Protein-inorganic array construction: Design and synthesis of the building blocks

Article abstract of DOI:10.1002/chem.200902649

Herein we describe the design and synthesis of the first series of di-functional ligands for the directed construction of inorganic-protein frameworks. The synthesized ligands are composed of a metal-ion binding moiety (terpyridine-based) conjugated to an epoxysuccinyl peptide, known to covalently bind active cysteine proteases through the active-site cysteine. We explore and optimize two different conjugation chemistries between the di-functionalized metal-ion ligand and the epoxysuccinyl-containing peptide moiety: peptide-bond formation (with limited success) and Cu'-catalysed click chemistry (with good results). Further, the complexation of the synthesized ligands with Fe" and Ni^II" ions is investigated: the di-functional ligands are confirmed to behave similarly to the parent terpyridine. As designed, the peptidic moiety does not interfere with the complexation reaction, in spite of the presence of two triazole rings that result from the click reaction. ES-MS together with NMR and UV/Vis stud-ies establish the structure, the stoichiometry of the complexation reactions, as well as the conditions under which chemically sensitive peptide-containing polypyridine ligands can undergo the self-assembly process. These results establish the versatility of our approach and open the way to the synthesis of di-functional ligands containing more elaborated polypyridine ligands as well as affinity labels for different enzyme families. As such, this paper is the first step towards the construction of robust supramolecular species that cover a size-regime and organization level previously unexplored.

Full text of DOI:10.1002/chem.200902649

DOI: 10.1002/chem.200902649
Protein–Inorganic Array Construction:
Design and Synthesis of the Building Blocks
Niculina D. Bogdan,[a, c] Mihaela Matache,[a, b, c] Veronika M. Meier,[a] Cristian Dobrota˘,[a]
Ioana Dumitru,[a, b] Gheorghe D. Roiban,[a] and Daniel P. Funeriu*[a]
Abstract: Herein we describe the
design and synthesis of the first series
of di-functional ligands for the directed
chemistry (with good results). Further,
the complexation of the synthesized
ies establish the structure, the stoichi-
ometry of the complexation reactions,
as well as the conditions under which
chemically sensitive peptide-containing
polypyridine ligands can undergo the
self-assembly process. These results es-
tablish the versatility of our approach
and open the way to the synthesis of
di-functional ligands containing more
elaborated polypyridine ligands as well
as affinity labels for different enzyme
families. As such, this paper is the first
step towards the construction of robust
supramolecular species that cover a
size-regime and organization level pre-
viously unexplored.
ligACHTNUTGRNEUNG
ands with FeII and NiII ions is investi-
construction
of
inorganic-protein
gated: the di-functional ligands are
confirmed to behave similarly to the
parent terpyridine. As designed, the
peptidic moiety does not interfere with
the complexation reaction, in spite of
the presence of two triazole rings that
result from the click reaction. ES-MS
together with NMR and UV/Vis stud-
frameworks. The synthesized ligands
are composed of a metal-ion binding
moiety (terpyridine-based) conjugated
to an epoxysuccinyl peptide, known to
covalently bind active cysteine prote-
ACHTUNGTRENNUNGases through the active-site cysteine.
We explore and optimize two different
conjugation chemistries between the
di-functionalized metal-ion ligand and
the epoxysuccinyl-containing peptide
moiety: peptide-bond formation (with
limited success) and CuI-catalysed click
Keywords: click chemistry · inor-
ganic–protein frameworks
· pep-
tides · self-assembly · terpyridines
Introduction
envisioned to combine the intrinsic efficiency of the self-as-
sembly process with the efficiency and specificity of the con-
jugation of active enzymes to affinity labels. Such a process
would result in supramolecular species, bearing both inor-
ganic and proteic frameworks,[2] reaching sizes and complex-
ity levels beyond the size-regime currently reached by
metal-ion self-assembled molecules (usually bellow 5 nm).[3]
To do so, we based our approach on ligands containing
metal-ion binding units known for their complexation prop-
erties,[1c,4] conjugated with affinity labels that are known to
bind specifically and covalently a specific amino acid in the
active site of an active enzyme.[5]
We describe herein, as a first example in this series, the
synthesis of ligands containing terpyridine as metal-ion com-
plexing unit and epoxysuccinyl-based peptides as affinity
labels well validated for cysteine proteases (Figure 1). The
general principle along which we intend to use these ligands
in order to build hybrid, protein–inorganic supramolecular
structures is depicted in Figure 1.
Metal-ion-directed self-assembly of polydentate ligands has
generated a variety of metallosupramolecular entities bear-
ing original physical and chemical properties, with discrete
sizes and well-defined geometric shapes.[1] To further broad-
en the scope of the construction of discrete supramolecular
species through metal-ion self-assembly-based methods we
˘
[a] Dr. N. D. Bogdan, M. Matache, Dr. V. M. Meier, Dr. C. Dobrota,
I. Dumitru, Dr. G. D. Roiban, Dr. D. P. Funeriu
Department of Chemistry, Marie Curie Excellence Team
Technical University Munchen, 4 Lichtenberg Str.
85748 Garching (Germany)
Fax : (+49)89-28-914-512
E-mail: daniel.funeriu@ch.tum.de
[b] M. Matache, I. Dumitru
Department of Chemistry, University of Bucharest
90-92 Panduri Str., 050663 Bucharest (Romania)
[c] Dr. N. D. Bogdan, M. Matache
These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902649.
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FULL PAPER
Results and Discussion
The first series of ligands on which we focused our attention
contain 2,2’-6’,2’’-terpyridine (terpy) as one of the most en-
countered units in the construction of supramolecular sys-
tems. Beyond supramolecular chemistry, compounds derived
from this versatile heterocycle system found applications in
molecular biology,[6] photochemistry,[7] polymer science,[8]
enantioselective synthesis,[9] and pharmaceutical research.[10]
This makes terpyridine (possibly bearing various functional
groups) a building unit of choice in the context of potential-
ly bio-compatible functions of the final assemblies. For co-
AHCTUNGTREGvNNUN alent binding of the ligands to proteins with the efficiency
of an enzymatic reaction we used epoxysuccinyl-terminated
peptides, known to bind and covalently link (activity depen-
dent) cysteine proteases of the papain family.[11] We chose to
use a cysteine-protease affinity label, since some enzymes of
this family, such as papain and bromelain, can be obtained
in large quantities and are relatively easy to handle. There-
fore, as a strong papain binding unit we used the short pep-
tide sequence (YL), terminated by the chemical “war head”,
epoxide. As a linker between the terpyridine units and the
YL–epoxide part, we investigated two spacer variants with
different properties: 1) a hydrophobic chain consisting of 6-
aminohexanoic acid (in peptide 1a); and 2) a hydrophilic
Figure 1. General principle for the construction of protein–inorganic
hybrid compounds. An affinity-label compound is conjugated to a ligand
known for its self-assembly properties. Complexation to metal-ions capa-
ble of expressing the molecular information encoded into the structure of
the ligand yields a supramolecular complex decorated with affinity-label-
ing units. Their reaction with enzyme produces well organized protein–in-
organic hybrid compounds.
