removed
by treatment with protic acid,
which attacks at the sulfonate
unit.[26] The methyl ethers on
the catechol groups, on the
other hand, are removed by
cleavage with Lewis acids.[21]
Our idea was to perform the
deprotection with only Lewis
acids. Hereby, the catechol
methyl ethers as well as the
tert-butyl group should be re-
moved. The Mtr group would
not be attacked at the sulfo-
nate unit but the Lewis acid
would cleave the methyl ether
and, thus, should labilize and
remove the protecting group.
The protected compound 16
was treated with BBr3 in di-
chloromethane and the Mtr
moiety, as well as the tert-butyl
group, was removed smoothly.
However, the methyl ethers
were not completely cleaved
under the mild conditions. Re-
action with 1m BBr3 led to the
isolation of a compound in
which the Mtr unit, tert-butyl
group, and only three of the
methyl ethers were cleaved.
Scheme 5. One-step deprotection of precursor 16 to form ligand 23-H4.
Table 1. 1H NMR spectroscopy data in [D4]-methanol for the ligand 23-H4 and the complex K2[(23)MoO2].
H-aryl
H-benzyl
Arg
a-H CH2
Gly
Asp
a-H CH2
23-H4
7.35, 6.95, 6.75, 6.69, 6.66, 6.61 4.35
4.80 2.01, 1.84, 1.70 3.90
4.60 2.88, 2.77
K2[(23)MoO2] 7.21, 6.72, 6.46, 6.39 (2H), 6.30 4.79, 4.34 4.90 2.96, 2.88, 1.87, 3.90, 3.78 4.61 3.18, 2.48
1.24, 1.15, 0.92
The last methyl group remained on the molecule. Upon ap-
plying harsher conditions, 16 was cleaved at the peptide
chain. Therefore, other cleavage conditions were tested. Re-
action of 16 with AlBr3 or AlCl3 also did not lead to the de-
sired ligand 23-H4. The deprotected RGD-bridged dicate-
chol 23-H4 was finally obtained in a yield of 45% by remov-
al of all protecting groups by treatment with AlCl3 and etha-
nethiol[27] in dichloromethane, followed by purification by
HPLC.
1
Compound 23-H4 was characterized by H NMR spectro-
scopy (see Table 1) and by high-resolution FAB-MS, which
shows the peak for [23-H5]+ at m/z 604.2367 (calcd: m/z
604.2392).
Scheme 6. Complexation of ligand 23-H4 to form K2[(23)MoO2].
Formation and characterization of K2[(23)MoO2]: The
metallacyclopeptide K2[(23)MoO2] was prepared by treat-
ment of ligand 23-H4 with MoO2(acac)2 and potassium car-
bonate in a ratio of 1:1.1:4 (Scheme 6). The reaction was
performed in methanol at room temperature and needed
about five days to obtain the thermodynamically favored
product. Shorter reaction times led to mixtures of oligomer-
ic metal complexes. The final product was purified by filtra-
tion over Sephadex LH20.
Negative ESI-MS peaks of the mononuclear metallacyclo-
peptide were observed at m/z 807 [{K2[(23)MoO2]}ÀH]À,
769 {K[(23)MoO2]}À, 730 {H[(23)MoO2]}À, and 383.5
[(23)MoO2]2À. All peaks showed the expected isotopic pat-
tern.
The 1H NMR spectroscopic data for K2[(23)MoO2] in
[D4]-methanol are shown in Table 1 and the spectrum (in-
cluding assignment of the signals) is presented in Figure 2.
Assignments were done by COSY, TOCSY, and NOE spec-
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Molybdenum Complexation of Tripeptides
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ligand 23-H4 in the solution phase or on solid support
showed us that the latter is more effective. Therefore, we in-
troduced the WKY sequence, which is a part of the natural
product urotensin II, by solid-phase synthesis (Scheme 7).
Again the polystyrene resin with an 4-Fmoc-hydrazino-
benzoyl linker, 17, was used in the synthesis of the protected
WKY-bridged ligand precursor 31.[24,25] Derivative 31 was
prepared as described for the solid-phase synthesis of the
protected RGD-bridged ligand precursor 16. First, the Fmoc
group of 17 was removed and then the protected Tyr(tBu),
Lys(Boc), and Trp(Boc) amino acids were introduced suc-
cessively in a repetitive activation coupling deprotection
cycle.[20] Finally 2,3-dimethoxybenzoic acid (1) was attached
to the N terminus and the peptide was cleaved from the
solid support by oxidation (copper(ii) acetate, air) and trap-
ping with 2,3-dimethoxybenzylamine (4). The ligand precur-
sor 31 was obtained in an overall yield of 67%. The depro-
tection of 31 by treament with BBr3 smoothly removed the
protecting groups and led to ligand 32-H4 with a WKY tri-
peptide moiety bridging the two catechol units.[21] The
1H NMR spectrum of 32-H4 in [D4]-methanol is depicted in
Figure 3.
Figure 2. 1H NMR spectrum for K2[(23)MoO2] in [D4]-
methanol including assignment of the signals.
troscopy. K2[(23)MoO2] led to a nicely re-
solved 1H NMR spectrum, which showed
some significant differences compared to the
spectrum of the free ligand 23-H4. In the free
ligand 23-H4, the resonances of the benzylic
and the glycine methylene units appeared as
singlets at d=4.35 and 3.90 ppm (2H each).
It had been expected that the protons of
each of the methylene units would result in a
separated signal because of diastereotopic
behavior. However, due to the high confor-
mational flexibility, the two proton resonan-
ces of each of the methylene units appeared
at the same (™isochronic∫) shift. Upon forma-
tion of the coordination compound
K2[(23)MoO2], the conformational flexibility
was restricted and each proton resulted in a
separate signal, at d=4.79 and 4.34 ppm for
the benzyl group and at d=3.90 and
3.78 ppm for the glycine hydrogen atoms.
Furthermore, the methylene units of the argi-
nine residue appeared as three multiplets in
the free ligand (d=2.01, 1.84, 1.70 ppm; 2H
each), whereas six separated resonances were
observed at d=2.96, 2.88, 1.87, 1.24, 1.15,
and 0.92 ppm in K2[(23)MoO2].
WKY-bridged dicatechol ligand 32-H4 and its
molybdenum(vi)dioxo complex K2[(32)MoO-
(acac)]: The synthesis of the RGD-bridged
Scheme 7. Solid-phase synthesis and complexation of 32-H4 to form K2[(32)MoO2].
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M. Albrecht et al.
