Zinc(II)cyclen-peptide conjugates interacting with the weak effector binding state of Ras

Article abstract of DOI:10.1016/j.ica.2010.07.067

Zinc(II)cyclen-peptide hybrid compounds and bis-zinc(II)cyclen complexes are prepared as potential binders of the guanine nucleotide binding protein Ras, an important molecular switch in cellular signal transduction. The design of the compounds is based on the previous observation that zinc(II)cyclen complexes could serve as lead compounds for inhibitors of Ras-effector interaction and thus be able to interrupt Ras induced signal transduction. Zinc(II)cyclen selectively stabilizes conformational state 1 of active Ras, a conformational state with drastically decreased affinity to effector proteins like Raf-kinase. To achieve higher binding affinities of such Ras-Raf interaction inhibitors, zinc(II)cyclen conjugates with short peptides, derived from the sequence of the Ras-activator SOS, were prepared by solid phase synthesis protocols. Dinuclear bis-zinc(II)cyclen complexes were obtained from alkyne-azide cycloaddition reactions. NMR investigations of the prepared compounds revealed that the peptide conjugates do not lead to an increase in Ras binding affinity of the metal complex-peptide conjugates. The dinuclear zinc complexes lead to an immediate precipitation of the protein prohibiting spectroscopic investigations of their binding.

Full text of DOI:10.1016/j.ica.2010.07.067

Inorganica Chimica Acta 365 (2011) 38–48
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Zinc(II)cyclen–peptide conjugates interacting with the weak effector binding state
of Ras
Florian Schmidt a, Ina C. Rosnizeck b, Michael Spoerner b, Hans Robert Kalbitzer b, Burkhard König a,
⇑
a Institute for Organic Chemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
b Institute for Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 April 2010
Received in revised form 20 July 2010
Accepted 24 July 2010
Available online 17 September 2010
Zinc(II)cyclen–peptide hybrid compounds and bis-zinc(II)cyclen complexes are prepared as potential
binders of the guanine nucleotide binding protein Ras, an important molecular switch in cellular signal
transduction. The design of the compounds is based on the previous observation that zinc(II)cyclen com-
plexes could serve as lead compounds for inhibitors of Ras-effector interaction and thus be able to inter-
rupt Ras induced signal transduction. Zinc(II)cyclen selectively stabilizes conformational state 1 of active
Ras, a conformational state with drastically decreased affinity to effector proteins like Raf-kinase. To
achieve higher binding affinities of such Ras–Raf interaction inhibitors, zinc(II)cyclen conjugates with
short peptides, derived from the sequence of the Ras-activator SOS, were prepared by solid phase synthe-
sis protocols. Dinuclear bis-zinc(II)cyclen complexes were obtained from alkyne-azide cycloaddition
reactions. NMR investigations of the prepared compounds revealed that the peptide conjugates do not
lead to an increase in Ras binding affinity of the metal complex–peptide conjugates. The dinuclear zinc
complexes lead to an immediate precipitation of the protein prohibiting spectroscopic investigations
of their binding.
Keywords:
Ras protein
Zinc cyclen
Protein–protein inhibition
Metal complex
NMR
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
concentrations. We could show, that the azamacrocyclic metal
complex Zn2+-1,4,7,10-tetraazacyclododecane (cyclen) selectively
The development of small molecules for the inhibition of pro-
tein–protein interactions is of high current interest due to possible
implications in medicinal chemistry. However, achieving sufficient
binding affinity and selectivity is still challenging [1–3]. A very
common approach improving the affinity and selectivity of a low
affinity binder is the introduction of additional binding sites. Such
bi- or multivalent ligands may lead to increased binding affinities
by additive or even cooperative action of the different binding sites
[4–6]. There are two principal ways to inhibit protein–protein
interactions by chemical inhibitors: They can either block the
interface (competitive inhibition) or induce changes in the proteins
conformation (allosteric inhibition) leading to an altered binding
ability of the surface of the protein interface [1].
Ras protein, which is known for several years to play an impor-
tant role in pathogenesis of certain human cancer types [7–9], ex-
ists in its active, GTP-bound form, in an equilibrium between at
least two conformational states (state 1 and state 2) [10]. Ras vari-
ants, which exist predominately in conformational state 1, show a
more than 20-fold decreased affinity to effector proteins [11]. This
affinity is too low in order to transduce the signal at physiological
binds state 1 [12], and that it is able to shift the equilibrium totally
towards that weak effector-binding conformation at higher con-
centration. Zn2+-cyclen shows affinity to phosphate ions [13–15]
and binds with millimolar affinity near the c-phosphate at the ac-
tive site of Ras stabilizing conformation of state 1. Ras in conforma-
tional state 2, which represents the strong effector binding mode,
and thus is responsible for Ras–effector-protein complex formation
in the cell, did not show significant interaction with Zn2+-cyclen.
The same results are obtained for GDP-bound, inactive Ras, here
again no indication for Zn2+-cyclen binding could be obtained in
the active site as studied by 31P NMR spectroscopy. Therefore,
Zn2+-cyclen can serve as lead for inhibitors of Ras–Raf interaction.
In this work we used the GTP analog GppNHp in order to resist
GTPase activity of Ras. The mutant Ras(T35A) was chosen because
it predominantly exists in the conformational state 1, which should
be recognized by the ligand, and because for this mutant in con-
trast to wild-type Ras all resonances can be obtained in the
[1H–15N]-HSQC NMR experiments. As the formation of such Ras–
effector-protein complexes induces certain cell signaling pathways
leading to unregulated cell growth in tumor cells, its selective inhi-
bition is of considerable interest [7]. However, a 20-fold excess of
Zn2+-cyclen is required to shift the equilibrium completely towards
⇑
Corresponding author. Tel./fax: +49 941 943 4575/1717.
E-mail address: burkhard.koenig@chemie.uni-regensburg.de (B. König).
0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.07.067

F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
39
state 1 due to the low binding affinity of the metal-complex to the
2.2. Protein preparation
active site of Ras [12].
The structure of the metal-cyclen complex with Ras was re-
cently solved [16] and different Zn2+-cyclen binding sites could
be identified. To increase the binding affinity, two types of biden-
tate ligands based on the Zn2+-cyclen binding motif were synthe-
sized: (1) Heterotopic peptide–metal complex hybrid-ligands, as
it was known that short peptides can interfere with the Ras–effec-
tor-protein interaction [17–19]. (2) Bidentate bis-(Zn2+-cyclen)
metal-complexes, as previous NMR-investigations showed that
there is more than one binding site for Zn2+-cyclen on the protein
surface of Ras [16].
The unlabeled C-terminal truncated T35A-mutant of human H-
Ras (amino acids 1–166) was expressed in Escherichia coli and puri-
fied as described before [20].
Uniformly 15N-labeled protein was obtained by growing the
bacteria in M9 minimal media containing 1 g Lꢀ1 15NH4Cl as the
sole nitrogen source [21]. The final purity of the proteins was
>95% as judged from the sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. Nucleotide exchange from GDP to GppNHp
was done using alkaline phosphatase treatment in the presence
of low excess GppNHp analogous to John et al. [22]. Free nucleo-
tides were removed by size exclusion chromatography.
2. Experimental procedures
2.3. STD NMR spectroscopy
2.1. General
NMR
samples
originally
contained
50
l
M
c’Ras(-
T35A)ꢁMg2+ꢁGppNHp in 40 mM Tris/HCl pH 7.4, 10 mM MgCl2,
25% D2O and 0.2 mM DSS as a reference standard. The spectra were
recorded on a Bruker Avance 600 MHz spectrometer equipped
with a 5 mm triple resonance cryo probe at 278 K. The ligands
were dissolved in the sample buffer and titrated to the protein
sample.
2.1.1. Absorption spectroscopy
Absorption spectra were recorded on a Varian Cary BIO 50 UV/
Vis/NIR Spectrometer by the use of 1 cm quartz cuvettes (Hellma)
and Uvasol solvents (Merck or Baker).