Abstract in German: Im Folgenden beschreiben wir das ge-
zielte Design und die Synthese di-funktionaler Liganden zum
erstmaligen Aufbau supramolekularer Metall-Protein-Hy-
bridarchitekturen. Die synthetisierten Liganden enthalten eine
Metallionen-Bindungsstelle (auf Terpyridin-Basis), die mit
einem Epoxysuccinyl-Peptid konjugiert wurde. Diese Peptide
binden bekannterweise an die aktiven Cysteine im katalyti-
schen Zentrum von Cystein-Proteasen. Wir untersuchen und
optimieren zwei verschiedene Arten chemischer Konjuga-
tions-Systeme zwischen den di-funktionalen Metallionen-Li-
ganden und dem Epoxysuccinyl-enthaltenden Peptidrest: Bil-
dung einer Peptid-Bindung (mit geringem Erfolg) und CuI-
katalysierte click Chemie (mit signifikantem Erfolg). Wie
beabsichtigt, erfolgt die Komplexierung von FeII- und NiII-
Ionen an den synthetisierten Liganden hochselektiv am Ter-
pyridylrest und nicht an den Triazol-Ringen des Peptidrests,
die aus der click Reaktion resultieren. Struktur, Stçchiometrie
der Komplexbildung und Bedingungen fꢀr den Selbstorgani-
sationsprozess der empfindlichen poly-Pyridyl-Peptid-Ligan-
den wurden durch ESI-MS-, NMR- und UV-VIS-Untersu-
chungen dokumentiert. Diese Ergebnisse demonstrieren die
Vielseitigkeit unseres neuartigen Ansatzes zur Synthese
maßgeschneiderter di-funktionaler Liganden mit poly-Pyrid-
yl-Resten und Affinity Label fꢀr unterschiedliche Enzymfa-
milien. Die Arbeit reprꢁsentiert somit den ersten Schritt in
der Entwicklung einer stabilen, supramolekularen Architek-
tur in bisher unerreichter Grçßenordnung und Organisations-
grad.
chain consisting of a tetraethylene glycol-based linker for
solubility and distance modulation reasons (in peptide 1b).
In order to find general conditions that allow a versatile syn-
thetic methodology we investigated two different coupling
chemistries for the conjugation between the metal-ion bind-
ing unit and the protein binding unit: amide bond forming
reactions for the amino-terminated epoxysucciACTHNUTRGENUGnN yl peptide
1a and click reactions for the azide-terminated epoxysuccin-
yl peptide 1b.
Terpyridine units 2a and 2b were synthesized through the
common intermediate diol 9 (Scheme 1). Optimized phase-
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D. P. Funeriu et al.
Scheme 2. Five-step synthesis of the polyethylene glycol spacer and
azide-decorated lysine: a) SOCl2, Py, CHCl3, 31%; b) NaN3, NH4Cl,
H2O, 73%; c) bromoacetic acid, aq NaOH 50%, TBAB, CH2Cl2, 76%;
d) H2, Pd/C, MeOH, 82%; e) Fmoc-Cl, NaHCO3, dioxane, H2O, 63%; f)
Fmoc-LysACTHUNTRGNE(UNG NH2)-OH, HBTU, HOBt, DIPEA, 55%.
Scheme 1. Synthesis of the decorated terpyridine motifs: a) i: POBr3
1708C, ii: MeOH, 90%; b) NaBH4, EtOH, 87%; c) DHP, PTSA, CHCl3,
81%; d) nBuLi, THF, À788C, (nBu)3SnCl, 75%; e) 2,6-dibromopyridine,
ed tetraethylene glycol amino acid 15 and, through standard
Fmoc protection conditions, compound 16. For the comple-
tion of the synthesis of 17, azide tetraethylene glycol 14 was
reacted with the side chain amine of Fmoc-protected lysine
to yield 17 in reasonable (55%) yield.
[PdACHTUNGTRENNUNG(PPh3)4], toluene, 54%; f) HCl, MeOH, 64%; g) for 10: tert-butyl bro-
moacetate, aq NaOH 50%, TBAB, CH2Cl2, 48%; for 2b propargyl bro-
mide, aq NaOH 50%, TBAB, CH2Cl2, 85%; h) 25% TFA in CH2Cl2,
85%.
With ligands 2a and 2b and peptides 1a and 1b in hand,
we proceeded to optimize the conditions of the coupling re-
actions between the carboxy-terminated terpyridine and
amino-terminated epoxysuccinyl peptide (Scheme 3). Amide
coupling of the amino-terminated affinity label 1a and the
carboxy-terminated ligand 2a was attempted by using differ-
ent activation conditions for the carboxylic groups. As stan-
dard coupling procedure for peptide synthesis we first used
stoichiometric amounts of activating reagents O-benzotria-
transfer reactions[12] with either tert-butyl bromoacetate or
propargyl bromide yielded (after TFA deprotection of the
tert-butyl group) the carboxy-functionalized terpyridine 2a
and the alkyne functionalized terpyridine 2b, respectively.
We found that the diol 9 was best synthesized through the
Pd-catalyzed coupling reaction[13] between the stannane 7
and 2,6-dibromopyridine followed by acid deprotection. This
procedure provided diol 9 in 64% isolated yield. In turn,
the stannane 7 was synthesized in four steps starting with
commercial hydroxynicotinic acid. Bromopyridine 4 was ob-
tained by melting at 1708C a mixture of neat hydroxynico-
tinic acid 3 with neat POBr3 and subsequent MeOH quench-
ing. NaBH4 reduction of 4 followed by THP protection of
zole-N,N,N’,N’-tetramethyluronium
(HBTU) and N-hydroxybenzotriazole (HOBt)) and three
equivalents of the tertiary base (N,N-diisopropylethylamine
hexafluorophosphate
AHCTUNGTRENNUNG
(DIPEA)) in anhydrous DMF under inert conditions. This
produced small amounts of the expected compound that
could be observed in a complex mixture of products as
judged from HPLC/MS data. Repeated attempts to synthe-
size the di-epoxide conjugates failed, despite using various
coupling reagents known to be highly efficient for amide
couplings (benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP), 2-(1H-7-azabenzotriazol-1-
the resulting alcohol 5 and the classical n-butyllithium/tri
butyl)tinchloride treatment yielded 7.
ACHTUNGERTN(NUNG n-
The affinity labels, 1a and 1b, used for the two coupling
chemistries investigated were synthesized by typical Fmoc-
peptide solid-phase synthesis using the appropriate spacer
units. Along with the commercially available amino acids
(lysine, tyrosine, leucine and 6-aminohexanoic acid), the
synthesis of 1a and 1b required the preparation of the epox-
ide unit and the azide-containing tetraethylene glycol spacer
17. Ethyl (2R,3R)-trans-2,3-epoxysuccinate was synthesized
starting from d-tartaric acid in a five-step synthesis as previ-
ously described.[14] Key compound 14 and the corresponding
Fmoc-protected amino acid 16 were synthesized starting
from tetraethylene glycol 11 (Scheme 2). Briefly, tetraethyl-
yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU)) as well as slightly modified peptides. Noticeably,
in the procedure using N,N’-diisopropylcarbodiimide (DIC)/
HOBt as activating agents only the mono-epoxide conjugate
compound 19 could be isolated in a modest yield (29%), to-
gether with unreacted starting materials. On the other hand,
exploration of the click reaction coupling chemistry, proved
to be very efficient for our particular purpose (for a detailed
study of the influence of polypyridine ligands on the click
reaction see work of Finn[16]). We employed click reaction
A
conditions using [CuACTHNGUTERNNU(G CH3CN)4]PF6 [tetrakis(acetonitrile)-
chloride and pyridine in chloroform[15] to obtain the mono-
chloro tetraethylene glycol 12. Substitution with sodium
azide and further reaction with bromoacetic acid under
phase-transfer conditions formed 14. Azide reduction yield-
copper(I) hexafluorophosphate] as catalyst, in degassed ace-
tonitrile under inert atmosphere. Due to strong affinity of
the terpyridine moiety towards copper ions, compound 2b
was first stirred for 30 min with one equivalent of the CuI
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Protein–Inorganic Arrays
FULL PAPER
Scheme 3. Amide coupling between the affinity label 1a and the carboxy-terminated terpyridine 2a yields: a) under HBTU, HOBt, DIPEA conditions:
low yield of di-epoxysuccinyl conjugated ligand 18 (traces), observed in HPLC-MS spectrum; or b) in DIC/HOBt conditions: mono-epoxysuccinyl conju-
gated ligand 19 in a modest yield (29%). Click reaction between the affinity label 1b and the terpyridine alkyne 2b yields (after decomplexation of the
[CuII
thane/acetonitrile, 10 h, 608C ([CuII
A
ACHTNUGTRNEN(NUG CH3CN)4]PF6, in dichlorome-
A
ACHTUNGTRENNUNG
salt.[17] Following addition of the azide–epoxysuccinyl pep-
tide, an extra 0.5 equivalents of copper(I) ions were added
and the reaction was allowed to stir overnight at 608C. The
reaction between the epoxysuccinyl peptide 1b and the di-
alkyne terpyridine 2b yielded the desired compound 20a as
a mixture of CuII complexes (copper(II) diterpyridine [CuII-
acid, a fact that is largely inconsequential to its reactivity to-
wards papain.[19] The structural characteristics of 20b were
confirmed by NMR spectroscopy (see Supporting Informa-
tion).