FULL PAPER
CD spectra of 23-H4, K2[(23)MoO2], 32-H4, and
K2[(32)MoO2] were taken in methanol. For the free ligands
23-H4 and 32-H4 no significant CD signals can be observed,
a result indicating a nonspecific ™random-coil∫ structure.
The metal complexes K2[(23)MoO2] and K2[(32)MoO2]
show very similar CD spectra with a very strong negative
band at about 230 nm and a strong negative band at 350 nm;
these bands are both due to transitions at the metal complex
moieties. This indicates that both complexes possess dicate-
chol molybdenumdioxo units with the same configuration.
Conclusion
In this paper we have presented the synthesis of three dica-
techol ligands which form metallamacrocycles with molyb-
denum(vi)dioxo moieties. One of the ligands is a simple
model system without any side chains or functionalities,
while the other two contain tripeptide sequences which
were adopted from natural products (RGD sequence of, for
example, echistatin or WKY sequence of urotensin II) with
an interesting biological activity. Our preferred synthetic
entry to prepare such tripeptide-bridged ligands is to use a
polystyrene resin with an Fmoc-hydrazidobenzoate linker,
because the cleavage of the residue from the solid support
proceeds simultaneously with the last coupling step.[24,25]
Multiple deprotection of the ligand precursors can be per-
formed in one step.
Coordination studies with the ligands and dioxomolybde-
num(vi) bis(acetylacetonate) shows that, in all cases, the 19-
membered macrocycle is formed. The functionalized side
chains of the amino acid residues (Tyr, Asp, Arg, Lys, Trp)
are tolerated and do not interfere with metal binding. The
1H NMR spectra of the obtained compounds show nicely re-
solved resonances, a result indicating a high conformational
fixation of the peptide.
Figure 3. 1H NMR spectra for 32-H4 and K2[(32)MoO2] in [D4]-methanol.
The spectrum for the conformationally constrained metallamacrocycle
K2[(32)MoO2] is more resolved than that for the free ligand 32-H4.
Reaction of ligand 32-H4 with [MoO2(acac)2] and potassi-
um carbonate in methanol for six days led to the macrocy-
clic complex K2[(32)MoO2]. Negative ESI-MS showed char-
acteristic signals of the coordination compound at m/z 916
{K[(32)MoO2]}À and 879 {H[(32)MoO2]}À. In addition, a
dominating peak is observed at m/z 1000, which was tenta-
tively assigned as {K(HBr)[(32)MoO2]}À. The characteristic
bands of the MoO2 unit were observed by IR spectroscopy
at n˜ =896 and 865 cmÀ1.[23]
Herein, we have presented a method to use metal coordi-
nation as a tool for the conformational fixation of short
cyclic peptide structures. However, before such derivatives
can be applied to binding studies with biological systems,
more knowledge of their properties and structures has to be
gained and some adjustments of the ligands and/or the
metal ions will surely have to be done.
1H NMR spectroscopy in [D4]-methanol (Figure 3) again
led to more resolved spectra in case of the conformationally
constrained metallamacrocycle K2[(32)MoO2] than for the
free ligand 32-H4. For example, the benzylic protons of the
2,3-dihydroxybenzylamide appeared for 32-H4 as one signal
at d=4.27 ppm. For the complex K2[(32)MoO2] the two dia-
stereotopic protons of this unit were observed as nicely sep-
arated doublets at d=4.81 and 4.42 ppm with a coupling
constant of J=13.8 Hz. Signals at d=5.50 and 1.97 ppm cor-
responded to acetylacetone, which was observed by elemen-
tal analysis as well and may be bound by hydrogen-bonding
interactions. The nicely resolved 1H NMR spectra of
K2[(32)MoO2] indicated that the conformation of the pep-
tide was constrained in the macrocycle and that only a low
degree of flexibility was left.
Experimental Section
NMR spectra were recorded on Bruker DRX 500 or WM 400, Varian
Inova 400, or Unity 500 spectrometers. FT-IR spectra were recorded by
diffuse reflection (KBr) on a Bruker IFSspectrometer. Mass spectra (EI,
70 eV; FAB with 3-nitrobenzoic acid (3-NBA) as the matrix) were taken
on Finnigan MAT 90, 95, or 212 mass spectrometers. UV/Vis spectra
were obtained with a Perkin Elmer Lambda2 spectrometer. Elemental
analyses were obtained with a Heraeus CHN-O-Rapid analyzer. Melting
points were measured on B¸chi B-540 apparatus and are uncorrected.
Compound 3: H¸nig×s base (118 mL, 0.69 mmol) and HBTU (286 mg,
0.75 mmol) were added to a solution of 2,3-dimethoxybenzoic acid (1;
114 mg, 0.63 mmol) in acetonitrile (10 mL). Before addition of 8-amino-
caprylic acid (2; 100 mg, 0.628 mmol), the mixture was stirred for 30 mi-
nutes. After stirring overnight, the solvent was distilled off under vacuum
and the crude product was dissolved in ethyl acetate and washed with
Attempts to crystallize the metallocyclopeptides to obtain
X-ray crystal structure analyses were unfortunately not suc-
cessful.
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Molybdenum Complexation of Tripeptides
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sat. aqueous NH4Cl, water, and brine. Compound 3 was obtained as a
yellow wax (200.5 mg, 0.62 mmol, 99%). 1H NMR (CDCl3, 300 MHz):
d=11.3 (brs, 1H, COOH), 8.04 (brs, 1H, NH), 7.54 (dd, J=7.9, 1.7 Hz,
1H), 7.02 (t, J=7.9 Hz, 1H), 6.94 (dd, J=7.9, 1.7 Hz, 1H), 3.79 (s, 6H,
OMe), 3.35 (q, J=6.9 Hz, 2H), 2.25 (t, J=7.4 Hz, 2H), 1.52 (m, 4H),
1.26 (m, 6H) ppm; 13C NMR (CDCl3, 75.4 MHz): d=178.5 (C), 165.7
(C), 152.6 (C), 147.4 (C), 140.0 (C), 124.5 (CH), 122.6 (CH), 115.5 (CH),
61.3 (CH3), 56.1 (CH3), 39.8 (CH2), 34.1 (CH2), 29.3 (CH2), 29.0 (CH2),
28.9 (CH2), 26.8 (CH2), 24.7 (CH2) ppm; MS(DIP): m/z: 323.09 [M]+,
324.12 [M+H]+; IR (KBr): n˜ =2935, 1721, 1626, 1577, 1542, 1477, 1386,
4.86 mmol) was added and the mixture was stirred overnight. The mix-
ture was diluted with dichloromethane and then washed with sat. aque-
ous NH4Cl solution, sat. aqueous NaHCO3 solution, water, and brine.