2.1.2. NMR spectroscopy
Saturation transfer difference (STD) NMR spectroscopy [23,24]
was used to detect the binding of the ligand to Ras. Compared to
non-NMR methods STD is especially well-suited for the detection
of the weak binding expected. The on-resonance irradiation fre-
quency was set to a chemical shift value of ꢀ2 ppm with an atten-
uation of 40 dB in the titration with Zn2+-cyclen and 1a or 30 dB in
the titration experiment with 1b. Off-resonance irradiation was
performed at +30 ppm. The protein was selectively saturated by
a train of 40 Gauss-shaped 90° pulses of 50 ms length giving a total
saturation time of 2 s. On- and off-resonance experiments have
been performed in an interlaced fashion. Water suppression was
Bruker Avance 600 (Cryo) (1H: 600.1 MHz, 13C: 150.1 MHz,
T = 300 K), Bruker Avance 400 (1H: 400.1 MHz, 13C: 100.6 MHz,
T = 300 K), Bruker Avance 300 (1H: 300.1 MHz, 13C: 75.5 MHz,
T = 300 K). The chemical shifts are reported in d [ppm] relative to
external standards (solvent residual peak). The spectra were ana-
lyzed by first order, the coupling constants are given in Hertz
[Hz]. Characterization of the signals: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, bs = broad singlet, dd = dou-
ble doublet, ddd = double double doublet. Integration is deter-
mined as the relative number of atoms. Assignment of signals in
13C-spectra was determined with DEPT-technique (pulse angle:
135°) and given as (+) for CH3 or CH, (ꢀ) for CH2 and (Cquat.) for
achieved using a delay of 100 ls for binomial water suppression.
Prior to acquisition, a T1 -filter consisting of a spinlock pulse with
q
quaternary Cquat.
. Error of reported values: chemical shift:
50 ms length and an attenuation of 15 dB was implemented in or-
der to suppress protein proton resonances, which eases data eval-
uation [25]. 32 k data points have been collected, zero-filled to 64 k
and multiplied by an exponential line broadening function of
0.3 Hz. After splitting the data into on- and off-resonance, both
spectra were processed and phased identically. Baseline correction
was done automatically using a polynomial of 5th degree. Process-
ing was performed using topspin 2.0 software (Bruker, Rheinstet-
ten, Germany).
0.01 ppm for 1H NMR, 0.1 ppm for 13C NMR and 0.1 Hz for coupling
constants. The solvent used is reported for each spectrum. Molec-
ular structures with atom numbers of the synthesized compounds
are provided in the Supporting Information to facilitate the assign-
ment traceability.
2.1.3. Mass spectrometry
Varian CH-5 (EI), Finnigan MAT 95 (CI), Finnigan MAT TSQ 7000
(ESI).
The STD amplification factor ASTD [24] is defined by
I0 ꢀ I
aSTD½Lꢂ
ASTD
¼
ð½Lꢂ ꢀ ½PꢂÞ ¼
ð1Þ
I0
½Lꢂ ꢀ Kd
2.1.4. IR spectrometry
Recorded with a Bio-Rad FTS 2000 MX FT-IR.
with [L] and [P] as the total ligand and protein concentrations,
respectively. I0 is the line intensity with off-resonance irradiation,
I the line intensity with saturation of the protein resonances (on-
2.1.5. Melting point
Melting points were determined on Büchi SMP or a Lambda
Photometrics OptiMelt MPA 100.
resonance), Kd the dissociation constant and
lue of ASTD
aSTD the maximum va-
.
2.1.6. TLC analysis and column chromatography
2.4. Syntheses
Analytical TLC plates (silica gel 60 F254) and silica gel 60 (70–
230 or 230–400 mesh) for column chromatography were
purchased from Merck. Spots were visualized by UV light and/or
staining with ninhydrine in EtOH.
Dry DMF was purchased from Fluka, dichloromethane (DCM)
was dried by adsorption and stored over molecular sieves. Solvents
for SPPS were all peptide grade. Petrol ether (PE) had a boiling
range of 70–90 °C. All other solvents and chemicals were of reagent
grade and used without further purification.
2.4.1. {2-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-ethoxy}-
acetic acid (7a)
{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-
butyl ester 6a (4.68 g, 14.8 mmol) was dissolved in 40 mL of THF/
toluene (1:1) and bromoacetic acid (6.16 g, 44.4 mmol) was added.
After heating the reaction mixture to 45 °C, powdered sodium
hydroxide (3.55 g, 88.7 mmol) was added and the mixture was
stirred over night. THF was evaporated and water was added to

40
F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
the toluene layer. After separating the aqueous layer, the organic
layer was extracted with aqueous sodium hydroxide solution
(5%; 3 ꢃ 40 mL) and the combined aqueous layers were washed
with DCM (3 ꢃ 40 mL). Conc. HClaq. was added slowly to the aque-
ous layer to reach pH 4 while stirring. By extraction with DCM
(3 ꢃ 50 mL) excess of bromoacetic acid could be removed. After
further addition of conc. HClaq. to reach pH 2, the product was iso-
lated by again extracting with DCM (5 ꢃ 50 mL). The combined or-
ganic layers were dried over MgSO4 and filtrated. Evaporation of
the solvent gave a yellowish oil (3.10 g, 68%).
13C NMR (150 MHz, COSY, HSQC, HMBC, CDCl3): d [ppm] = 174.7
(Cquat., 1C, 3), 156.7 (Cquat., 1C, 15), 144.0 (Cquat., 1C, 19), 141.2
(Cquat., 1C, 24), 127.6 (+, 2C, 22), 127.0 (+, 2C, 21), 125.1 (+, 2C,
20), 119.9 (+, 2C, 23), 70.1–69.8 (ꢀ, 6C, 4 + 6 + 7 + 9 + 10 + 12),
66.5 (ꢀ, 1C, 17), 47.2 (+, 1C, 18), 40.6 (ꢀ, 1C, 13) – IR (ATR)
~
[cmꢀ1]:
m = 3057, 2880, 1706, 1608, 1531, 1449, 1247, 1089,
1023, 740 – UV (MeOH) [nm]: k (e) = 265 (7600), 269 (10 900),
272 (11 300), 289 (4000), 300 (4900)
–
ESI-MS (DCM/
MeOH + 10 mmol/L NH4Ac): m/z (%) = 430.2 (100) [MH+], 447.3
(12) [MNH4+], 452.2 (50) [MNa+] – HR-MS (MeOH/glycerine): calcd.
for C23H28NO7 [MH+] (430.1866); found: 430.1865 (PI-LSIMS) –
Molecular Formula: C23H27NO7 – Molecular Weight: 429.2 g/mol.
1H NMR (400 MHz, COSY, HSQC, CDCl3): d [ppm] = 8.68 (s, 1H,
1), 5.12 (s, 1H, 14), 4.15 (s, 2H, 4), 3.76–3.71 (m, 2H, 6), 3.71–
3
3.63 (m, 4H, 7 + 9), 3.63–3.58 (m, 2H, 10), 3.52 (t, JH,H = 5.2 Hz,
2H, 12), 3.35–3.19 (m, 2H, 13), 1.42 (s, 9H, 18) – 13C NMR
(100 MHz, COSY, HSQC, CDCl3): d [ppm] = 172.8 (Cquat., 1C, 3),
156.2 (Cquat., 1C, 12), 79.3 (Cquat., 1C, 17), 71.2 (ꢀ, 1C, 6), 70.4 (ꢀ,
1C, 9), 70.2 (ꢀ, 2C, 7 + 12), 70.0 (ꢀ, 1C, 10), 68.6 (ꢀ, 1C, 4), 40.3
~
2.4.4. (2-{2-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-
ethoxy}-ethoxy)-acetic acid (7b)
(2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl)-carbamic
acid tert-butyl ester 6b (0.46 g, 1.6 mmol) was dissolved in 5 mL of
THF/toluene (1:1), then bromoacetic acid (0.66 g, 4.8 mmol) was
added. After heating the reaction mixture to 45 °C, powdered so-
dium hydroxide (0.38 mg, 9.5 mmol) was added and the mixture
was stirred over night. THF was evaporated and water (10 mL)
was added to the toluene layer. After separating the aqueous layer,
the organic layer was extracted with aqueous sodium hydroxide
solution (5%; 3 ꢃ 15 mL) and the combined aqueous layers were
washed with DCM (3 ꢃ 30 mL). Conc. HClaq. was added slowly to
the aqueous layer while stirring to reach pH 4, and was then
washed with DCM (3 ꢃ 50 mL). After further addition of conc.
HClaq. to reach pH 2, the product was isolated by extraction with
DCM (5 ꢃ 50 mL). Drying over MgSO4, filtration and evaporation
of the solvent yielded a slightly yellow oil with high viscosity
(0.52 g, 93%).