With compounds 19 and 20b in hand, we set to test a cru-
cial element of design in our overall strategy, namely to
ensure that there is no interference relative to the metal-ion
complexation between the metal-ion and protein-binding
centers. Indeed, both amide-bond formation chemistry and
click chemistry that we explored create, in close proximity
to the terpyridine unit, functionalities that are potentially
metal-ion complexing. Moreover, the importance of these
control studies is underlined by recent reports[20] of the com-
plexation of triazoles formed by click reactions with a varie-
ty of metal ions. With particular relevance to our work are
the complexation studies performed by the Schibli group[21]
on triazoles linked to biomolecules. In the light of these re-
sults, we set to demonstrate that the metal-ion complexing
ACHTUNGTRENNUNG ACHUTNGTREN(NGUN 20a)]) in a
(20a)2] and copper(II) monoterpyridine [CuII
ratio of 1:12 as inferred from the HPLC/ES-MS spectrum of
the crude of reaction (see Supporting Information). The
complexes were separated by preparative HPLC and charac-
terized as unique compounds. To obtain the free terpyri-
dine–di-epoxide, the copper(II) complex [CuII
ACTHUNTGRENNG(U 20a)] was
subjected to decomplexation by using a powerful chelating
agent namely HEEDTA (2-(carboxymethyl-{2-[carboxy-
methyl-(2-hydroxyethyl)amino]ethyl}amino) acetic acid).[18]
This yielded the metal-free elaborated affinity label 20b, pu-
rified by HPLC. It should be noted that during the decom-
plexation step the ester groups were hydrolyzed to the free
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D. P. Funeriu et al.
properties of the model terpyridine triazole 21 are similar to
those of terpyridine. Although, a priori, less susceptible of
complexation interference, besides the complexing proper-
ties of 21, we also studied the complexing properties of ter-
pyridine diacid 2a and terpyridine–di-alkyne 2b (see Sup-
porting Information). The simple triazole–terpyridine mole-
cule (21, Figure 2) was synthesized by reaction of the triple-
bond decorated terpyridine with a simple azide (N3-CH2-
COOtBu) in a manner analog to compounds 20a and 20b.
The obtained product 21 was characterized by UV/Vis and
NMR spectroscopy and ES-MS.
the formed complexes, we performed UV/Vis spectroscopy
titration experiments. In the case of the triazole-bearing ter-
pyridine ligand 21 complexation with FeII we also performed
1H NMR titration experiments in order to gather structural
information about the structure of the complex. The general
conclusion is that, as expected, all terpyridine-based ligands
form [MII
ACHTNUTRGNE(NUG terpy)2] complexes, thus validating our approach.
When treated with increasing concentrations of FeII, ligand
21 displayed two absorption maxima at 325 nm (band char-
acteristic for the conformational change in terpyridine from
trans/trans conformation in ligand to the planar cis/cis con-
formation in the complex) and at 554 nm (band assigned to
the metal–ligand charge transfer, MLCT) characteristic for
Terpyridine-based ligands are known to produce [MII-
ACHTUNGTRENNUNG(terpy)2] complexes with octahedral metal ions. In particu-
lar, we studied the complexes of those ligands with NiII and
FeII ions, because they complex terpyridine strongly and
with different kinetic properties,[22] the FeII being particular-
ly inert, whereas NiII complexes display a somewhat faster
exchange between ligands (as a rule of thumb, a 5.4 mm so-
ACTHNGUTERNNUG
[FeII(terpy)2] complexes.[22] The increase in absorbance
reached a plateau at a ratio of FeII/21=1/2, indicating that
the formed complex is composed of one FeII ion and two lig-
AHCTUNGTREGaNNUN nds 21. ES-MS of the characteristic violet-colored complex
displays a main peak at m/z=711.5, corresponding to the
molecular mass of [FeII(21)2]2+/2, a peak at m/z=1509.2 that
is due to the monocharged species [FeII(21)2BF4]+, as well
as lower mass fragments at m/z=683.6, 655.5, 627.5 and
599.6 respectively, corresponding to the successive loss of
lution of [NiII
ACHTUNGTRENNUNG(terpy)2] in acetonitrile requires 12 h at room
temperature to reach the exchange equilibrium). The
formed complexes were characterized by NMR and UV/Vis
spectroscopy, and ES-MS. To establish the stoichiometry of
1
Figure 2. a) UV/Vis titration of 21, 2a, and 2b, with FeII at 554 nm; b) H NMR spectra of free ligand 21 (top) and of the [FeII(21)2] complex (bottom);
c) ES-MS of the [FeII(21)2] complex. Further data (NMR, UV/Vis and ES-MS) corresponding to the complexation of 2a and 2b with FeII and NiII can be
found in the Supporting Information.
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Protein–Inorganic Arrays
FULL PAPER
the four tert-butyl groups from the ligands 21. The same
Conclusions
phenomenon could be observed in the case of the complex
[FeII
A
In conclusion we have developed an efficient method of syn-
thesizing nitrogen-based ligands decorated with affinity
labels. Although the initial attempts to couple the terpyri-
dine moiety with the peptide-based affinity label by classical
amide-bond formation chemistry yielded non-convincing re-
sults, a click reaction provided the desired ligand 20b as
well as, based on the new spacer 17, the possibility to easily
modulate the distance between the metal-ion complexing
unit and the affinity labeling unit. This last fact is of particu-
lar importance considering the steric constraints that are re-
lated to our goal of attaching several enzyme molecules to
one ligand. Setting the stage for the enzyme-reaction stud-
ies, we clearly established that the complexation of transi-
tion-metal ions, such as FeII, to ligand 21 does not interfere
with the chemical functionalities (i.e., epoxysuccinyl pep-
tides) introduced to covalently react in an activity-depen-
dent manner with enzymes (in our case cysteine proteases
of the papain family). Based on the synthetic methods de-
veloped herein we are currently expanding the repertoire of
ligands coupled with affinity labels as well as studying the
reaction of enzymes such as papain with the described lig-
ACHTUNGTRENNUNG
[FeII(21)2] complex in [D3]acetonitrile reveals several diag-
nostic characteristics. Noticeably, FeII complexation results
in downfield shifts of protons H3’/H5’ (Dd=0.37 ppm) and
H4’ (Dd=0.57 ppm), which belong to the central pyridine
ring, and a significant upfield shift (Dd=1.70 ppm) of the
protons H6/H6’’ next to the nitrogen atoms N1/N1’’ (Figure 2).