The organic layer was dried over MgSO4 and the solvent was removed
under reduced pressure. The product was recrystallized from dichlorome-
thane and hexane. Compound 8 was obtained as a white solid (2.46 g,
4.39 mmol, 90%). M.p. 1258C (decomp); 1H NMR (CDCl3, 500 MHz):
d=7.75 (m, 2H, Fmoc), 7.67 7.54 (m, 2H, Fmoc), 7.38 (m, 2H, Fmoc),
7.31 7.26 (m, 2H, Fmoc), 6.96 (t, J=7.9 Hz, 1H, aryl), 6.88 (brs, 1H,
NH), 6.83 (m, 2H, aryl), 5.98 (d, J=8.1 Hz, 1H, NH), 4.53 (brs, 1H, a-
H), 4.46 (m, 2H, CH2 Fmoc), 4.44 4.35 (m, 2H, CH2 benzyl), 4.19 (t, J=
7.0 Hz, 1H, CH Fmoc), 3.83 (s, 3H, OMe), 3.82 (s, 3H, OMe), 2.69 (dd,
J=16.9, 6.4 Hz, 1H, CH2 Asp), 2.91 2.81 (m, 1H, CH2 Asp), 1.41 (s, 9H,
tBu) ppm; 13C NMR (CDCl3, 125.7 MHz): d=171.1 (C), 170.1 (C), 152.6
(C), 147.1 (C), 143.8 (2 C), 143.7 (C), 141.3 (2 C), 131.4 (C), 127.7 (2
CH), 127.1 (2 CH), 125.0 (2 CH), 124.1 (CH), 121.0 (CH), 120.0 (2 CH),
111.9 (CH), 81.8 (C), 67.2 (CH2), 60.7 (CH3), 55.7 (CH3), 51.2 (CH), 47.1
(CH), 38.9 (CH2), 37.5 (CH2), 28.0 (3CH3) ppm; FAB-MS(3-NBA/
DMSO): m/z: 561.3 [M+H]+, 583.3 [M+Na]+; elemental analysis calcd
(%) for C32H36N2O7¥H2O: C 66.42, H 6.62, N 4.84; found: C 66.53, H
6.49, N 4.77; IR (KBr, drift): n˜ =3295, 2977, 2936, 1728, 1482, 1274,
1155 cmÀ1; UV (CHCl3): lmax =227, 267, 289, 301 nm.
1311, 1266, 1234, 1086, 997, 845, 753 cmÀ1
.
Protected ligand 5: Derivative (1.02 g, 3.14 mmol), H¸nig×s base
3
(590 mL, 3.45 mmol) and HBTU (1.43 g, 3.77 mmol) were dissolved in
acetonitrile (70 mL) and stirred for 30 min. Then, 2,3-dimethoxybenzyl
amine (4; 465 mL, 3.14 mmol) was added to the activated acid and the re-
action mixture was stirred overnight. The solvent was removed under
vacuum and the residue was dissolved in ethyl acetate. The organic layer
was washed with sat. aqueous NH4Cl, sat. aqueous NaHCO3, water, and
brine. The by-products were removed by silica gel chromatography with
CH2Cl2 as the eluent and the product was obtained by eluting with
CH2Cl2/methanol 4:1. Compound 5 was obtained as a yellow wax (1.43 g,
3.01 mmol, 96%). 1H NMR (CDCl3, 400 MHz): d=7.91 (t, J=5.4 Hz,
1H, NH), 7.50 (dd, J=7.9, 1.7 Hz, 1H), 6.99 (t, J=8.2 Hz, 1H), 6.91 (dd,
J=8.2, 1.6 Hz, 1H), 6.86 (t, J=7.9 Hz, 1H), 6.75 (m, 2H), 6.70 (dd, J=
8.2, 1.6 Hz, 1H), 4.33 (d, J=5.8 Hz, 2H), 3.77 (s, 3H, OMe), 3.76 (s, 3H,
OMe), 3.72 (s, 3H, OMe), 3.71 (s, 3H, OMe), 3.30 (m, 2H), 2.09 (t, J=
7.56 Hz, 2H), 1.50 (m, 4H), 1.22 (m, 6H) ppm; 13C NMR (CDCl3,
100.6 MHz): d=173.0 (C), 165.0 (C), 164.8 (C), 152.5 (C), 152.4 (C),
147.2 (C), 132.2 (C), 26.8 (C), 124.2 (CH), 124.0 (CH), 122.4 (CH), 121.0
(CH), 115.3 (CH), 11.9 (CH), 61.2 (CH3), 60.6 (CH3), 56.0 (CH3), 55.8
(CH3), 39.6 (CH2), 39.1 (CH2), 36.5 (CH2), 29.5 (CH2), 29.2 (CH2), 29.0
Amine 9: Piperidine (0.36 mL, 3.67 mmol) was added to a solution of 8
(1.71 g, 3.06 mmol) in dichloromethane (45 mL) and the mixture was stir-
red overnight. The solvents were distilled off and the residue was washed
with hexane overnight. Compound 9 was obtained as a bright yellow
solid (237 mg, 0.67 mmol, quantitative). 1H NMR (CDCl3, 500 MHz): d=
7.77 (s, 1H, NH), 6.99 (t, J=7.9 Hz, 1H, aryl), 6.88 6.83 (m, 2H, aryl),
4.46 (m, 2H, CH2 benzyl), 3.86 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.14
(m, 1H, a-H), 2.89 (dd, J=16.8, 4.4 Hz, 1H, CH2 Asp), 2.61 (dd, J=16.8,
7.8 Hz, 1H, CH2 Asp), 1.42 (s, 9H, tBu) ppm; 13C NMR (CDCl3,
125.7 MHz): d=172.1 (C), 171.1 (C), 152.6 (C), 147.2 (C), 131.8 (C),
124.1 (CH), 121.2 (CH), 111.9 (CH), 81.4 (C), 60.7 (CH3), 55.7 (CH3),
51.8 (CH), 39.8 (CH2), 38.6 (CH2), 28.0 (3CH3); FAB-MS(DMSO/3-
NBA): m/z: 339.3 [M+H]+, 361.3 [M+Na]+; IR (KBr, drift): n˜ =2948,
1725, 1668, 1482, 1274, 1154 cmÀ1; UV (CHCl3): lmax =228, 249, 258,
247 nm.
(CH2), 26.9 (CH2), 25.7 (CH2) ppm; LC-MS(EIS):
m/z: 472.3 [M]+,
495.5 [M+Na]+; IR (KBr, drift): n˜ =3000, 2934, 1647, 1578, 1537, 1477,
1431, 1308, 1269, 1223, 1083, 1002, 846, 754 cmÀ1; elemental analysis
calcd (%) for C26H36N2O6¥1.5H2O: C 62.51, H 7.87, N 5.61; found: C
62.58, H 7.65, N 5.64.