(ꢀ, 1C, 13), 28.3 (+, 3C, 18) – IR (ATR) [cmꢀ1]:
m = 3468, 3346,
1722, 1625, 1532, 1478, 1449, 1378, 1345, 1263, 1211, 1161,
1080, 1009, 916, 833, 759, 737 – ESI-MS (DCM/MeOH + 10 m-
mol/L NH4Ac): m/z (%) = 308.1 (44) [MH+], 352.2 (100) [MNH4+],
330.1 (22) [MNa+]
– HR-MS (MeOH/glycerine): calcd. for
C13H26NO7 [MH+] (308.1709); found: 308.1717 (PI-EIMS) – Ele-
mental Analysis: Anal. Calc. (%) for C13H25NO7 (307.34) + 4/5 H2O:
C 48.53, H 8.33, N 4.35; found: C 48.51, H 8.32, N 4.60 – Molecular
Formula: C13H25NO7 – Molecular Weight: 307.2 g/mol.
2.4.2. 2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-acetic acid
hydrochloride (8a)
{2-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-ethoxy}-
acetic acid 7a (3.00 g, 9.8 mmol) was diluted with 10 mL of water,
then half concentrated HClaq. (97.6 mmol) was added to the solu-
tion until CO2 evolution was observed. After stirring for 1 h the
reaction was complete. The solvent was removed by lyophilizing
the aqueous solution to obtain the product as highly viscous, yel-
lowish oil (2.40 g, quant.).
1H NMR (600 MHz Cryo, COSY, HSQC, HMBC, CDCl3):
d
[ppm] = 4.15 (s, 2H, 4), 3.81–3.73 (m, 2H, 6), 3.73–3.59 (m, 10H,
3
7 + 9 + 10 + 12 + 13), 3.55 (t, JH,H = 5.1 Hz, 2H, 11), 3.31 (t,
3JH,H = 4.8 Hz, 2H, 12), 1.43 (s, 9H, 15) – 13C NMR (150 MHz, COSY,
1H NMR (400 MHz, D2O): d [ppm] = 4.28 (s, 2H, 4), 3.84–3.75
HSQC, HMBC, CDCl3): d [ppm] = 172.5 (Cquat., 1C, 3), 156.1 (Cquat.,
3
(m, 10H, 6 + 7 + 9 + 10 + 12 + 14), 3.27 (t, JH,H = 5.0 Hz, 2H, 13) –
1C, 18), 79.2 (Cquat., 1C, 20), 71.1 (ꢀ, 1C, 6), 70.5–69.8 (ꢀ, 6C,
13C NMR (100 MHz, D2O): d [ppm] = 174.3 (Cquat., 1C, 3), 71.1,
69.7, 69.6, 69.5, 67.6, 66.4 (ꢀ, 6C, 4 + 6 + 7 + 9 + 10 + 12), 39.2 (ꢀ,
1C, 13) – ESI-MS (TFA/MeCN): m/z (%) = 207.9 (100) [MH+], 229.9
(17) [MNa+] – Molecular Formula: C8H17NO5 – Molecular Weight:
207.0 g/mol.
7 + 9 + 10 + 12 + 13 + 15), 68.7 (ꢀ, 1C, 4), 40.2 (ꢀ, 1C, 16), 28.3 (+,
3C, 21) – IR (ATR) [cmꢀ1]:
= 3346, 2972, 2927, 2870, 1712,
~
m
1521, 1454, 1366, 1277, 1251, 1122, 631, 535, 499, 408 – ESI-MS
(DCM/MeOH + 10 mmol/L NH4Ac): m/z (%) = 369.1 (100) [MNH4+],
352.0 (76) [MH+], 259.9 (20) [MH+ ꢀ C4H8], 251.9 (57) [MH+
ꢀ
Boc], 374.0 (12) [MNa+] – HR-MS (MeOH/glycerine): calcd. for
2.4.3. (2-{2-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-ethoxy]-
ethoxy}-ethoxy)-acetic acid (9a)
C
15H29NO8 [MH+] (352.1966); found: 352.1969 (EI-MS) – Elemen-
tal Analysis: Anal. Calc. (%) for C15H29NO8 (351.40) + 3/5 H2O: C
49.74, H 8.40, N 3.87; found: C 49.76, H 8.10, N 4.64 – Molecular
Formula: C15H29NO8 – Molecular Weight: 351.2 g/mol.
2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-acetic acid hydrochlo-
ride 8a (2.45 g, 10.1 mmol) was diluted with 5 mL of water and
potassium carbonate (4.17 g, 30.2 mmol) was added. After 15 min
stirring at room temperature, N-(9H-fluoren-2-ylmethoxycarbon-
yloxy)-succinimide (5.09 g, 15.1 mmol) was added in portions over
30 min and stirring was continued for 16 h. The solution was ad-
justed to pH 9 by adding aqueous sodium hydroxide solution
(2 M) and the aqueous layer was washed with DCM
(3 ꢃ 200 mL). Conc. HClaq. was added while stirring to reach pH 2
and the crude product was extracted with DCM (5 ꢃ 100 mL). After
drying of the organic phase over MgSO4, filtration and evaporation
of the solvent, the product was isolated by flash column chroma-
tography (DCM ? DCM:MeOH = 95:5 ? DCM:MeOH = 9:1; Rf,
DCM:MeOH=8:2 = 0.23) as a greenish-yellow solid (2.76 g, 64%).
2.4.5. (2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-acetic acid
hydrochloride (8b)
(2-{2-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-eth-
oxy}-ethoxy)-acetic acid 7b (3.00 g, 8.5 mmol) was diluted with
10 mL of water, then half concentrated HClaq. (14.2 mL, 85.3 mmol)
was added until formation of CO2 was observed. After stirring for
1 h the reaction was complete. The solvent was removed by lyoph-
ilization of the aqueous solution to obtain the product as highly
viscous, yellowish oil (2.50 g, quant.).
1H NMR (400 MHz, D2O): d [ppm] = 4.28 (s, 2H, 4), 3.84–3.75
MP: 46 °C – 1H NMR (600 MHz Cryo, COSY, HSQC, HMBC,
(m, 14H, 6 + 7 + 9 + 10 + 12 + 13 + 15), 3.28 (t, JH,H = 5.0 Hz, 2H,
3
3
CDCl3):
d
[ppm] = 7.72 (d, JH,H = 7.5 Hz, 2H, 23), 7.59 (d,
16) – 13C NMR (100 MHz, D2O): d [ppm] = 174.4 (Cquat., 1C, 3),
70.1, 69.7, 69.6, 69.5, 67.7, 66.4 (ꢀ, 8C, 4 + 6 + 7 + 9 + 10 + 12 +
13 + 15), 39.2 (ꢀ, 1C, 16) – ESI-MS (H2O/MeOH + 10 mmol/L
NH4Ac): m/z (%) = 252.0 (100) [MH+], 274.0 (9) [MNa+] – Molecular
Formula: C10H21NO6 – Molecular Weight: 251.1 g/mol.
3
3JH,H = 7.3 Hz, 2H, 20), 7.36 (dd, JH,H = 7.4 Hz, 7.5 Hz, 2H, 22), 7.28
3
(dd, JH,H = 7.4 Hz, 7.5 Hz, 2H, 21), 5.97 (bs, 1H, 14), 4.46–4.31 (m,
2H, 17), 4.19 (t, JH,H = 6.8, 1H, 18), 3.99 (s, 2H, 4), 3.74–3.53 (m,
3
8H, 6 + 7 + 9 + 10), 3.53–3.47 (m, 2H, 12), 3.39–3.29 (m, 2H, 13) –

F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
41
2.4.6. [2-(2-{2-[2-(9H-Fluoren-9-yl-methoxycarbonylamino)-ethoxy]-
2.4.8. 10-Carboxymethyl-1,4,7,10-tetraaza-cyclododecane-1,4,7-
ethoxy}-ethoxy)-ethoxy]-acetic acid (9b)
tricarboxylic acid tri-tert-butyl ester (13)
2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-acetic acid
hydrochloride 8b (2.25 g, 7.8 mmol) was diluted in 5 mL of water,
then potassium carbonate (3.24 g, 23.5 mmol) was added and
deprotonation was observed as gas formation occurred. After
15 min stirring at room temperature, N-(9H-fluoren-2-ylmethoxy-
carbonyloxy)succinimide (3.96 g, 11.7 mmol) was added in por-
tions over 30 min and stirring was continued for 16 h. The
solution was adjusted to pH 9 by adding aqueous sodium hydrox-
ide solution (2 M) and the aqueous layer was washed with DCM
(3 ꢃ 200 mL). Conc. HClaq. was added while stirring to reach pH 2
and the crude product was extracted with DCM (5 ꢃ 100 mL). After
drying of the organic layer over MgSO4, filtration and evaporation,
the product was purified by flash column chromatography
10-Methoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododecane-
1,4,7-tricarboxylic acid tri-tert-butyl ester 12 (1.26 g, 2.3 mmol)
was dissolved in 30 mL of acetone/water (3:1), lithium hydroxide
monohydrate (0.29 g, 6.9 mmol) was added and the mixture was
stirred over night at 50 °C. Afterwards, acetone was evaporated
and the aqueous layer was acidified with aqueous KHSO4 (5%).