This behavior,[23] especially the significant upfield shift of
the H6/H6’’ protons, is characteristic for the formation of a
2:1 complex and is due to the ring current of the second,
perpendicularly placed terpyridine unit. This conclusion is
further reinforced by the observation[23a] that the absence of
the second terpyridine unit, in a 1:1 complex, results in a
downfield shift of those protons. Moreover, following the
FeII complexation of 21 we observe the upfield shift of the
triazole proton (Dd=0.17 ppm) as well as the upfield shift
of the protons that belong to the two methylene groups (a
and b, Figure 2) next to the terpyridine (Dd=0.19 and
0.33 ppm). This indicates that these protons are in close
proximity to the aromatic ring current (as would be the case
in a classical [FeII
G
ACHTUNGTNERaNUNG nds.
cal position of the triazole ring that excludes the nitrogen
1
complexation to a FeII ion. Additionally, H NMR titration
of 21 with FeII reveals that the complexation reaction is
complete at a molar ratio of FeII/21=1/2. Furthermore,
during the titration experiments we could only observe sig-
nals belonging to the uncomplexed terpyridine and the
[FeII(21)2] complex. Taken together, these data prove that
the presence of the triazole in close proximity to the terpyri-
dine does not affect the complexation of terpyridine with
FeII. Recent data[24] show that somewhat similar compounds
Experimental Section
General: All water-sensitive reactions were performed in anhydrous sol-
vents and under a positive pressure of argon. Dry tetrahydrofuran (THF)
and toluene were obtained by distillation under argon over sodium and
benzophenone, while dimethylformamide (DMF) was distilled over di-
phosphorus pentoxide under argon. All other solvents, reagents and
resins were purchased from commercial suppliers and used without fur-
ther purification. Melting points were determined in open capillary tubes
using an electric melting point STUART SMP3 apparatus and are uncor-
rected. The NMR spectra were recorded at room temperature on a
JEOL-Delta 400, Bruker DPX-400 or Bruker-360 spectrometers. Chemi-
cal shifts (d) are reported in parts per million (ppm) values using residual
solvent peak as internal reference. Multiplicities are abbreviated as fol-
lows: br broad; s singlet; d doublet; t triplet; q quadruplet; and m multi-
form [EuIII
ACHTUNGTRENNUNG(terpy)2] complexes, in which the nitrogen atoms
of a tetrazole ring close to terpyridine are involved in the
complexation of EuIII, a fact that is not surprising in the
light of the high coordination number of EuIII.
Based on these results we performed the complexation of
ligands 19 and 20b with FeII. The reaction mixtures were an-
alyzed by ES-MS and the complexes [FeII(19)2] as well as
AHCTUNGERTGpNNUN let. The mass spectra were obtained by electrospray ionization (ES)
technique using a Finnigan LCQ instrument. The UV-Vis spectra were
recorded on a Jasco V-630 spectrophotometer, using 10 mm quartz cell.
Elemental analyses were performed on a CHN Elementar Vario EL ap-
paratus. Thin-layer chromatography (TLC) was performed on aluminum
oxide 60 F254 neutral plates or silica-gel-coated aluminium F254 plates
from Merck. All plates were visualized by UV irradiation at 254 nm and/
or staining with potassium permanganate. Preparative column chroma-
tography was performed on Merck Aluminum oxide 60 [active basic (ac-
tivity I) 70–230 mesh] or silica Merck 230. Reverse-phase HPLC analyses
[FeII
ACHTUNGTRENNUNG(20b)2] were purified by preparative HPLC. The ES-MS
spectrum of FeII complex of 19 shows a main peak at m/z=
1095.9 corresponding to the double charged species
[FeII(19)2]2+. The ES-MS spectrum of FeII complex of ligand
20b is more complex and exhibits peaks corresponding to
triply charged species [FeII
G
,
[FeII
E
,
,
[FeII
G
,
[FeII
R
,
[FeII(20b)2NaK]3+
ACHTUNGTRENNUNG
and purifications of the peptidic products were carried out on
a
VARIAN Pro Star 210 system, equipped with UV detection, using an an-
alytical C18 VYDAC (300 ꢁ, 4.6 mmꢂ150 mm, 5 mm) or a preparative
C18 VYDAC (300 ꢁ, 20 mmꢂ250 mm, 10 mm) columns, respectively and
eluting with gradient mixtures of water (containing 0.1% TFA) and ace-
tonitrile. The retention time (Rt) is given in minutes with the gradient in
percentage of acetonitrile.
[FeII
A
,
E
(ClO4)]3+ at m/z:
1475.5, 1482.6, 1488.1, 1488.9, 1495.4, 1499.9 and 1507.4, re-
spectively, as well as quadruply charged species [FeII-
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
Methyl 6-bromo-nicotinate (4): Compound 3 (1 equiv, 10 g, 71.88 mmol)
was mixed in a mortar with POBr3 (2 equiv, 41.25 g, 143.77 mmol). The
mixture was transferred to a round-bottomed flask and heated to melting
(approx 1708C, for 10 min) using a heat gun until it became a yellow-red-
dish oil. The resulted oil solidified at cooling and was carefully dissolved
G
ACHTUNGTRENNUNG
Chem. Eur. J. 2010, 16, 2170 – 2180
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2175

D. P. Funeriu et al.
in MeOH. The solvent was removed under reduced pressure and the resi-
due was neutralized with NaHCO3 and subsequently extracted with
CH2Cl2. The organic phase was dried on MgSO4 and evaporated to give 4
as light yellow solid (14 g, 90%). Rf =0.63 (silica, AcOEt/pentane 4:1);
1H NMR (400 MHz, MeOD): d=8.16 (d, 4J=2.4 Hz, 1H; H2), 7.99 (dd,
3J=9.6 Hz, 4J=2.4 Hz, 1H; H4), 6.52 (d, 3J=9.6 Hz, 1H; H5), 3.85 ppm
(s, 3H; CH3).
sis calcd (%) for C27H31N3O4: C 70.26, H 6.77, N 9.10; found: C 69.95, H
6.79, N 8.98.
5,5’’-Bis(hydroxymethyl)-2,2’-6’,2’’terpyridine (9): Compound 8 (1 equiv,
100 mg, 0.34 mmol) was suspended in MeOH (20 mL) and 2 drops of
37% HCl were added. Reflux overnight followed by evaporation of the
solvent (to 5 mL) and addition of 10% NaHCO3 (5 mL) gave 9 as a
white precipitate that was filtered and washed with water (120 mg, 64%).
1H NMR (400 MHz, MeOD): d=8.64 (s, 2H; H6, H6’’), 8.59 (d, 3J=
8.0 Hz, 2H; H3, H3’’), 8.35 (d, 3J=8.0 Hz, 2H; H3’, H5’), 8.02 (t, 3J=
8.0 Hz, 1H; H4’), 7.96 (d, 3J=8.0 Hz, 2H; H4, H4’’), 4.74 ppm (s, 4H;
CH2OH).
2-Bromo-5-hydroxymethyl pyridine (5): Compound
4 (1 equiv, 5 g,
23 mmol) was dissolved in absolute ethanol (100 mL) and NaBH4
(12 equiv, 10.5 g, 276 mmol) was slowly added. The reaction was stirred
overnight at RT. The solvent was removed in vacuo and the reaction mix-
ture was treated with KOH (1m, 100 mL). Extraction with CH2Cl2 (8ꢂ
100 mL) afforded after drying over MgSO4, evaporation and flash chro-
matography pure alcohol 5 (3.8 g, 87%). Rf =0.45 (silica, AcOEt/pentane
2:1); 1H NMR (400 MHz, MeOD): d=8.89 (d, 4J=2.4 Hz, 1H; H6), 8.21
(dd, 3J=8.4 Hz, 3J=2.4 Hz, 1H; H4), 7.74 (d, 3J=8.4 Hz, 1H; H5),
3.94 ppm (s, 2H, CH2).