Ligand 6-H4: BBr3 (0.65 mL, 6.81 mmol) was added to a solution of 5
(128.7 mg, 0.27 mmol) in dichloromethane (17 mL). The mixture was stir-
red for two days at room temperature and then was hydrolyzed with
methanol (10 mL). If the ether cleavage reaction was not complete, the
residue was once again suspended in CH2Cl2, mixed with BBr3 (0.65 mL,
6.81 mmol), and stirred overnight. After quenching with methanol, the
solvents were removed under vacuum. The residue was dissolved in
methanol and then the solvent was distilled off to remove all BBr3.
Compound 11: Fmoc-Gly-OH (10; 295 mg, 0.99 mmol) was dissolved in a
mixture of CH2Cl2 (10 mL) and DMF (2.4 mL). Then, H¸nig×s base
(0.19 mL, 1.09 mmol) und a solution of HBTU (452 mg, 1.19 mmol) in
DMF (2.6 mL) were added and the mixture was stirred together for two
hours. Meanwhile 9 (352 mg, 1.04 mmol) was suspended in DMF (7 mL)
and CH2Cl2 (2 mL) was added, followed by the activated amino acid mix-
ture. The reaction mixture was stirred for three days, filtered, and then
diluted with CH2Cl2. The organic layer was washed with sat. aqueous
NH4Cl solution, sat. aqueous NaHCO3 solution, water, and brine. It was
dried over MgSO4 and the solvent was distilled off under vacuum. The
crude product was purified by chromatography over silica gel with ethyl
acetate/hexane 2:1. Compound 11 was obtained as a white solid (481 mg,
0.78 mmol, 79%). Rf =0.29 (ethyl acetate/hexane 2:1); m.p. 1158C
(decomp); 1H NMR (CDCl3, 500 MHz): d=7.76 (d, J=7.5 Hz, 2H,
Fmoc), 7.56 (d, J=7.3 Hz, 2H, Fmoc), 7.40 (t, J=7.4 Hz, 2H, Fmoc),
7.35 7.30 (brs, 1H, NH), 7.30 (m, 2H, Fmoc), 7.08 (brs, 1H, NH), 6.90
(t, J=8.0 Hz, 1H, aryl), 6.80 (m, 1H, aryl), 6.75 (d, J=8.0 Hz, 1H, aryl),
5.50 (brs, 1H, NH), 4.79 (d, J=2.7 Hz, 1H, a-H), 4.44 (t, J=6.1 Hz, 2H,
CH2 benzyl), 4.33 (d, J=7.1 Hz, 2H, CH2 Fmoc), 4.18 (t, J=6.9 Hz, 1H,
CH Fmoc), 3.87 (d, J=5.0 Hz, 2H, CH2 Gly), 3.82 (s, 6H, OMe), 2.95
(dd, J=16.9, 3.4 Hz, 1H, CH2 Asp), 2.56 (dd, J=16.9, 6.5 Hz, 1H, CH2
Asp), 1.39 (s, 9H, tBu) ppm; 13C NMR (CDCl3, 125.7 MHz): d=171.3
(C), 169.8 (C), 168.7 (C), 156.8 (C), 152.5 (C), 147.0 (C), 143.7 (C), 141.3
(C), 131.5 (C), 127.7 (2CH), 127.1 (2CH), 125.1 (2CH), 124.1 (CH),
120.8 (CH), 120.0 (2CH), 111.8 (CH), 81.9 (C), 67.4 (CH2), 60.6 (CH3),
55.7 (CH3), 49.3 (CH), 47,0 (CH), 44.7 (CH2), 38.8 (CH2), 36.8 (CH2),
Ligand 6-H4 was obtained as
a yellow wax (113 mg, quantitative);
1H NMR (CD3OD, 400 MHz): d=7.24 (dd, J=8.0, 1.4 Hz, 1H), 7.00 (dd,
J=8.0, 1.4 Hz, 1H), 6.81 (dd, J=7.7, 1.7 Hz, 1H), 6.75 (t, J=8.0 Hz,
1H), 6.72 (dd, J=7.7, 1.7 Hz, 1H), 6.65 (t, J=7.8 Hz, 1H), 4.52 (s, 2H),
3.42 (t, J=7.1 Hz, 2H), 2.57 (m, 2H), 1.64 (m, 3H), 1.36 (m, 7H) ppm;
MS(DIP): m/z: 416.4 [M]+, 417.5 [M+H]+; IR (KBr): n˜ =3461, 2934,
2859, 1636, 1593, 1547, 1331, 1259, 742 cmÀ1
.
Complex K2[(6)MoO2]: Ligand 6-H4 (56.6 mg, 0.14 mmol) was dissolved
in methanol (10 mL) and K2CO3 (75 mg, 0.54 mmol) and [MoO2(acac)2]
(53 mg, 0.163 mmol) were added. The reaction mixture was stirred over-
night, then the solvent was distilled off and the residue was filtrated over
Sephadex LH20 with methanol. The coordination compound
K2[(6)MoO2] was obtained as a yellow wax (63 mg, 0.12 mmol, 86%).
1H NMR (CD3OD, 400 MHz): d=7.20 (brs, 1H), 6.70 (m, 1H), 6.46 (m,
4H), 4.44 (m, 1H), 4.31 (m, 1H), 3.15 (m, 2H), 2.20 (m, 1H), 1.95 (m,
1H), 1.63 (m, 1H), 1.50 (m, 1H), 1.20 (m, 8H) ppm; negative ESI-MS:
m/z: 543 {H[(6)MoO2]}À, 581 {K[(6)MoO2]}À; IR (KBr): n˜ =3461, 2934,
2859, 1636, 1593, 1547, 1331, 1259, 896, 863, 742 cmÀ1; elemental analysis
calcd (%) for C22H24N2O8MoK¥4H2O: C 38.26, H 4.67, N 4.06; found: C
38.50, H 4.44, N 3.60.
27.9 (3CH3) ppm, two
C signals are not visible; FAB-MS(3-NBA/
DMSO): m/z: 617.1 [M]+, 618.1 [M+H]+, 640.2 [M+Na]+; elemental
analysis calcd (%) for C34H39N3O8: C 66.11, H 6.36, N 6.80; found: C
65.78, H 6.39, N 6.69; IR (KBr): n˜ =3304, 2978, 2937, 1727, 1660, 1533,
1273 cmÀ1; UV (CHCl3): lmax =227, 267, 289, 301 nm.