The product was extracted with EtOAc and isolated after drying
over MgSO4, filtration and evaporation of the solvent as yellowish
solid (1.10 g, quant.).
MP: 138 °C – 1H NMR (600 MHz, NOESY, HSQC, HMBC, CDCl3): d
[ppm] = 3.61–3.21 (m, 14H, 4 + 7 + 9 + 10), 2.85 (bs, 4H, 6), 1.45 (s,
9H, 15), 1.43 (s, 18H, 19) – 13C NMR (150 MHz, NOESY, HSQC,
HMBC, CDCl3): d [ppm] = 173.4 (Cquat., 1C, 3), 156.2 (Cquat., 2C,
16), 155.5 (Cquat., 1C, 12), 80.1 (Cquat., 2C, 18), 79.7 (Cquat., 1C, 14),
54.3 (ꢀ, 2C, 6), 52.4 (ꢀ, 1C, 4), 49.9 (ꢀ, 2C, 9), 47.5 (ꢀ, 4C,
(DCM ? DCM: MeOH = 95:5 ? DCM:MeOH = 9:1; Rf,
DCM:MeOH:
TFA=9:0.5:0.5 = 0.22) to obtain a greenish-yellow, hygroscopic solid
(1.70 g, 46%).
7 + 10), 28.6 (+, 3C, 15), 28.4 (+, 6C, 19)
– ESI-MS (DCM/
MP: 51 °C – 1H NMR (400 MHz, COSY, NOESY, HSQC, HMBC,
MeOH + 10 mM NH4OAc): m/z (%) = 531.4 (100) [MH+], 475.3 (10)
[MH+ ꢀ C4H8], 431.3 (52) [MH+ ꢀ Boc] – Molecular Formula:
3
CDCl3):
d
[ppm] = 7.73 (d, JH,H = 7.5 Hz, 2H, 26), 7.60 (d,
3
3JH,H = 7.3, 2H, 23), 7.37 (dd, JH,H = 7.4 Hz, 7.4 Hz, 2H, 25), 7.29
C25H46N4O8 – Molecular Weight: 530.4 g/mol.
3
4
(ddd, JH,H = 7.4 Hz, 7.4 Hz, JH,H = 1.1 Hz, 2H, 24), 5.90 (bs, 1H,
3
3
17), 4.37 (d, JH,H = 6.9, 2H, 20), 4.20 (t, JH,H = 6.8, 1H, 21), 3.98
(s, 2H, 4), 3.68–3.47 (m, 14H, 6 + 7 + 9 + 10 + 12 + 13 + 15),
3.42–3.29 (m, 2H, 16) – 13C NMR (100 MHz, COSY, NOESY, HSQC,
HMBC, CDCl3): d [ppm] = 174.6 (Cquat., 1C, 3), 156.7 (Cquat., 1C,
18), 144.0 (Cquat., 2C, 22), 141.2 (Cquat., 2C, 27), 127.6 (+, 2C,
25), 127.0 (+, 2C, 24), 125.1 (+, 2C, 23), 119.8 (+, 2C, 26), 70.1
(ꢀ, 1C, 15), 70.0–69.8 (ꢀ, 6C, 6 + 7 + 9 + 10 + 12 + 13), 69.6 (ꢀ,
1C, 4), 66.5 (ꢀ, 1C, 20), 47.2 (+, 1C, 21), 40.7 (ꢀ, 1C, 16) – IR
~
2.4.9. Solid-phase peptide–cyclen conjugate synthesis (17a and 17b)
The peptides were synthesized on an Advanced Chemtech
496 MOS synthesizer. Rink Amide MBHA resin and Fmoc pro-
tecting group strategy were used. Coupling was achieved by
TBTU/HOBt/DIPEA. HOBt was used as a 0.45 M solution, TBTU
as a 0.44 M solution and DIPEA as a 1.2 M solution, all in DMF.
The Fmoc-protected amino acids as well as the Fmoc-protected
amino-PEG-acid spacer and also the cyclen building block were
dissolved in NMP as 0.4 M solutions. The syntheses were carried
out in a 96 well reactor block where wells were left empty in
between used ones due to the danger of resin being blown
across the block, thus contaminating other wells. The peptides
were synthesized on 50 mg resin. The lot of the resin used had
a loading of 0.72 mmol/g (manufacturer’s claims). Before each
synthesis the resin was allowed to preswell in DMF for 30 min.
Each coupling was done twice using a 5-fold excess of HOBt
and slightly less than 5-fold excess of TBTU. DIPEA was used
in 10-fold excess. Fmoc deprotection was done by shaking the
resin with 40% piperidine in DMF for 3 min, subsequent washing
and addition of 20% piperidine in DMF followed by shaking for
10 min. After completion, the resin was washed with MeOH,
DCM and Et2O (5 ꢃ 2 mL each). Cleavage from the resin was
afforded by shaking the resin for 3 h after addition of 1.5 mL
of TFA/TIS/water (90:5:5). After filtering off the resin, the TFA
solution was reduced in volume to about 0.5 mL. It was then
transferred to a Falcon tube and precipitated with cold Et2O.
The precipitate was centrifuged at ꢀ4 °C for 10 min. The solution
was then carefully decanted off and the precipitate resuspended
in cold Et2O before being centrifuged again. This resuspending/
centrifuging step was repeated five times. Finally, Et2O was dec-
anted off again and the peptide dried under vacuum. The pep-
tides were analysed by ESI-MS and LC–MS or HPLC or both
and purified by preparative HPLC.
(ATR) [cmꢀ1]:
m = 3049, 2874, 1710, 1607, 1538, 1449, 1349,
1246, 1092, 1030, 945, 858, 760, 740 – UV (MeOH) [nm]: k
) = 269 (12 700), 272 (12 800), 288 (4700), 300 (5300) – ESI-
MS (DCM/MeOH + 10 mmol/L NH4Ac): m/z (%) = 474.2 (100)
[MH+], 491.3 (15) [MNH4+], 496.2 (45) [MNa+]
HR-MS
(MeOH/glycerine): calcd. for
25H32NO8 [MH+] (474.2128);
(e
–
C
found: 474.2136 (PI-LSIMS) – Molecular Formula: C25H31NO8
Molecular Weight: 473.2 g/mol.
–
2.4.7. 10-Methoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododecane-
1,4,7-tricarboxylic acid tri-tert-butyl ester (12)
(Boc)3cyclen 11 (1.00 g, 2.1 mmol) was dissolved in 20 mL of
MeCN, then potassium carbonate (0.35 g, 2.6 mmol) and methyl
bromoacetate (0.36 g, 2.4 mmol) were added. The mixture was
stirred over night at 50 °C, then solids were filtered off and the sol-
vent was evaporated to obtain the crude product, which was puri-
fied by column chromatography (EtOAc:PE = 1:1; Rf,
=
EtOAc:PE=7:3
0.63) yielding a pale yellow solid (1.16 g, quant.).
MP: 69 °C – 1H NMR (600 MHz, NOESY, HSQC, HMBC, CDCl3): d
[ppm] = 3.66 (s, 3H, 1), 3.60–3.11 (m, 14H, 4 + 7 + 9 + 10), 2.99–
2.75 (m, 4H, 6), 1.45 (s, 9H, 15), 1.42 (s, 18H, 19) – 13C NMR
(100 MHz, NOESY, HSQC, HMBC, CDCl3): d [ppm] = 171.1 (Cquat.
,
1C, 3), 156.1, 155.7, 155.3 (Cquat., 3C, 12 + 16 + 160), 79.7 (Cquat.
,
1C, 18), 79.5 (Cquat., 1C, 180), 79.2 (Cquat., 1C, 14), 54.9 (ꢀ, 1C, 6),
53.6 (ꢀ, 1C, 6’), 51.2 (+, 1C, 1), 50.9 (ꢀ, 1C, 4), 50.0 (ꢀ, 2C, 9 + 9’),
47.7 (ꢀ, 2C, 7 + 70), 47.3 (ꢀ, 1C, 10), 47.0 (ꢀ, 1C, 100), 28.7 (+, 3C,
~
Peptide-cyclen conjugate 17a: ESI-MS: m/z (%) = 305.8 (100)
[M + 3 H+]3+, 458.3 (74) [M + 2 H+]2+, 573.6 (4) [M + 2 H+ + TFA]2+
,
915.7 (1) [MH+] – Molecular Formula: C40H78N14O10 – Molecular
Weight: 914.6 g/mol.