5,5’’-Bis{[(tert-butoxycarbonyl)methoxy]methyl}-2,2’-6’,2’’-terpyridine
(10): The diol 9 (1 equiv, 140 mg, 0.48 mmol) was suspended in dichloro-
methane (2 mL) and the resulting mixture was cooled to 108C. Aqueous
solution of NaOH 50% (24 equiv, 240 mg, 11.52 mmol) and tetra n-butyl-
AHCTUNGTREGaNNUN mmonium bromide (25 mol%) were added and the solution was al-
lowed to stir for 30 min at 108C. Bromo-tert-butylacetate (3 equiv,
294 mg, 1.44 mmol) was then added and the reaction was allowed to stir
overnight at RT. The reaction mixture was diluted with water and
CH2Cl2, and the two phases were separated. The organic phase was
washed with brine, dried on MgSO4, and evaporated. The residue was
subjected to chromatography on aluminium oxide to yield the pure white
compound 10 (120 mg, 48%). Rf =0.47 (alox, AcOEt/petroleum ether
1:3); m.p. 140–1428C; 1H NMR (400 MHz, CDCl3): d=8.67 (d, 4J=
2.4 Hz, 2H; H6, H6’’), 8.61 (d, 3J=8.8 Hz, 2H; H3, H3’’), 8.44 (d, 3J=
8.8 Hz, 2H; H3’, H5’), 7.94 (t, 3J=8.8 Hz, 1H; H4’), 7.92 (dd, 3J=8.8 Hz,
4J=2.4 Hz, 2H; H4, H4’’), 4.72 (s, 4H; CH2O), 4.05 (s, 4H;
OCH2COOtBu), 1.49 ppm (s, 18H; tBu); 13C NMR (100 MHz, CDCl3):
d=169.3, 155.9, 155.1, 148.6, 137.9, 136.7, 132.9, 121.0, 120.9, 81.8, 70.6,
67.9, 28.1 ppm; MS (ES+): m/z: 522.2 [M+H]+, 466.2 [MÀtBu+2H]+,
410.3 [MÀ2tBu+3H]+, 544.1 [M+Na]+; elemental analysis calcd (%)
for C29H35N3O6: C 66.78, H 6.76, N 8.06; found: C 66.94, H 6.52, N 7.81.
2-Bromo-5-{[(tetrahydro-2H-pyran-2-yl)oxy]methyl}pyridine (6): Com-
pound 5 (1 equiv, 2.5 g, 13.3 mmol) was suspended in CHCl3 (15 mL) and
3,4-dihydro-2H-pyran (DHP) (1.3 equiv, 1.58 mL, 17.3 mmol) was added.
A catalytic amount of p-TsOH (10 mol%) was also added to the reaction
mixture. The reaction was stirred overnight at reflux. The solvent was re-
moved in vacuo and the residue was subjected to column chromatogra-
phy to afford the pure compound 6 as an yellow oil (2.9 g, 81%). Rf =
0.68 (silica, AcOEt/pentane 1:1); 1H NMR (400 MHz, [D6]DMSO): d=
8.36 (d, 4J=2.4 Hz, 1H; H6), 7.73 (dd, 3J=8.0 Hz, 4J=2.4 Hz, 1H; H4),
7.64 (d, 3J=8.0 Hz, 1H; H5), 4.68–4.65 (overlapped peaks, 2H; CH2-O-
2
CH-O, CHH-OTHP), 4.46 (d, J=12.0 Hz, 1H; CHH-OTHP), 3.74 (ddd,
3
3
2
2J=10.4 Hz, J=5.2 Hz, J’=3.2 Hz, 1H; O-CH-O-CHH), 3.45 (ddd, J=
10.4 Hz, 3J=5.2 Hz, 3J’=5.2 Hz, 1H; O-CH-O-CHH), 1.70–1.63 (over-
lapped peaks, 2H; OTHP), 1.53–1.45 ppm (overlapped peaks, 6H;
OTHP).
2-[Tri
dine (7): nBuLi (1.12 equiv, 2.55 mL, 4.4 mmol) was added in dry THF
(10 mL) at À788C, under Ar atmosphere. The protected alcohol
G
5,5’’-Bis
AHCTUNGTRENNUNG
ACHTUNGERTN{NUNG [(carboxy)methoxy]methyl}-2,2’-6’,2’’-terpyridine (2a): The di-
6
acid in dichloromethane (10 mL) were stirred at RT overnight. The sol-
vent was then removed in vacuo and the reaction mixture was triturated
with ethyl ether. The precipitate was filtered and dried under reduced
(1 equiv, 1 g, 3.67 mmol) was added dropwise to this solution. The mix-
ture was allowed to stir for 45 min at À788C and nBu3SnCl (1.2 equiv,
1.2 mL, 4.41 mmol) was added. The reaction mixture was allowed to
warm to RT overnight and the solvent was removed in vacuo. The result-
ed residue was partitioned between CH2Cl2 (20 mL) and water (10 mL).
The aqueous phase was washed three times with CH2Cl2 (20 mL), then
the combined organic phases were dried on MgSO4 and evaporated. The
product 7 (1.6 g, 75%) was used in the next step without any further pu-
rification. 1H NMR (400 MHz, CDCl3): d=8.74 (s, 1H; H6), 7.51 (d, 3J=
7.6 Hz, 1H; H4), 7.39 (d, 3J=7.6 Hz, 1H; H5), 4.76 (d, 2J=12.0 Hz, 1H;
CHH-OTHP), 4.71 (t, 3J=3.6 Hz, 1H; CH2-O-CH-O), 4.47 (d, 2J=
12.0 Hz, 1H; CHH-OTHP), 3.90 (ddd, 2J=10.8 Hz, 3J=5.6 Hz, 3J’=
3.2 Hz, 1H; O-CH-O-CHH), 3.45 (ddd, 2J=10.8 Hz, 3J=5.6 Hz, 3J’=
4.8 Hz, 1H; O-CH-O-CHH), 1.8–1.46 (overlapped peaks, 18H; OTHP,
1
pressure to yield 2a (80 mg, 85%). M.p. 190–1928C; H NMR (400 MHz,
MeOD): d=8.74 (d, 4J=2.0 Hz 2H; H6, H6’’), 8.69 (d, 3J=8.0 Hz, 2H;
H3, H3’’), 8.49 (d, 3J=7.6 Hz, 2H; H3’, H5’), 8.11 (t, 3J=7.6 Hz, 1H; H4’),
8.05 (dd, 3J=8.0 Hz, 4J’=2.0 Hz, 2H; H4, H4’’), 4.77 (s, 4H; CH2O), 4.25
(s, 4H; OCH2COOH), 3.18 ppm (br, 1H; COOH); 13C NMR (100 MHz,
MeOD): d=173.7, 152.9, 152.3, 146.4, 142.3, 141.1, 138.5, 124.5, 124.2,
70.7, 68.7 ppm; UV/Vis (MeCN) lmax(e)=285 (16000), 242 nm
(15800 molÀ1 m3 cmÀ1); MS (ES+): m/z: 410.3 [M+H]+ ; elemental analy-
sis calcd (%) for C21H19N3O6: C 61.61, H 4.78, N 10.26; found: C 61.33, H
4.47, N 9.96.