Preparation of RGD-bridged compounds
Solution-phase synthesis
Compound 8: H¸nig×s base (0.92 mL, 5.35 mmol) and a solution of
HBTU (2.21 g, 5.83 mmol) in DMF (20 mL) was added to a solution of
Fmoc-Asp(OtBu)-OH (7; 2.00 g, 4.86 mmol) in dichloromethane
(100 mL). After two hours 2,3-dimethoxybenzylamine (4; 0.72 mL,
Amine 12: Piperidine (0.37 mL, 3.76 mmol) was added to a solution of 11
(1.94 g, 3.14 mmol) in CH2Cl2 (30 mL). The mixture was stirred over-
night, then the solvent was removed under reduced pressure and the resi-
Chem. Eur. J. 2004, 10, 3657 3666
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3663

M. Albrecht et al.
FULL PAPER
due was dried under vacuum. The obtained solid was washed overnight
with hexane and then dried under vacuum. Compound 12 was obtained
as a bright yellow solid (1.09 g, 2.76 mmol, 88%). 1H NMR (DMSO,
500 MHz): d=8.26 (brs, 1H, NH), 8.00 (brs, 1H, NH), 6.89 (t, J=7.9 Hz,
1H, aryl), 6.77 6.73 (m, 2H, aryl), 4.67 (d, J=4.4 Hz, 1H, a-H), 4.22 (d,
J=5.9 Hz, 2H, CH2 benzyl), 3.75 (s, 3H, OMe), 3.71 (s, 3H, OMe), 2.61
(d, J=6.2 Hz, 2H, CH2 Asp), 1.33 (s, 9H, tBu) ppm, the CH2 Gly signal
is covered by the H2O signal at 3.4 ppm; 13C NMR (CDCl3, 125.7 MHz):
d=171.8 (C), 170.6 (C), 169.9 (C), 152.4 (C), 146.5 (C), 132.4 (C), 123.9
(CH), 120.3 (CH), 111.5 (CH), 80.8 (C), 60.4 (CH3), 55.8 (CH3), 49.4
(CH), 44.4 (CH2), 37.7 (CH2), 28.04 (3 CH3), 22.6 (CH2) ppm; FAB-MS
(3-NBA/DMSO): m/z: 396.3 [M+H]+, 418.3 [M+Na]+, 396.3
[MÀtBu+2H]+; IR(KBr): n˜ =3305, 2940, 2937, 1727, 1660, 1481, 1273,
1156, 727 cmÀ1; UV (CH2Cl2): lmax =217, 272 nm.
(7.9 mL). After two hours a suspension of 15 (1.78 g, 1.97 mmol) in
CH2Cl2 (12 mL) and DMF (2.5 mL) was added. The reaction mixture was
stirred overnight and then washed with sat. aqueous NH4Cl, sat. aqueous
NaHCO3, water, and brine. After drying over MgSO4, the solvent was
distilled off. The by-products were removed by chromatography over
silica gel with ethyl acetate/hexane (2:1 v/v) and the pure product was
obtained by eluting with ethyl acetate/methanol (2:1 v/v). Compound 16
was obtained as a white solid (1.56 g, 1.68 mmol, 86%). Rf =0.72 (ethyl
acetate/methanol 2:1). Analytical data: see below.
Solid-phase syntheses
General synthesis of protected tripeptide-bridged ligand precursors on
the solid support: The synthesis of protected tripeptide-bridged ligand
precursors was performed on the 4-Fmoc-hydrazinobenzoyl resin 17 (4-
Fmoc-hydrazinobenzoyl AM resin, Novabiochem) by using Fmoc-pro-
tected amino acids. Initially the resin was swollen in dichloromethane.
After washing with DMF, the Fmoc group was cleaved with a 20% solu-
tion of piperidine in DMF to yield 18.[24,25]
Compound 14: Fmoc-Arg(Mtr)-OH (13; 1.68 g, 2.76 mmol) was dissolved
in CH2Cl2 (30 mL) and then H¸nig×s base (0.52 mL, 3.04 mmol) and a
solution of HBTU (1.26 g, 3.31 mmol) in DMF (8.5 mL) were added.
After 8 min, a suspension of 12 (1.09 g, 2.76 mmol) in a mixture of DMF
(4 mL) and CH2Cl2 (16 mL) was added. The mixture was stirred over-
night and then washed with sat. aqueous NH4Cl, sat. aqueous NaHCO3,
water, and brine. After drying over MgSO4, the solvent was distilled off
and the residue was purified by chromatography over silica gel with ethyl
acetate/hexane 2:1. Product 14 was obtained as a white solid (2.16 g,
2.19 mmol, 79%) by eluting with ethyl acetate/methanol 2:1. Rf =0.65
(ethyl acetate/methanol 2:1); m.p. 1208C (decomp); 1H NMR (CDCl3,
500 MHz): d=7.93 (brs, 1H, NH), 7.72 (d, J=7.5 Hz, 2H, Fmoc), 7.56
(m, 2H, Fmoc), 7.42 (brs, 1H, NH), 7.35 (t, J=7.3 Hz, 2H, Fmoc), 7.23
(m, 2H, Fmoc), 6.84 (t, J=8.0 Hz, 1H, aryl), 6.73 (m, 1H aryl), 6.69 (d,
J=8.0 Hz, 1H, aryl), 6.45 (s, 1H, aryl Mtr), 6.37 (brs, 1H, NH), 6.05
(brs, 1H, NH), 4.77 (m, 1H, a-H Asp), 4.40 4.24 (m, 5H, CH2 benzyl,
CH2 Fmoc, a-H Arg), 4.13 (t, J=6.7 Hz, 1H, CH Fmoc), 3.88 (m, 2H,
CH2 Gly), 3.75 (s, 3H, OMe), 3.75 (s, 3H, OMe), 3.72 (s, 3H, OMe Mtr),
3.32 3.24 (m, 1H, CH2 Asp), 3.14 3.04 (m, 1H, CH2 Asp), 2.63 (s, 3H,
CH3 Mtr), 2.58 (s, 3H, CH3 Mtr), 2.06 (s, 3H, CH3 Mtr), 1.84 1.75 (m,
1H, CH2 Arg), 1.62 1.44 (m, 5H, CH2 Arg), 1.32 (s, 9H, tBu) ppm;
13C NMR (CDCl3, 125.7 MHz): d=173.5 (C), 170.8 (C), 170.5 (C), 169.6
(C), 156.6 (C), 152.4 (C), 146.6 (C), 143.8 (C), 143.7 (C), 141.2 (2C),
131.6 (C), 127.7 (2CH), 127.1 (2CH), 125.1 (2CH), 124.0 (CH), 120.6
(CH), 120.0 (2CH), 111.8 (CH), 111.6 (CH), 81.7 (C), 67.0 (CH2), 60.5
(CH3), 55.6 (CH3), 55.4 (CH3), 54.1 (CH), 49.7 (CH), 47.1 (CH), 43.3
(CH2), 38.4 (CH2), 37.0 (CH2), 29.5 (CH2), 27.9 (3CH3), 24.2 (CH3), 18.4
(CH3), 11.9 (CH3) ppm, the missing C and CH2 signals could not be ob-
For the preparation of the peptide sequences the following protocol was
used: The C-terminal-unprotected N-Fmoc amino acid (2 equiv) was acti-
vated with H¸nig×s base (4 equiv) and HBTU (2 equiv) in DMF. After
ten minutes, this solution was added to the N-terminal-unprotected resin
and the mixture was shaken for one hour. Before attaching the next
amino acid, the Fmoc group had to be cleaved by treating the resin with
a 20% solution of piperidine in DMF for 15 min.[20] In a final step, 2,3-di-
methoxybenzoic acid (1) was attached and the resin was washed with
DMF, CH2Cl2, and methanol. After drying under vacuum the peptide
was cleaved from the resin with simultaneous formation of the amide
with 2,3-dimethoxybenzyl amine (4). This was achieved by treatment
with copper acetate (1 equiv) in DMF and bubbling air through the mix-
ture for 4 h in the presence of 4.[24] The resin is filtered off and washed
with dichloromethane, then the combined organic layers are washed with
aqueous 1m KHSO4, water, and brine. After drying with NaSO4, the or-
ganic solvents are distilled off under vacuum.