15), 28.4 (+, 6C, 19) – IR (ATR) [cmꢀ1]:
m = 2974, 2933, 2874,
1739, 1692, 1456, 1413, 1364, 1246,1180, 978, 859, 772 – ESI-
MS (DCM/MeOH + 10 mmol/L NH4Ac): m/z (%) = 545.4 (100)
[MH+], 445.3 (38) [MH+ ꢀ Boc], 345.2 (10) [MH+ ꢀ 2 Boc], 389.2
(10) [MH+ ꢀ Boc ꢀ C4H8], 489.3 (7) [MH+ ꢀ C4H8] – Elemental Anal-
ysis: Anal. Calc. for C26H48N4O8 (544.69): C 57.33, H 8.88, N 10.29;
found: C 57.04, H 8.83, N 10.05 – Molecular Formula: C26H48N4O8 –
Molecular Weight: 544.4 g/mol.
Peptide–cyclen conjugate 17b: ESI-MS: m/z (%) = 480.3 (100)
[M + 2 H+]2+
C
,
1073.9 (9) [MH+ + TFA]
–
Molecular Formula:
42H82N14O11 – Molecular Weight: 958.6 g/mol.
2.4.10. General procedure hybrid-ligands 1a and 1b
Hybrid-ligand precursors were dissolved in a small amount of
water and pH 8 was adjusted by addition of LiOH (1 M) or NaHCO3

42
F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
(10% w/v). Then, 1 eq of ZnCl2 was added as 1 M aqueous solution,
which was prepared in advance. After heating the mixtures to re-
flux for 2 h, the products were obtained by lyophilization as color-
less, amorphous solids in sufficient purities and with quantitative
yields.
2.4.13. Boc-N-[2-(1,4,7,10-tetraazacyclododec-1-yl)-ethyl]-3-{1-[2-
(2-{2-[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-
ethylcarbamoyl]-ethyl}-[1,2,3]triazol-1-yl)ethoxy]-ethoxy}-ethoxy)-
ethyl]-1H-[1,2,3]triazol-4-yl}-propionamide (25)
N-10-(2-aminoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-tri-
carbonylicacid-tri-tert-butyl-ester-pent-4-ynamide 24 (0.50 g,
0.84 mmol) was dissolved in 20 mL of MeOH. Then, copper(II)-sul-
fate hexahydrate (0.03 g, 0.13 mmol) and sodium ascorbic acid
(0.05 g, 0.25 mmol) were dissolved in a small amount of water –
resulting in a dark brown solution, which turned slowly into beige
– and poured into the reaction flask while stirring. When the reac-
tion solution’s color turns from beige to light blue within the first
minute’s further sodium ascorbic acid was added. The mixture was
then warmed to 40 °C and stirred for 20 min followed by the addi-
tion of 1-{2-[2-(2-azidoethoxy) ethoxy]ethoxy}-2-azido-ethane
22b (0.10 g, 0.42 mmol). Reaction was complete after stirring at
50 °C for 30 min. Subsequently, MeOH was evaporated resulting
in a brown, viscous residue insoluble in water. After addition of
water (15 mL) and chloroform (20 mL), the organic layer was sep-
arated and the aqueous solution was extracted with chloroform
(3 ꢃ 20 mL). The combined organic layers were dried over MgSO4,
filtered and evaporated. The resulting residue was cleaned by a
short column filtration using flash silica gel (1.5 cm ꢃ 4 cm; EtOA-
c:EtOH = 95:5 ? 7:3) to gain a colorless, resin-like solid (0.55 g,
91%).
Hybrid-ligand 1a: ESI-MS (MeOH + 10 mmol/L NH4Ac): m/z
(%) = 489.4 (100) [M2+], 519.4 (74) [M2+ + CH3COOH]2+, 546.4 (20)
[M2+ + TFA]2+
C
,
977.8 (1.2) [M2+
ꢀ
H+]
–
Molecular Formula:
40H78N14O10Zn2+ – Molecular Weight: 978.6 g/mol.
Hybrid-ligand 1b: ESI-MS (H2O/MeOH + 10 mmol/L NH4Ac): m/
z (%) = 511.3 (100) [M2+], 541.3 (12) [M2+ + CH3COOH], 568.3 (8)
[M2+ + TFA]2+, 1021.5 (0.3) [M2+ ꢀ H+], 1057.6 (0.2) [M2+ + Clꢀ],
1035.6 (0.1) [M2+
ꢀ
H+ + TFA]
–
Molecular Formula:
C42H82N14O11Zn2+ – Molecular Weight: 1022.6 g/mol.
2.4.11. 2-{2-[2-(2-{[(4-Methylphenyl)sulfonyl]oxy}ethoxy)ethoxy]-
ethoxy}ethyl-4-methyl-benzene sulfonate (tetra(ethylene glycol)-
ditosylate) (21b)
Tetra(ethylene glycol) 20b (5.00 g, 4.40 mL, 25.7 mmol) and 4-
methylbenzene-1-sulfonyl chloride (14.7 g, 77.2 mmol) were dis-
solved in about 100 mL of THF and cooled to 0 °C in an ice-bath.
Then, potassium hydroxide (10.1 g, 180.2 mmol) dissolved in
25 mL of water was added dropwise (ꢄ1 h). After additional
2 h of stirring at room temperature the mixture was poured into
water/Et2O (50 mL/150 mL). The organic layer was separated and
the aqueous layer was re-extracted three times with Et2O. The
combined organic layers were washed with saturated NH4Cl-
solution and water and were dried over MgSO4 before evaporat-
ing the solvent. The clean product was obtained by column-chro-
MP: 89 °C – 1H NMR (400 MHz, COSY, HSQC, HMBC, CDCl3): d
3
[ppm] = 7.45 (s, 2H, 8), 6.76 (bs, 2H, NH), 4.40 (t, JH,H = 5.1 Hz,
3
4H, 6), 3.77 (t, JH,H = 5.1 Hz, 4H, 5), 3.55–3.39 (m, 16H,
3
2 + 3 + 20), 3.38–3.10 (m, 20H, 14 + 18 + 21), 2.93 (t, JH,H = 7.4 Hz,
4H, 10), 2.74–2.33 (m, 16H, 11 + 15 + 17), 1.38 (s, 18H, 26), 1.36
matography (EtOAc:MeOH = 95:5; Rf, EtOAc:MeOH=9:1 ꢄ 0.6) as
a
(s, 36H, 30) – 13C NMR (100 MHz, COSY, HSQC, HMBC, CDCl3): d
orange-brown, viscous oil (12.9 g, quant.) identical in every as-
pect to that prepared by the literature procedure [26].
[ppm] = 172.9 (Cquat., 2C, 12), 155.8 (Cquat., 4C, 27), 155.3 (Cquat.,
2C, 23), 146.4 (Cquat., 2C, 9), 122.2 (Cquat., 2C, 8), 79.5 (Cquat., 4C,
29), 79.3 (Cquat., 2C, 25), 70.3 (ꢀ, 4C, 2 + 3), 69.3 (ꢀ, 2C, 5), 55.6–
53.7 (ꢀ, 4C, 17), 51.8 (ꢀ, 2C, 15), 49.9 (ꢀ, 2C, 6), 49.7 (ꢀ, 4C, 21),
48.2 (ꢀ, 4C, 20), 47.8 (ꢀ, 4C, 18), 35.8 (ꢀ, 2C, 14), 35.3 (ꢀ, 2C,
11), 28.5 (+, 6C, 26), 28.3 (+, 12C, 30), 21.3 (ꢀ, 2C, 10) – IR (ATR)
~
2.4.12. N-10-(2-aminoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-
tricarbonylicacid-tri-tert-butyl ester-pent-4-ynamide (24)
4-Pentynoic-acid (0.04 g, 0.4 mmol) was dissolved in a mix-
ture of DCM and DMF in a nitrogen flushed round bottom flask,
[cmꢀ1]:
m = 2972, 2931, 1681, 1543, 1459, 1414, 1364, 1247,
1153, 1107, 1050, 976, 858, 773 – UV (MeCN) [nm]: k (e) = 215
then DIPEA (0.24 g, 250
(0.08 g, 0.5 mmol) were added. When the mixture was cooled
to 0 °C, EDC (0.08 g, 87 L, 0.5 mmol) and 10-(2-aminoethyl)-
l
L, 1.8 mmol) and HOBt monohydrate
(4800) – Elemental Analysis: Anal. Calc. (%) for C68H122N16O17
(1435.83) + 4H2O: C 54.16, H 8.69, N 14.86; found: C 54.11, H
8.50, N 14.70 – ESI-MS (DCM/MeOH + 10 mmol/L NH4Ac): m/z
(%) = 718.7 (100) [M + 2 H+]2+, 1436.2 (13) [MH+], 1458.3 (4)
[MNa+] – Molecular Formula: C68H122N16O17 – Molecular Weight:
1434.9 g/mol.
l
1,4,7,10-tetraazacyclododecan-1,4,7-tricarbonylic-acid-tri-tert-
butylester 23 (0.32 g, 0.6 mmol) – in portions – were added.