5,5’’-Bis[(propynyloxy)methyl]-2,2’-6’,2’’-terpyridine (2b): The diol
9
(1 equiv, 140 mg, 0.48 mmol) was suspended in dichloromethane (2 mL)
and the resulted mixture was cooled to 108C. Aqueous solution of
NaOH 50% (24 equiv, 240 mg, 11.52 mmol,) and tetra-n-butylammonium
bromide (25 mol%) were added and the solution was allowed to stir for
30 min at 108C. Propargyl bromide (3 equiv, 168.5 mg, 1.44 mmol) was
then added, and the reaction was allowed to stir overnight at RT. The re-
action mixture was diluted with water and CH2Cl2, and the two phases
were separated. The organic phase was washed with brine, dried on
MgSO4, and evapotated. The residue was subjected to chromatography
on aluminium oxide to yield the pure white compound 2b (150 mg,
85%). M.p. 105–1068C; Rf =0.50 (alox, AcOEt/petroleum ether 1:3);
1H NMR (400 MHz, CDCl3): d=8.66 (s, 2H; H6, H6’’), 8.60 (d, 3J=
8.8 Hz, 2H; H3, H3’’), 8.44 (d, 3J=8.8 Hz, 2H; H3’, H5’), 7.95 (t, 3J=
8.8 Hz, 1H; H4’), 7.85 (d, 3J=8.8 Hz, 2H; H4, H4’’), 4.71 (s, 4H; CH2O),
4.24 (d, 4J=2.4 Hz, 4H; OCH2), 2.51 ppm (t, 4J=2.4 Hz, 2H; CH);
13C NMR (100 MHz, CDCl3): d=155.9, 155.1, 148.8, 137.9, 136.6, 132.9,
121.1, 120.9, 79.2, 75.1, 68.8, 57.4 ppm; UV/Vis (MeCN) lmax(e)=285
(18500), 242 nm (18000 molÀ1 m3 cmÀ1); MS (ES+): m/z: 370.3 [M+H]+,
185.8 [M+2H]2+, 392.2 [M+Na]+; elemental analysis calcd (%) for
C23H19N3O2: C 74.78, H 5.18, N 11.37; found: C 74.97, H 5.41, N 11.04.
3
3
nBu), 1.32 (q, J=7.2 Hz, 6H; Bn), 0.9 ppm (t, J=7.2 Hz, 9H; nBu).
5,5’’-Bis{[(tetrahydro-2H-pyran-2-yl)oxy]methyl}-2,2’-6’,2’’-terpyridine
(8): Dry toluene (50 mL) was added to a mixture of 2,6-dibromopyridine
(1 equiv, 1.5 g, 6.3 mmol), stannane 7 (3 equiv, 9.5 g, 18.9 mmol) and tet-
rakis(triphenylphosphine)palladium(0) (1.8 g, 1.5 mmol, 24 mol%) under
Ar atmosphere, and the mixture was refluxed overnight. The reaction
was evaporated and treated with a mixture of AcOEt (50 mL) and con-
centrated aqueous KF (15 mL) under vigurous stirring for an hour. The
white precipitate that formed was filtered and the biphasic system was
separated. The organic layer was evaporated and subjected to chromatog-
raphy (alox, CHCl3/pentane 7:3) to afford 1.56 g (54%) of pure com-
pound 8. 1H NMR (200 MHz, CDCl3): d=8.68 (d, 4J=1.6 Hz, 2H; H6,
H6’’), 8.60 (d, 3J=8.2 Hz, 2H; H3, H3’’), 8.43 (d, 3J=8.0 Hz, 2H; H3’, H5’),
7.94 (t, 3J=7.8 Hz, 1H; H4’), 7.86 (d, 3J=8.2 Hz, 4J=1.6 Hz, 2H; H4,
H4’’), 4.88 (d, 2J=13.5 Hz, 2H; CHH-OTHP), 4.75 (t, 3J=3.3 Hz, 2H;
CH2-O-CH-O), 4.61 (d, 2J=13.5 Hz, 2H; CHH-OTHP), 4.01–3.91 (m,
2H; OTHP), 3.68–3.60 (m, 2H; OTHP), 1.76–1.68 ppm (m, 12H;
OTHP); 13C NMR (50 MHz, CDCl3): d=155.3, 155.0, 148.0, 137.6, 136.1,
133.7, 120.7, 120.6, 97.7, 66.0, 62.0, 30.3, 25.2, 19.0 ppm; elemental analy-
2176
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 2170 – 2180

Protein–Inorganic Arrays
FULL PAPER
5,5’’-Bis[({[(1-tert-butoxycarbonyl)methyl]-1H-1,2,3-triazol-4-yl}meth-
ACHTUNGTRENNUNGoxy)methyl]-2,2’-6’,2’’-terpyridine (21): [CuACHTUNGTRNEN(GUN CH3CN)4]PF6 (1 equiv, 40 mg,
was extracted with ethyl acetate (5ꢂ50 mL). The combined organic
phases were washed with brine, dried on MgSO4, and evaporated. The
residue was subjected to chromatography on silica gel (DCM/MeOH 9:1)
to yield 14 as colorless oil (76%). Rf =0.42 (silica, CH2Cl2/MeOH 9:1);
1H NMR (400 MHz, CDCl3): d=5.38 (brs, 1H; COOH), 3.94 (s, 2H;
H2), 3.70–3.60 (overlapped peaks, 14H; H4, H5, H7, H8, H10, H11, H13),
3.38 ppm (t, 3J=5.2 Hz, 2H; H14); 13C NMR (100 MHz, CDCl3): d=
172.5, 70.5, 70.3, 70.2, 70.12, 70.08, 70.0, 69.6, 68.4, 50.3 ppm; MS (ES+):
m/z: 276.8 [M+H]+.
0.108 mmol) was dissolved in degassed acetonitrile (1 mL) and added,
under argon, to a solution of compound 1b (39.8 mg, 0.108 mmol) in de-
gassed acetonitrile (1 mL). The reaction mixture was allowed to stir for
30 min at RT. Upon addition of the copper(I) salt, the solution turned
immediately to dark red, color characteristic for copper(I) complexes of
terpyridine ligands. Azide (tert-butyl-2-azidoacetate, 2 equiv, 61.8 mg,
0.432 mmol) and copper(I) catalyst [CuACHTUNRTGNE(UNG CH3CN)4]PF6, (0.5 equiv,
20.1 mg, 0.56 mmol) were added to this solution containing the copper(I)
complex of 2b, generated in situ. The reaction mixture was heated over-
night at 608C. After cooling at RT, aqueous solution of hydroxy-2-
14-Amino-3,6,9,12-tetraoxa-1-tetradecanoic acid (15): Compound 14
(6.5 g, 23.5 mmol) was dissolved in methanol (25 mL), flushed with nitro-
gen, and Pd/C (10 mol%, 10% w/w) was added. Hydrogen filled balloons
were used as hydrogen provider until completion of the reaction as moni-
tored by TLC (CH2Cl2/MeOH 1:1). The reaction mixture was then fil-
tered over Celite and the solvent removed in vacuo. The residue was sub-
jected to column chromatography on silica gel (CH2Cl2/MeOH 1:1) to
yield amino acid 15 as colorless oil (4.6 g, 82%). Rf =0.15 (silica, CH2Cl2/
MeOH 1:1); 1H NMR (400 MHz, CDCl3): d=3.92 (s, 2H; CH2COOH),
3.79 (t, 3J=4.4 Hz, 2H; H13), 3.67–3.60 (overlapped peaks, 12H; H4, H5,
ethyleneACHTUNGTRENNUNGdiaminetriacetic acid (HEEDTA) 50% w/w (1 mL) was added
and the mixture was stirred at RT for 1 h. The two phases were separat-
ed, and the organic phase evaporated in vacuo. The residue was parti-
tioned between water (10 mL) and CH2Cl2 (20 mL). The organic phase
was washed with water (10 mL) and brine (10 mL), dried over MgSO4,
and evaporated. The crude product was further purified by flash chroma-
tography to yield 21 as a white powder (53 mg, 72%). Rf =0.23 (alox,
AcOEt/petroleum ether 1:1); m.p. 126–1288C; 1H NMR (400 MHz,
3
H7, H8, H10, H11), 3.13 ppm (t, J=4.4 Hz, 2H; H14); 13C NMR (100 MHz,
3
CD3CN): d=8.63 (s, 2H; H6, H6’’), 8.60 (d, J=8.4 Hz, 2H; H3, H3’’), 8.43
CDCl3): d=175.6, 71.0, 70.1, 69.59, 69.57, 69.47, 69.35, 69.2, 67.3,
39.3 ppm; MS (ES+): m/z: 252.3 [M+H]+, 274.3 [M+Na]+, 296.3 [M-
H+2Na]+.