Compound 16: This was prepared following the general method from
resin 17 (305 mg, 0.30 mmol), HBTU (228 mg, 0.60 mmol), H¸nig×s base
(205 mL, 1.20 mmol), Fmoc-Asp(OtBu)-OH (7; 247 mg, 0.60 mmol),
Fmoc-Gly-OH (10; 187 mg, 0.60 mmol), Fmoc-Arg(Mtr)-OH (13;
365 mg, 0.60 mmol), 2,3-dimethoxybenzoic acid (1; 109 mg, 0.60 mmol),
and 2,3-dimethoxybenzylamine (4; 1.00 mL, 6.76 mmol). The by-products
were removed by chromatography over silica gel with ethyl acetate and
the pure product was obtained by eluting with ethyl acetate/methanol
4:1. Compound 16 was obtained as a white solid (179 mg, 0.19 mmol,
64%). M.p. 1258C (decomp); 1H NMR (CDCl3, 500 MHz): d=8.70 (brs,
1H, NH), 8.09 (brs, 1H, NH), 7.67 (brs, 1H, NH), 7.55 (d, J=7.5 Hz,
1H, aryl), 7.47 (brs, 1H, NH), 7.09 (t, J=8.0 Hz, 1H, aryl), 7.03 (m, 1H,
aryl), 6.40 (brs, 1H, NH), 6.87 (t, J=8.0 Hz, 1H, aryl), 6.75 7.71 (m, 2H,
aryl), 6.47 (s, 1H, Mtr), 4.82 4.72 (m, 2H, a-H Asp, a-H Arg), 4.35 (m,
2H, CH2 benzyl), 3.97 3.91 (m, 2H, CH2 Gly), 3.89 (s, 3H, OMe), 3.87
(s, 3H, OMe), 3.78 (6H, OMe), 3.73 (s, 3H, OMe), 3.38 (m, 1H, CH2
Asp), 3.15 (m, 1H, CH2 Asp), 2.63 (s, 3H, CH3 Mtr), 2.58 (s, 3H, CH3
Mtr), 2.06 (s, 3H, CH3 Mtr), 1.74 (m, 2H, CH2 Arg), 1.58 (m, 4H, CH2
Arg), 1.31 (s, 9H, tBu) ppm; 13C NMR (CDCl3, 125.7 MHz): d=172.9
(C), 170.8 (C), 170.7 (C), 169.7 (C), 165.6 (C), 162.6 (C), 152.7 (C), 152.4
(C), 148.0 (C), 146.6 (C), 131.6 (C), 124.3 (CH), 124.0 (CH), 122.4 (CH),
120.3 (CH), 115.9 (CH), 111.8 (CH), 81.5 (C), 61.5 (CH3), 60.4 (CH3),
56.1 (CH3), 55.7 (CH3), 55.4 (CH3), 53.4 (CH2), 49.8 (CH), 43.2 (CH2),
38.4 (CH2), 36.9 (CH2), 29.7 (CH2), 27.9 (3 CH3), 24.2 (CH3), 18.4 (CH3),
11.9 (CH3) ppm, the missing signals could not be observed; FAB-MS
(DMSO/3-NBA): m/z: 872.4 [MÀtBu+H]+, 928.5 [M+H]+, 951.4
[M+Na]+; elemental analysis calcd (%) for C44H61N7O13S¥4H2O: C 52.84,
H 6.95, N 9.80; found: C 52.98, H 6.56, N 9.87; IR (KBr): n˜ =3342, 2936,
1657, 1650, 1548, 1265, 1121 cmÀ1; UV (CHCl3): lmax =227, 245 nm.
served; FAB-MS(3-NBA/DMOS):
m/z: 930.5 [MÀtBu+H]+, 952.4
[MÀtBu+H+Na]+, 985.5 [M]+, 986.5 [M+H]+, 1008.5 [M+Na]+; ele-
mental analysis calcd (%) for C50H63N7O12S¥3H2O: C 57.73, H 6.69, N
9.43; found: C 57.88, H 6.31, N 9.54; IR (KBr): n˜ =3338, 2939, 1666,
1548, 1260, 1121 cmÀ1; UV (CHCl3): lmax =227, 255, 289, 301 nm.
Amine 15: Compound 14 (1.90 g, 1.97 mmol) was dissolved in CH2Cl2
(20 mL) and piperidine (0.23 mL, 2.36 mmol) was added. The reaction
mixture was stirred overnight. The solvent was removed under reduced
pressure and the residue was dried under vacuum. The solid was washed
with hexane overnight and dried once again under vacuum. Compound
15 was obtained as a bright yellow solid (1.78 g, 2.33 mmol, quantitative).