After 12 h of stirring at room temperature, water was added.
Then, the organic layer was separated and the aqueous layer
was extracted with DCM. The combined organic layers were
washed with citric acid solution (10%), dried over MgSO4
and the solvent was evaporated. The crude product was puri-
fied by flash chromatography (EtOAc:MeOH = 95:5, Rf = 0.4) to
obtain a colorless solid (0.20 g, 82%).
2.4.14. N-[2-(1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]-3-{1-[2-(2-
{2-[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-ethylcarbamoyl]-
ethyl}-[1,2,3]triazol-1-yl)ethoxy]ethoxy}-ethoxy)-ethyl]-H-
[1,2,3]triazol-4-yl}-propionamide octa-hydrochloride (26 ꢁ 8 HCl)
Boc-protected N-[2-(1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]
-3-{1-[2-(2-{2-[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-eth-
ylcarbamoyl]-ethyl}-[1,2,3]triazol-1-yl)ethoxy]ethoxy}-ethoxy)-
ethyl]-1H-[1,2,3]triazol-4-yl}-propionamide 25 (0.10 g, 0.07 mmol)
was dissolved in a small amount of DCM and cooled down to 0 °C.
While stirring rapidly, HCl saturated Et2O (2.7 mL) was dropped into
the reaction mixture. After 12 h of stirring at room temperature, the
solvent was evaporated in vacuo to dryness to obtain the product
(0.07 g, 99%) as a greenish-yellow solid.
MP: 66–68 °C – 1H NMR (600 MHz, COSY, HSQC, HMBC, CDCl3):
d [ppm] = 6.76 (bs, 1H, NH), 3.44 (s, 4H, 13), 3.32 (s, 4H, 14), 3.29–
3.15 (m, 6H, 7 + 11), 2.73–2.43 (m, 6H, 8 + 10), 2.43–2.39 (m, 2H,
3
3
3), 2.35 (t, JH,H = 7.1 Hz, 2H, 4), 1.90 (t, JH,H = 2.5 Hz, 1H, 1), 1.38
(s, 9H, 19), 1.36 (s, 18H, 23) – 13C NMR (150 MHz, COSY, HSQC,
HMBC, CDCl3): d [ppm] = 172.9 (Cquat., 1C, 5), 155.8 (Cquat., 2C,
20), 155.4 (Cquat., 1C, 16), 83.0 (Cquat., 1C, 2), 79.6 (Cquat., 3C,
18 + 22), 69.0 (+, 1C, 1), 55.0 (ꢀ, 2C, 10), 52.3 (ꢀ, 1C, 8), 49.8 (ꢀ,
2C, 14), 48.5 (ꢀ, 2C, 13), 47.9 (ꢀ, 2C, 11), 36.4 (ꢀ, 1C, 7), 35.0 (ꢀ,
1C, 4), 28.4 (+, 9C, 19 + 23), 14.8(ꢀ, 1C, 3) – IR (ATR) [cmꢀ1]:
~
MP: 85 °C – 1H NMR (600 MHz, COSY, HSQC, HMBC, D2O): d
3
[ppm] = 8.10 (s, 2H, 8), 4.55 (t, JH,H = 4.9 Hz, 4H, 6), 3.80 (t,
m
= 2975, 2927, 1763, 1543, 1458, 1413, 1364, 1246, 1152, 1039,
3JH,H = 4.9 Hz, 4H, 5), 3.46–3.42 (m, 4H, 2), 3.41–3.35 (m, 4H, 3),
3
3
975, 858, 773 – ESI-MS (DCM/MeOH + 10 mmol/L NH4Ac): m/z
3.18 (t, JH,H = 6.5 Hz, 14), 3.13 (t, JH,H = 5.0 Hz, 8H, 19), 3.10–3.05
(%) = 596.4 (100) [MH+] – HR-MS: calcd. for C30H53N5O7 [MH+]
(m, 8H, 18), 3.10–3.05 (m, 8H, 20), 2.93 (3JH,H = 7.3 Hz, 4H, 10),
3
(595.3945); found: 595.3937 (EI-MS)
–
Molecular Formula:
2.86 (3JH,H = 4.7 Hz, 8H, 17), 2.64 (t, JH,H = 6.4 Hz, 4H, 15), 2.51 (t,
C30H53N5O7 – Molecular Weight: 595.4 g/mol.
3JH,H = 6.4 Hz, 4H, 11) – 13C NMR (150 MHz, COSY, HSQC, HMBC,

F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
43
D2O): d [ppm] = 174.0 (Cquat., 2C, 12), 143.2 (Cquat., 2C, 9), 126.5
3. Results and discussion
(Cquat., 2C, 8), 69.5 (ꢀ, 2C, 2), 69.3 (ꢀ, 2C, 3), 67.8 (ꢀ, 2C, 5), 52.3
(ꢀ, 2C, 6), 51.9 (ꢀ, 2C, 15), 48.2 (ꢀ, 4C, 17), 43.4 (ꢀ, 4C, 20), 42.4
(ꢀ, 4C, 18), 41.8 (ꢀ, 4C, 21), 35.1 (ꢀ, 2C, 11), 33.6 (ꢀ, 2C, 14),
19.2 (ꢀ, 2C, 10) – ESI-MS (DCM/MeOH + 10 mmol/L NH4Ac): m/z
(%) = 279.2 (100) [M + 3 H+]3+, 418.7 (96) [M + 2 H+]2+, 835.7 (5)
[MH+] – Molecular Formula: C38H74N16O5 – Molecular Weight:
834.6 g/mol.
A column was filled with a strong basic anion exchange resin
(OHꢀ-form; 1.5 cm ꢃ 3 cm) and rinsed first with a mixture of
water/MeOH (1:1) then followed by pure water. The above ob-
tained compound was dissolved in a small amount of water and
eluted from the column thus yielding the salt-free ligand 26
(0.05 g, quant.).
3.1. Ligand design
Fig. 1 shows the solution structure of the Ras.Mg2+.GppNHp
complex in the conformational state that is only weakly interacting
with effectors. This state is stabilized by Zn2+-cyclen bound to the
c
-phosphate of GppNHp [16].
In the approach presented here Zn2+-cyclen bound to its site
close to the c-phosphate group of GppNHp should be linked with
a second interacting group via a flexible linker. State 1 is assumed
to be closely related to the conformation of Ras found in the com-
plex with its nucleotide exchange factor SOS (son of sevenless)
[27,28]. Therefore, the crystal structure of the Ras–SOS complex
[19] was used to derive Zn2+-cyclen–peptide ligands (Fig. 2) con-
taining a possible additional interacting site with Ras. The penta-
peptide contains three amino acids (Leu822, Ile825, Arg826) from
the sequence of the exchange factor SOS, which directly interact
with Ras. The amino acids that do not contribute to the interaction
with Ras have been replaced by glycines in order to allow for more
2.4.15. Di-Zn2+-{N-[2-(1,4,7,10-Tetraaza-cyclododec-1-yl)-ethyl]-3-
{1-[2-(2-{2-[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-
ethylcarbamoyl]-ethyl}-[1,2,3]triazol-1-yl)ethoxy]ethoxy}-ethoxy) -
ethyl]-1H-[1,2,3]triazol-4-yl}-propionamide} tetrachloride (18c)
N-[2-(1,4,7,10-Tetraaza-cyclododec-1-yl)-ethyl]-3-{1-[2-(2-{2-
[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-ethylcarbamoyl]-
ethyl}-[1,2,3]triazol-1-yl) ethoxy]ethoxy}-ethoxy)-ethyl]-1H-[1,2,
3]triazol-4-yl}-propionamide 26 (60.0 mg, 0.07 mmol) and zin-
c(II)-chloride (19.6 mg, 0.14 mmol) were dissolved separately in a
small amount of water and then dropped simultaneously in 2 mL
of warm water while stirring. The pH was adjusted to 7–8 with
aqueous lithium hydroxide (1 M). After 7 h of reflux, the mixture
was cooled down to ꢄ5 °C in the fridge. As no precipitation oc-
curred, also when MeOH or EtOH were added, the solvent was
evaporated and subsequently lyophilized to obtain the product
(62 mg, 92%) as a light brown solid.
flexibility. Next, the distance between the binding position of Zn2+
-
cyclen at the protein’s active centre and the peptide was estimated
and the peptide was tethered to Zn2+-cyclen by spacers of different
lengths.