(d, 3J=7.6 Hz, 2H; H3’, H5’), 7.99 (t, 3J=7.6 Hz, 1H; H4’), 7.88 (d, 3J=
8.4 Hz, 2H; H4, H4’’), 7.84 (s, 2H; CHtriazole), 5.09 (s, 4H; N-CH2), 4.70 (s,
4H; CH2O), 4.66 (s, 4H; OCH2), 1.46 ppm (s, 18H; tBu); 13C NMR
(100 MHz, CDCl3): d=167.0, 156.1, 149.6, 145.4, 139.1, 137.5, 135.3,
125.6, 121.6, 121.4, 118.2, 83.7, 70.0, 64.3, 52.2, 28.1 ppm; UV/Vis
(MeCN) lmax(e)=285 (24500), 242 nm (23600 molÀ1 m3 cmÀ1); MS (ES+):
m/z: 684.4 [M+H]+, 706.3 [M+Na]+, 628.4 [MÀtBu+H]+, 572.5
[MÀ2tBu+H]+; elemental analysis calcd (%) for C35H41N9O6: C 61.48, H
6.04, N 18.44; found: C 61.76, H 6.42, N 18.45.
14-N-{[(9’H-Fluoren-9’-yl)methoxy]carbonylamino}-3,6,9,12-tetraoxa-1-
tetradecanoic acid (16): Compound 15 (1 equiv, 5 g, 21 mmol) was dis-
solved in a mixture of water/dioxane (1:1, v/v) and the pH was brought
to about 8 using solid sodium bicarbonate (1.5 equiv, 2.65 g, 31.5 mmol).
The mixture was then cooled to 08C and
a
solution of
fluorenylmethyloxycarbonyl chloride (Fmoc-Cl, 1.2 equiv, 6.5 g,
AHCTUNGTRENNUNG
25.2 mmol,) in 1,4-dioxane (10 mL) was added dropwise. The reaction
was allowed to stir at RT overnight. The solvent was evaporated in vacuo
and the remained aqueous phase was washed with ethyl acetate and
acidified with HCl (1n). Extraction with ethyl acetate (5ꢂ30 mL) was
followed by drying of the combined organic phases over MgSO4 and
evaporation of the solvent. The residue was subjected to chromatography
on silica gel (CH2Cl2/MeOH 9:1) to yield Fmoc-protected amino acid 16
as colorless oil (6.3 g, 63%). The compound was azeotroped with toluene
prior to use in SPPS. Rf =0.21 (silica, CH2Cl2/MeOH 9:1); 1H NMR
11-Chloro-3,6,9-trioxa-1-undecanol (12): Tetraethylene glycol 11 (1 equiv,
50 g, 0.25 mol) was dissolved in chloroform (50 mL). Pyridine (1 equiv,
19.75 g, 0.25 mol) and thionyl chloride (1 equiv, 19 mL, 0.25 mol) were
added to this solution, and the mixture was refluxed overnight. The sol-
vent was removed in vacuo and the residue was partitioned between
water (30 mL) and dichloromethane (100 mL). The two phases were sep-
arated; the organic phase was dried over MgSO4 and evaporated. The re-
sulted oil was distilled under reduced pressure (100 mbar). The fraction
between 110–1158C was then subjected to chromatography on silica gel
(AcOEt) to yield 12 as colorless oil (16.4 g, 31%). Rf =0.35 (silica,
AcOEt/petroleum ether 1:2); 1H NMR (360 MHz, CDCl3): d=3.84–3.67
(overlapped peaks, 4H; H10, H11), 3.66–3.64 (overlapped peaks, 8H; H4,
H5, H7, H8), 3.61–3.57 ppm (overlapped peaks, 4H; H1, H2); 13C NMR
(90 MHz, CDCl3): d=72.4, 71.3, 70.6, 70.5, 70.4, 70.2, 61.6, 42.6 ppm; MS
(ES+): m/z: 213.1 [M+H]+, 235.2 [M+Na]+.
3
3
(400 MHz, CDCl3): d=7.76 (d, J=7.6 Hz, 2H; HAr), 7.61 (d, J=7.6 Hz,
2H; HAr), 7.39 (t, 3J=7.6 Hz, 2H; HAr), 7.31 (t, 3J=7.6 Hz, 2H; HAr.),
CHTUNGTRENNUNG
5.53 (br, 1H; NH), 4.40 (d, 3J=7.2 Hz, 2H; CH (Fmoc)), 4.22 (t, 3J=
14H; H4, H5, H7, H8, H10, H11, H13), 3.39 ppm (q (overlapped td), 3J=
9.6 Hz, 3J=4.8 Hz, 2H; H14); 13C NMR (100 MHz, CDCl3) d=175.8,
159.0, 145.4, 142.7, 128.8, 128.2, 126.2, 120.9, 72.1, 71.9, 71.5, 71.4, 71.2,
71.1, 70.7, 67.6, 58.4, 41.7 ppm; MS (ES+): m/z: 473.3 [M+H]+, 496.4
[M+Na]+.
11-Azido-3,6,9-trioxa-1-undecanol (13): Compound 12 (1 equiv, 9.5 g,
45 mmol) was dissolved in water (10 mL). Sodium azide (10 equiv,
29.25 g, 450 mmol) and NH4Cl (12 equiv, 28.9 g, 540 mmol) were added
to this solution and the mixture was heated to 60–808C, until complete
conversion of the chloro-alcohol 12, as monitored by 13C NMR spectros-
copy. The reaction mixture was then diluted with water and the product
was extracted with ethyl acetate (7ꢂ50 mL). The product 13 (colorless
oil) was used in the next step without any further purification (73%).
1H NMR (360 MHz, CDCl3): d=3.71 (overlapped peaks, 3H; H10, OH),
3.67–3.65 (overlapped peaks, 10H; H2, H4, H5, H7, H8), 3.60 (dt, 3J=
N-a-{[(9’H-Fluoren-9’-yl)methoxy]carbonyl}-N-e-[1’’-azido-14’’-oxo-
3’’,6’’,9’’,12’’-tetraoxatetradecan]-l-lysine (17): Compound 14 (1 equiv,
400 mg, 1.3 mmol) was dissolved in anhydrous DMF, under argon, and
HBTU (1 equiv, 544 mg, 1.3 mmol), HOBt (1 equiv, 194 mg, 1.3 mmol)
and DIPEA (3 equiv, 0.70 mL, 3.9 mmol) were added and allowed to stir
at RT for 10 min. Fmoc-Lys-COOH was then added (1.2 equiv, 590 mg,
1.56 mmol) and the reaction was allowed to stir overnight. The solvent
was removed in vacuo and the product was purified by column chroma-
tography (CH2Cl2/MeOH 9:1) to yield 17 as colorless oil (448 mg, 55%).
Rf =0.22 (silica, CH2Cl2/MeOH 9:1); 1H NMR (400 MHz, CDCl3): d=
7.74 (d, 3J=7.2 Hz, 2H; HAr), 7.58 (d, 3J=7.2 Hz, 2H; HAr), 7.38 (t, 3J=
7.2 Hz, 2H; HAr), 7.29 (t, 3J=7.2 Hz, 2H; HAr), 7.08 (br, 1H; NH), 5.77
3
3
5.0 Hz, J=1.8 Hz, 2H; H1), 3.38 ppm (t, J=5.0 Hz, 2H; H11); 13C NMR
(90 MHz, CDCl3): d=72.5, 70.7, 70.6, 70.5, 70.3, 70.0, 61.7, 50.6 ppm; MS
(ES+): m/z: 220.1 [M+H]+, 242.2 [M+Na]+.