1H NMR (CDCl3, 500 MHz): d=8.28 (brs, 1H, NH), 7.54 (brs, 1H, NH),
6.91 (t, J=7.6 Hz, 1H, aryl), 6.76 (brs, 2H, aryl), 6.66 (brs, 1H, NH),
6.48 (brs, 1H, Mtr), 4.76 (brs, 1H, a-H), 4.36 (brs, 2H, CH2 benzyl), 3.96
(brs, 2H, CH2 Gly), 3.79 (s, 6H, OMe), 3.79 (s, 3H, OMe), 3.46 (brs,
1H, CH2 Asp), 3.15 (brs, 1H, CH2 Asp), 2.63 (s, 3H, CH3 Mtr), 2.57 (s,
3H, CH3 Mtr), 2.08 (s, 3H, CH3 Mtr), 1.59 1.40 (m, 6H, CH2 Arg), 1.36
(s, 9H, tBu) ppm, the second a-H cannot be assigned; 13C NMR (CDCl3,
125.7 MHz): d=176.4 (C), 171.0 (C), 170.5 (C), 169.7 (C), 158.4 (C),
156.7 (C), 152.4 (C), 146.6 (C), 138.4 (C), 136.4 (C), 133.4 (C), 131.7 (C),
128.7 (CH), 124.8 (C), 124.1 (CH), 120.5 (CH), 111.7 (CH), 81.7 (C), 60.6
(CH3), 55.7 (CH3), 55.4 (CH3), 54.1 (CH), 49.6 (CH), 44.7 (CH2), 43.3
(CH2), 38.4 (CH2), 36.9 (CH2), 27.9 (3CH3), 24.2 (CH3), 22.6 (CH2), 22.4
Preparation of the RGD-bridged ligand 23-H4: AlCl3 (65 mg, 0.49 mmol)
was dissolved in ethanethiol (0.5 mL) under ice cooling. Then, a solution
of 16 (15 mg, 0.02 mmol) in CH2Cl2 (1 mL) was added to the AlCl3 solu-
tion.[27] The mixture was allowed to warm to room temperature, stirred
overnight, and then poured into cold water (7 mL). The solution was
acidified with 1m aqueous HCl. The water layer was separated and was
distilled to dryness. The crude product was purified by HPLC (Nucleosil,
250î20 mm, 100C18, 7 mm, 5 mLminÀ1) with acetonitrile (containing
(CH2), 18.4 (CH3), 11.9 (CH3) ppm; FAB-MS(3-NBA/DMOS ):
m/z:
764.3 [M+H]+, 786.3 [M+Na]+; IR (KBr): n˜ =3324, 2938, 1669, 1551,
1270, 1112 cmÀ1; UV (CH2Cl2): lmax =219, 247, 256, 305 nm.
Protected ligand 16: 2,3-Dimethoxybenzoic acid (1; 358 mg, 1.97 mmol)
was dissolved in CH2Cl2 (30 mL) and mixed with H¸nig×s base (0.37 mL,
2.16 mmol) and
a
solution of HBTU (895 mg, 2.36 mmol) in DMF
3664
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Molybdenum Complexation of Tripeptides
3657 3666
0.1% trifluoroacetic acid (TFA)) and water (containing 0.1% TFA).
Compound 23-H4 was obtained as a white solid (4.4 mg, 45%). 1H NMR
(CD3OD, 400 MHz): d=7.35 (dd, J=7.9, 1.6 Hz, 1H, aryl), 6.95 (dd, J=
7.9, 1.6 Hz, 1H, aryl), 6.75 (t, J=7.9 Hz, 1H, aryl), 6.69 (dd, J=7.6,
2.0 Hz, 1H, aryl), 6.66 (dd, J=7.6, 2.0 Hz, 1H, aryl), 6.61 (t, J=7.6 Hz,
1H, aryl), 4.80 (dd, J=7.6, 5.6 Hz, 1H, a-H), 4.60 (m, 1H, a-H), 4.35 (s,
2H, CH2 benzyl), 3.90 (s, 2H, CH2 Gly), 3.21 (m, 2H, CH2 Arg), 2.88
(dd, J=16.3, 5.6 Hz, 1H, CH2 Asp), 2.77 (dd, J=16.3, 7.6 Hz, 1H, CH2
Asp), 2.01 (m, 1H, CH2 Arg), 1.84 (m, 1H, CH2 Arg), 1.70 (m, 2H, CH2
Arg) ppm; 13C NMR (CD3OD, 125 MHz): d=173.7 (C), 171.9 (C), 171.5
(C), 170.6 (C), 169.8 (C), 157.6 (C), 148.2 (C), 146.2 (C), 143.5 (C), 125.1
(C), 118.9 (CH), 116.4 (C), 114.5 (CH), 53.5 (CH), 50.3 (CH), 42.6
(CH2), 41.0 (CH2), 39.1 (CH2), 35.9 (CH2), 29.0 (CH2), 25.2 (CH2) ppm;
1.5 Hz, 1H, aryl), 7.19 (s, 1H, indole H), 7.09 (m, 1H), 6.99 (m, 1H),
6.94 (dd, J=7.8, 1.5 Hz, 1H, aryl), 6.89 (m, 2H, aryl), 6.72 (m, 2H, aryl),
6.59 (m, 3H, aryl), 6.54 (d, J=14.6 Hz, 1H, aryl), 4.82 (t, J=6.5 Hz, 1H),
4.50 (t, J=7.6 Hz, 1H, a-H), 4.27 (m, 3H, a-H Lys, CH2 benzyl), 3.31 (m,
2H, CH2), 2.99 (m, 1H, CH2), 2.76 (m, 3H, CH2, e-CH2 Lys), 1.58 (m,
1H, b-CH2 Lys), 1.49 (m, 3H, b- and g-CH2 Lys), 1.13 (m, 2H, d-CH2
Lys) ppm; elemental analysis calcd (%) for C40H44N6O9¥2H2O¥2HBr: C
49.60, H 5.41, N 8.68; found: C 49.91, H 5.24, N 8.28; positive FAB-MS
(3-NBA/DMSO): m/z: 754.4 [M+H]+; negative FAB-MS(3-NBA/
DMSO): m/z: 752.3 [MÀH]À; LC-MS(ESI): m/z: 753.19 [M]+; IR (KBr):
n˜ =3364, 2943, 1641, 1534, 1252, 746 cmÀ1
.
Formation of K2[(32)MoO2]: Ligand 32-H4 (22 mg, 0.03 mmol), K2CO3
(16 mg, 0.12 mmol), and [MoO2(acac)2] (9.6 mg, 0.03 mmol) were dis-
solved in methanol (11 mL). The mixture was stirred for six days. The
solvent was distilled off under vacuum and the crude product was filtered
over Sephadex LH20 with methanol. Coordination complex
K2[(32)MoO2] was obtained as a red solid (15 mg, 0.02 mmol, 67%).