Two binding sites for Zn2+-cyclen complexes had been identi-
fied on the surface of the Ras protein [16]. One is located close to
the c-phosphate of the bound nucleotide, the other is located close
to the C-terminus of Ras. In binding site 1 the Zn2+ ion is directly
coordinated to the phosphate group, in binding site 2 to the imid-
azole nitrogen of His166 of Ras. Dinuclear bis(Zn2+-cyclen) com-
plexes (Fig. 4) that recognize the two binding sites should
therefore show an increased affinity to Ras-GTP, if they act syner-
gistic or even cooperative. However, if the PEG linker would be
smaller than the distance between the Ras GTP binding site and
the Zn2+-cyclen surface binding site, the compound should be able
to cross-link the proteins. PEG-linked bis(Zn2+-cyclen) complexes
based on tri- and tetra(ethylene glycol) units should be suitable
to test the hypothesis.
MP: 109 °C – 1H NMR (400 MHz, COSY, HSQC, HMBC, D2O): d
3
[ppm] = 7.88 (s, 2H, 8), 4.64 (dd, JH,H = 4.9 Hz, 5.2 Hz, 4H, 6), 3.99
3
(dd, JH,H = 4.9 Hz, 5.2 Hz, 4H, 5), 3.93–3.85 (m, 2H, 13), 3.68–3.63
3
(m, 4H, 2), 3.62–3.56 (m, 4H, 3), 3.18 (dd, JH,H = 6.9 Hz, 7.3 Hz,
14), 3.13–2.78 (m, 40H, 10 + 15 + 17 + 16 + 20 + 21), 2.65 (dd,
3JH,H = 7.2 Hz, 7.3 Hz, 4H, 11) –13C NMR (100 MHz, COSY, HSQC,
HMBC, D2O): d [ppm] = 175.0 (Cquat., 2C, 12), 146.4 (Cquat., 2C, 9),
123.7 (Cquat., 2C, 8), 69.7 (ꢀ, 2C, 2), 69.5 (ꢀ, 2C, 3), 67.8 (ꢀ, 2C,
5), 51.3 (ꢀ, 2C, 15), 50.0 (ꢀ, 2C, 6), 49.5 (ꢀ, 4C, 17), 44.4 (ꢀ, 4C,
21), 43.5 (ꢀ, 4C, 20), 42.2 (ꢀ, 4C, 18), 35.2 (ꢀ, 2C, 11), 33.6 (ꢀ,
3.2. Synthesis and characterization of Zn2+-cyclen–peptide hybrid
ligands
2C, 14), 21.0 (ꢀ, 2C, 10)
– ESI-MS (H2O/MeOH + 10 mmol/L
NH4Ac): m/z (%) = 480.4 (70) [M + 2 H+]2+, 498.4 (17) [M + 3
Solid phase peptide synthesis (SPPS) and Fmoc-strategy were
used to prepare ligands 1 in analogy to our previously published
protocols [29,30]. The appropriate PEG spacers, assuring sufficient
water solubility of the target molecules, were synthesized as
shown in Scheme 1.
Ethylene glycols were mono-tosylated according to literature
known procedures giving compounds 3a and b [31,32]. For the
preparation of azides 4a and b two different reaction conditions
have been reported [32–34]. In our hands, the room temperature
reactions gave higher yields compared to heating to 80 °C. The
clean catalytic reduction of 4a was performed analogously to the
reported conversion of 4b [34] and the amines were Boc protected
to give 6a/b [35,36]. The subsequent alkylation step with 2-bromo-
acetic acid in the presence of sodium hydroxide provided the cor-
responding acids in moderate yields. Finally, the Boc-protection
groups were replaced by Fmoc.
An N-protected cyclen carboxylic acid was prepared for later
use in the Fmoc SPPS protocol (Scheme 2). Compound 13 [37]
was obtained in almost quantitative overall yield starting from
compound 11, which was prepared according to a literature known
procedure [38]. The pentapeptide was synthesized by standard
Fmoc-based SPPS protocols using an automated peptide synthe-
sizer on Rink amide MBHA resin. Subsequently, it was coupled to
H+ + Clꢀ]2+, 517.4 (23) [M + 4H+ + 2 Clꢀ]2+ – Molecular Formula:
4+
C
38H74N16O5Zn2 – Molecular Weight: 962.4 g/mol.
2.4.16. Di-Cu2+-{N-[2-(1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]-3-
{1-[2-(2-{2-[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-ethylcar-
bamoyl]-ethyl}-[1,2,3]triazol-1-yl)ethoxy]ethoxy}-ethoxy)-ethyl]-1H-
[1,2,3]triazol-4-yl}-propionamide} tetrachloride (19c)
N-[2-(1,4,7,10-tetraaza-cyclododec-1-yl)-ethyl]-3-{1-[2-(2-{2-
[2-(4-{2-[2(1,4,7,10-tetraaza-cyclododec-1-yl)-ethylcarbamoyl]-
ethyl}-[1,2,3]triazol-1-yl)ethoxy]ethoxy}-ethoxy)-ethyl]-1H-[1,2,
3]triazol-4-yl}-propionamide 26 (68.0 mg, 0.07 mmol) and cop-
per(II)-chloride (18.6 mg, 0.14 mmol) were dissolved separately
in a small amount of water and then dropped simultaneously in
2 mL of warm water while stirring resulting in a deep blue solu-
tion. The pH was adjusted to 7–8 with aqueous lithium hydroxide
(1 M). After 7 h of reflux, the mixture was cooled down to ꢄ5 °C in
the fridge. The solvent was evaporated and subsequently lyophi-
lized to obtain the product (68 mg, 89%) as a dark green solid.
MP: 126 °C – ESI-MS (H2O/MeOH + 10 mmol/L NH4Ac): m/z
(%) = 479.3 (95) [M + 2 H+]2+, 497.3 (35) [M + 3 H+ + Clꢀ]2+, 516.3
(19) [M + 4 H+ + 2 Clꢀ]2+ – Molecular Formula: C38H74N16O5Cu2
4+
– Molecular Weight: 960.4 g/mol.

44
F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
Fig. 1. Solution structure of Zn2+-cyclen complexed to Ras.Mg2+ GppNHp in conformational state 1. The solution structure of Ras complexed with Zn2+-cyclen was taken from
Rosnizeck et al. [16] Zn2+-cyclen is depicted in white. The second binding site of Zn2+-cyclen is indicated in red, the amino acids of Ras interacting in the crystal structure of
the Ras–SOS complex with the SOS-pentapeptide from amino acid 822–826 (Leu-Lys-Met-Ile-Arg) [18] are labeled in green. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
H
H
N
Zn2+
N
O
O
O
O
N
H
N
H
H
O
N
N
N
N
H
O
N
N
H
NH2
NH
H
H
n
O
O
O
2 Cl-
1a:
1b:
n = 2
n = 3
H2N
NH
Fig. 2. Zn2+-cyclen–peptide hybrid ligands 1 for Ras binding.
a
O
O
b
O
OH
OTs
HO
HO
HO
N3
n
n
n
2a: n = 2
2b: n = 3
3a: n = 2, 96%
3b: n = 3, 81%
4a: n = 2, quant.
4b:
n = 3, 89%
c
d
Boc
O
O
NH2
HO
N
n
HO
HO
n
H
5a: n = 2, 94%
6a: n = 2, 68%
5b:
6b:
n = 3, 96%
n = 3, 80%
Boc
R
O
f
HO
O
e
O
N
O
N
n
n
H
H
O
O
7a: n = 2, 68%
7b:
8a: n = 2, quant.
8b: n = 3, quant.
n = 3, 93%
R = H
g
9a:
9b:
n = 2, 64%
n = 3, 46% R = Fmoc
Scheme 1. Synthesis of Fmoc-protected amino-PEG-acid spacers 9. (a) TsCl, NaOH; b) NaN3, DMF; (c) Pd/C, H2, THF; (d) Boc2O, DCM; e) 2-bromoacetic acid, NaOH, THF/
toluene; (f) HClaq. (ꢄ18%); (g) Fmoc-succinimide, K2CO3, water.
spacers 9 and finally, compound 13 was N-terminally added to
yield the resin bound peptide-(Boc)3cyclen conjugate 16
(Scheme 3). TFA treatment cleaved the peptide-(Boc)3cyclen conju-
gate from the resin and removed cyclen and the arginine side-
chain protecting groups. Compounds 17a and b were purified by
HPLC.