1H NMR (CD3OD, 400 MHz): d=7.72 (d, J=7.4 Hz, 1H, aryl), 7.33 (d,
J=7.4 Hz, 1H, aryl), 7.12 (m, 3H, aryl), 6.92 (s, 1H, indole), 6.70 (dd,
J=7.6, 1.6 Hz, 3H, aryl), 6.59 (m, 4H, aryl), 6.42 (m, 2H, aryl), 5.16 (t,
J=6.7 Hz, 1H, a-H), 4.81 (d, J=13.8 Hz, 1H, CH2), 4.51 (dd, J=11.4,
3.7 Hz, 1H, a-H), 4.42 (d, J=13.8 Hz, 1H, CH2), 3.89 (t, J=6.3 Hz, 1H,
a-H), 3.21 (m, 1H), 3.08 (m, 2H), 2.73 (dd, J=15.1, 6.8 Hz, 1H, CH2),
2.48 (m, 1H, CH2), 1.51 (m, 1H, CH2), 1.32 (m, 4H, CH2), 0.71 (m,
2H) ppm and signals of CH3COCH2COCH3 at d=5.50 and 1.97 ppm; el-
emental analysis calcd (%) for C40H40K2N6O9MoO2¥(C5H8O2)¥5H2O: C
47.20, H 5.11, N 7.34; found: C 47.12, H 5.06, N 7.23; ESI-MS: m/z: 916
{K[(32)MoO2]}À, 879 {H[(32)MoO2]}À, 1000 {K(HBr)[(32)MoO2]}À; IR
high-resolution FAB-MS(3-NBA/DMOS): calcd for
C
26H34N7O10
[M+H]+: m/z: 604.2392; found: 604.2367.
Formation of K2[(23)MoO2]: Ligand 23-H4 (13.9 mg, 0.02 mmol) was dis-
solved in methanol (8 mL) and mixed with K2CO3 (12.4 mg, 0.09 mmol)
and MoO2(acac)2 (8.3 mg, 0.03 mmol). This solution was stirred for five
days. The solvent was distilled off under vacuum and the crude product
was filtered over Sephadex LH20 with methanol. K2[(23)MoO2] was ob-
1
tained as a red solid (14 mg, 0.02 mmol, quantitative). H NMR (CD3OD,
400 MHz): d=7.21 (dd, J=8.2, 1.5 Hz, 1H, benzoic acid), 6.72 (dd, J=
7.3, 1.5 Hz, 1H, benzoic acid), 6.46 (m, 1H, benzoic acid), 6.39 (m, 2H,
benzyl amine), 6.30 (m, 1H, benzyl amine), 4.90 (covered a-H Arg), 4.79
(d, J=14.0 Hz, 1H, CH2 benzyl), 4.61 (m, 1H, a-H Asp), 4.34 (d, J=
14.0 Hz, 1H, CH2 benzyl), 3.90 (d, J=17.0 Hz, 1H, CH2 Gly), 3.78 (d, J=
17.0 Hz, 1H, CH2 Gly), 3.18 (dd, J=16.7, 2.4 Hz, 1H, CH2 Asp), 2.96 (m,
1H, d-CH2 Arg), 2.88 (m, 1H, d-CH2 Arg), 2.48 (dd, J=16.7, 5.2 Hz,
CH2 Asp), 1.87 (m, 1H, b-CH2 Arg), 1.24 (m, 1H, b-CH2 Arg), 1.15 (m,
1H, g-CH2 Arg), 0.92 (m, 1H, g-CH2 Arg) ppm; ESI-MS: m/z: 807
[{K2[(23)MoO2]}ÀH]À, 769 {K[(23)MoO2]}À, 730 {H[(23)MoO2]}À, 383.5
(KBr): n˜ =3386, 2929, 1633, 1541, 1449, 1234, 896, 865, 746 cmÀ1
.
[(23)MoO2]2À
.
Preparation of WKY-bridged compounds: The same solid-phase protocol
as was described for the preparation of 16 was used.
Acknowledgement
Ligand precursor 31: This was prepared following the general method
from resin 17 (536 mg, 0.53 mmol), HBTU (398 mg, 1.12 mmol), H¸nig×s
base (360 mL, 2.24 mmol), Fmoc-Tyr(tBu)-OH (24; 487 mg, 1.12 mmol),
Fmoc-Lys(Boc)-OH (26; 497 mg, 1.12 mmol), Fmoc-Trp(Boc)-OH (28;
558 mg, 1.12 mmol), 2,3-dimethoxybenzoic acid (1; 193 mg, 1.12 mmol),
and 2,3-dimethoxybenzylamine (4; 50 mL, 5.30 mmol). The by-products
were removed by chromatography over silica gel with ethyl acetate/
hexane 2:1 and the pure product 31 was obtained as a white solid by elut-
ing with ethyl acetate (379.1 mg, 0.36 mmol, 67%). M.p. 1308C
We thank Prof. Dr. M. Kappes and the Nanotechnology Institute, For-
schungszentrum Karlsruhe, for facilitating the ESI-MS measurements,
Dr. O. Spie˚ for his orientating investigations to build up the RGD-
bridged ligand, and Prof. G. Raabe for the CD measurements.
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1
(decomp); H NMR (CDCl3, 400 MHz): d=8.62 (d, J=5.2 Hz, 1H, NH),
8.09 (d, J=8.6 Hz, 1H, NH), 7.60 (dd, J=7.8, 1.8 Hz, 1H, aryl), 7.54 (m,
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7.07 (dd, J=8.2, 1.6 Hz, 1H, aryl), 7.04 7.00 (m, 3H, aryl), 6.98 (m, 1H,
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6.74 (d, J=8.52 Hz, 2H, aryl), 6.51 (brs, 1H, NH), 4.86 (brs, 1H, NH
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3H, OMe), 3.59 (s, 3H, OMe), 3.37 (dd, J=14.1, 5.4 Hz, 1H, CH2), 3.26
(pseudo-t, J=6.3, 4.4 Hz, 2H, CH2), 2.92 (dd, J=13.9, 9.9 Hz, 1H, CH2),
2.81 (brs, 2H, e-CH2 Lys), 1.67 (s, 9H, tBu), 1.41 (s, 11H, tBu, g-CH2
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1265, 1164 cmÀ1
.
WKY-bridged ligand 32-H4: At À188C, BBr3 (0.23 mL, 2.35 mmol) was
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7.60 (d, J=8.0 Hz, 1H, aryl), 7.34 (d, J=8.0 Hz, 1H), 7.26 (dd, J=8.1,
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Received: February 12, 2004
Published online: June 9, 2004
3666
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3657 3666