As an example Fig. 3 shows the results obtained for compound
1b. Clearly the largest STD enhancement factor ASTD is observed
for the cyclen moiety and a lower value for the peptide moiety.
This suggests that Zn2+-cyclen is involved in a direct interaction,
but that the linker is too short in compound 1b to allow for a direct
interaction of the peptide with Ras. In line with this observation,
STD titration experiments show that the affinity of these com-
pounds is not increased relative to Zn2+-cyclen alone.
Compounds 1a and b were characterized by STD-experiments.
All compounds show a positive STD effect and thus bind to Ras.

F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
45
Scheme 2. Synthesis of cyclen carboxylic acid 13. (a) Boc2O, CHCl3; (b) methyl bromoacetate, K2CO3, MeCN; (c) LiOH, acetone/water.
O
O
O
H
H
a, b
N
N
NH2
MBHA N
H
N
H
N
H
MBHA NH2
peptide
O
O
coupling
cycle (5x):
14
HN
Pbf
N
NH
H
O
O
O
H
N
H
H
c, d
N
N
O
MBHA N
N
H
N
O
NH2
H
H
n
O
O
O
15a:
n = 2
HN
15b: n = 3
Pbf
N
NH
O
H
Boc
Boc
N
N
O
O
O
H
N
H
N
H
N
e
O
N
N
MBHA N
H
N
H
N
H
O
N
n
H
Boc
O
O
O
HN
16a:
n = 2
16b: n = 3
Pbf
N
H
NH
H
H
N
N
O
O
O
O
H
H
H
f
N
N
N
O
N
N
H2N
HN
N
N
O
N
H
H
n H
H
O
O
O
17a: n = 2
17b: n = 3
5 TFA
H2N
NH
H
H
N
N
O
O
O
O
Zn2+
N
g
H
H
N
H
O
N
N
quant.
N
N
O
N
N
NH2
NH
H
n
H
H
H
O
O
O
2 Cl-
1a: n = 2
1b:
n = 3
HN
NH2
Scheme 3. SPPS synthesis of peptide-cyclen conjugates. (a) Fmoc-AA, TBTU, HOBt, DIPEA (2ꢃ); (b) piperidine (40%); (c) 9a/b, TBTU, HOBt, DIPEA (2ꢃ); (d) piperidine (40%);
(e) 13, TBTU, HOBt, DIPEA (2x); (f) TFA/TIS/water (90:5:5); (g) ZnCl2, water, pH 7–8.
3.3. PEG-linked bis(Zn2+-cyclen) complexes
21 was prepared adapting a published synthesis protocol for the
corresponding mono-tosylates [31].
Ethylene glycol based spacers with corresponding lengths were
azide functionalized on each terminus by substitution of a previ-
ously synthesized bis-tosylate with sodium azide following a liter-
ature procedure (Scheme 4) [33]. Tetra(ethylene glycol)-ditosylate
Alkyne cyclen 24 was prepared as reactant for a copper catalyzed
alkyne–azide [39,40] cycloaddition reaction (Scheme 5) with 22.
Therefore, N-2-aminoethyl-(Boc)3-cyclen (10-(2-aminoethyl)-1,
4,7,10-tetraazacyclododecan-1,4,7-tricarbonylic-acid-tri-tert-

46
F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
8.5
7.5
6.5
5.5
4.5
3.5
2.5
1.5 ppm
Fig. 3. STD experiment with the Zn2+-cyclen hybrid-ligand 1b in the presence of Ras. Off-resonance spectrum (upper trace) and STD spectrum (lower trace) of a sample
containing 50
M Ras(T35A)ꢁGppNHp in 40 mM Tris/HCl pH 7.4 and 10 mM MgCl2 and 4.84 mM compound 1b.
l
a
b
O
O
O
TsO
OTs
N3
N3
HO
OH
n
n
n
20:
21:
22:
n = 3, 80%
n = 3
n = 3, quant.
Scheme 4. Preparation of tetra(ethylene glycol)-diazide 22. (a) TsCl, KOH, THF; (b) NaN3, DMF.
Scheme 5. Synthesis of alkyne (Boc)3cyclen 24. (a) Boc2O, CHCl3; (b) bromoacetonitrile, K2CO3, MeCN; (c) Raney-Ni, H2, EtOH(NH3sat.); (d) 4-pentynoic acid, EDC, HOBt,
DIPEA.
plex formation under slightly basic conditions with zinc(II)-chloride
and copper(II)-chloride, gave complexes 18 and 19, respectively.
O
3
N
N
(Scheme 6).
N
Addition of the PEG-linked bis(M2+-cyclen) complexes
N
N
H
H
H
(M2+ = Zn2+, Cu2+) (compounds 18 and 19) led to a immediate pre-
cipitation of Ras(T35A)ꢁGppNHp present in a concentration of
H
H
N
N
N
N
N
M2+
N
N
M2+
N
50 lM). This effect is clearly due to a direct interaction with the
O
N
N
4 Cl-
H
protein, since compound 26 lacking the metal ions does not induce
a precipitation, but also does not bind Ras as indicated by the miss-
ing STD.
N
O
H
H
M
2+ = Zn2+
M2+ = Cu2+
19:
18:
Fig. 4. Bis(Zn2+-cyclen) complex 18 and bis(Cu2+-cyclen) complex 19.
4. Conclusions
We have reported two different types of artificial ligands for
state 1, the low affinity state for effectors of active Ras based on
the known Zn2+-cyclen binding motif. It was the intention to
strengthen the binding to the protein by additive or even coopera-
tive effects due to the introduction of a second binding site for Ras.
Hybrid peptide–(Zn2+-cyclen) ligands were synthesized on solid-
phase using Fmoc-based standard protocols for SPPS, whereas
symmetrical PEG-linked bis(Zn2+/Cu2+-cyclen) ligands were pre-
pared in solution using a twofold ‘‘click”-reaction as the key step
in the reaction sequence. Unfortunately, the peptide-(Zn2+-cyclen)
ligands did not show a significantly increased binding affinity to
Ras compared to the parent compound Zn2+-cyclen. The
butylester) 23 was synthesized in three steps from threefold
Boc protected cyclen 11 [38]. The free amino group was alkyl-
ated by bromoacetonitrile and hydrogenated to the corre-
sponding amine 23 [41]. Compound 23 was then reacted
under peptide coupling conditions with 4-pentynoic acid to
give N-10-(2-aminoethyl)-1,4,7,10-tetraazacyclododecan-1,4,7-tri-
carbonylicacid-tri-tert-butylester-pent-4-ynamide 24. The twofold
Cu(I)-catalyzed 1,3-dipolar cycloaddition of 22 and 24 worked best
using copper(II)sulfate/sodium ascorbate in aqueous methanolic
solution providing 25 in sufficient purity and good yield after filtra-
tion over flash silica gel. Acidic cleavage of the Boc groups and com-

F. Schmidt et al. / Inorganica Chimica Acta 365 (2011) 38–48
47
Boc
Boc
Boc
N
N
N
2
a
O
+
N3
N3
N
91%
3
N
H
O
24
22
O
3
N
N
N
N
N
Boc
Boc
Boc
H
N
Boc
N
N
N
N
N
N
N
O
N
N
Boc
N
H
25
O
Boc
O
3
N
N
N
N
N
N
H
H
H
H
H
N
N
N
N
b, c
quant.
N
N
N
O
N
N
H
N
26
O
H
H
O
3
N
N
4Cl-
N
N
N
N
H
H
H
H
N
H
N
N
N
M2+
N
d
N
N
M2+
O
N
N
H
N
H
O
H
18: M2+ = Zn2+, 92%
19: M2+ = Cu2+, 89%
Scheme 6. Click-reaction yielding sixfold Boc protected metal chelator and synthesis of PEG-linked bis(M2+-cyclen) complexes 18 and 19. (a) CuSO4, sodium ascorbate,
MeOH/water; (b) HCl-Et2O, DCM; (c) basic anion exchanger; (d) ZnCl2 or CuCl2, water, pH 7-8.
PEG-linked bis(Zn2+/Cu2+-cyclen) ligands precipitated the protein
immediately, thus preventing any spectroscopic studies. Most
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probably this effect is due to a cross-linking of Ras-proteins by
the bivalent ligand. Overall, rationally designed bivalent ligands
for Ras protein binding are synthetically accessible, but among
the so far investigated examples no increase in binding affinity
could be observed.
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