14-Azido-3,6,9,12-tetraoxa-1-tetradecanoic acid (14): Compound 13
(1 equiv, 7 g, 32 mmol,) was dissolved in dichloromethane (10 mL) and
the solution was cooled to 108C. Aqueous solution of NaOH 50%
(12 equiv, 12 mmol) and tetra-n-butylammonium bromide (25 mol%)
were added and the solution was allowed to stir for 30 min at 108C. Bro-
moacetic acid (1.5 equiv, 6.6 g, 48 mmol) was then added, and the reac-
tion was allowed to stir overnight at RT. The reaction mixture was dilut-
ed with water, and three washings with dichloromethane (20 mL) fol-
lowed by two washings with ethyl acetate (20 mL) were performed. The
aqueous solution was acidified with concentrated HCl and the product
(br, 1H; NH), 4.35 [d, 3J=6.4 Hz, 2H; CH
(Fmoc)], 4.19 [t, 3J=6.4 Hz,
2ACHTUNGTRENNUNG
1H; CH(Fmoc)], 3.97 (s, 2H; H13), 3.71–3.61 (overlapped peaks, 15H;
AHCTUNGTRENNUNG
H2, H4, H5, H7, H8, H10, H11, H20), 3.37 (t, 3J=5.2 Hz, 2H; H1), 3.16 (td,
3J=7.2 Hz, 3J’=2 Hz, 2H; H16), 1.82–1.40 ppm (overlapped peaks, 6H;
H17, H18, H19); 13C NMR (100 MHz, CDCl3): d=174.9, 170.9, 161.9, 143.8,
141.2, 127.7, 127.1, 125.1, 119.9, 70.4, 70.3, 70.1, 69.85, 69.7, 69.5, 55.5,
50.5, 47.1, 43.5, 27.4, 31.5, 18.4, 17.0 ppm; MS (ES+): m/z: 628.2
AHCTUNGTRENNUNG
[M+H]+.
Chem. Eur. J. 2010, 16, 2170 – 2180
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2177

D. P. Funeriu et al.
Solid-phase synthesis of the amino-terminated peptide H2N-Lys-Ahx-Tyr-
Leu-epoxide 1a: Solid-phase peptide synthesis was performed on Rink
amide MBHA (100 mg, loading 0.72 mmolgÀ1). Unless otherwise stated
the subsequent building blocks were introduced using Fmoc-amino acid/
HBTU/HOBt/DIPEA (4:4:4:12 equiv) with respect to the loading report-
ed by the supplier) in DMF (approximately 0.2m) for 1–2 h at RT. Fmoc
protecting groups were removed with piperidine/DMF 1:4 (2ꢂ10 min).
Ethyl (2R,3R)-trans-2,3-epoxysuccinate (2 equiv) was coupled with DIC/
HOBt (2:2 equiv) in DMF and the reaction shaken for 1 h. The peptide
was cleaved from the resin using a cleavage cocktail (1 mL; 95% TFA,
2.5% water, 2.5% triisopropylsilane) and allowed to shake for 2 h. Ice-
cold diethyl ether (15 mL) was used to precipitate the product. The resin
was washed with TFA (1.5 mL) and the solution was collected separately
in ice-cold diethyl ether. The solid was centrifuged, separated from dieth-
yl ether and dissolved in a minimal amount of acetonitrile–water (0.1%
TFA). The product was analyzed on a C18 reverse phase HPLC column
using a linear gradient of 15–90% water (0.1% TFA)/acetonitrile and pu-
rified on a corresponding preparative C18 reverse-phase HPLC column,
using the same gradient. Fractions containing the product were pooled,
frozen and lyophilized to obtain 1a as a white powder (31 mg, 63%).
Rt =8.62 min (RP-C18, 15–90% water (0.1% TFA)/acetonitrile); MS
(ES+): m/z calcd for for C33H52N6O9 [M+H]+: 677.38; found: 677.4
[M+H]+, 700.5 [M+Na]+, 810.2 [M+Na+TFA]+, 829.0 [M+K+TFA]+.
1b (1.18 equiv, 6.2 mg, 6.64 mmol) and copper(I) catalyst [Cu-
AHCTNUGTREN(GUNN CH3CN)4]PF6, (0.5 equiv, 0.5 mg, 1.4 mmol) were added to the solution
containing the copper(I) complex of 2b, generated in situ. The reaction
mixture was allowed to stir at 608C until complete conversion of the cop-
per(I) complex of 2b (10 h), as monitored by HPLC. The solvent was re-
moved in vacuo and the crude product was purified by preparative
HPLC on a C18 reverse phase column using a linear gradient of water
(0.1% TFA)/acetonitrile.
Complex [CuII
(RP-C18, 25–50% water (0.1% TFA)/acetonitrile); MS (ES+): m/z calcd
for [CuII
(20a)]=[C111H159CuN19O30] 2303.1; found m/z: 1152.0 [(20a)+
CuII]2+, 1162.9 [(20a)+CuII +Na]2+, 1228.5 [(20a)+CuII +H+(PF6)]2+
762.9 [(20a)+CuII +2H]3+
Complex [CuII
(20a)2][PF6]2: Green powder (0.8 mg, 6%); Rt =7.56 min
(RP-C18, 25–50% water (0.1% TFA)/acetonitrile); MS (ES+): m/z calcd
for [CuII
(20a)2]=[C222H318CuO60N38] 4542.6; found m/z: 1137.9 [(20a)2 +
CuII +4H]4+, 1148.6 [(20a)2 +CuII +2H+2Na]4+, 1156.8 [(20a)2 +CuII +
2K+H]4+, 766.7 [(20a)2 +Cu CuII +Na+K+2H]6+
Decomplexation of [CuII
(20a)][PF6]2: HEEDTA (100 mL, of 5% solution
ACHTUNGTREN(NUG 20a)]CAHTUTGNRNEN[GUN PF6]2: Green powder (5.3 mg, 79%); Rt =10.09 min
AHCTUNGTRENNUNG
,
.
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
.
A
ACHTUNGTRENNUNG
w/w) was added to a solution of complex [CuII(20)] in acetonitrile/water
(0.72 mm, 900 mL, 2:8) and the reaction was monitored by HPLC. Com-
plete decomplexation was achieved in 30 min. at RT. The reaction was
quenched with a solution of 5% TFA in water (10 mL). Without any
workup, the reaction mixture was immediately purified by preparative
HPLC on a C18 reverse phase column using a linear gradient of water
(0.1% TFA)/acetonitrile to yield the pure white compound 20 (92%).
Rt =10.6 min (RP-C18, 15–60% water (0.1% TFA)/acetonitrile);
1H NMR (400 MHz, MeOD/D2O=8:2): d=8.72 (s, 2H; H6, H6’’), 8.65 (d,
3J=7.6 Hz, 2H; H3, H3’’), 8.44 (d, 3J=8.0 Hz, 2H; H3’, H5’), 8.15–8.00
(overlapped peaks, 5H; H4’, H4, H4’’, CHtriazole), 7.04 (d, 3J=8.4 Hz, 2H;
Solid-phase synthesis of the azide-terminated peptide Lys
ACHTUNGRTENUN(NG TegN3)-Ahx-
Tyr-Leu-epoxide 1b
Procedure A: Peptide 1b was synthesized on of Rink amide MBHA
(100 mg, loading 0.72 mmolg)À1, as described for peptide 1a. We used as
first amino acid commercial Fmoc-LysACTHNUGTRNEUNG(Mtt)-OH and the corresponding
amount of coupling reagents. Prior to the N-terminal Fmoc group depro-
tection, the Mtt group was deprotected using a solution 1% TFA in
DCM (2 mL, 2 minꢂ7) and compound 14 was coupled with HBTU,
HOBt, DIPEA (2:2:2:4 equiv) for 2 h. The synthesis was further per-
formed as described for peptide 1a; (36 mg, 53%).
H
ArÀTyr), 7.02 (d, 3J=8.4 Hz, 2H; HArÀTyr), 6.71 (d, 3J=8.4 Hz, 2H;
HArÀTyr), 6.69 (d, 3J=8.4 Hz, 2H; HArÀTyr), 5.0–0.8 (overlapped peaks,
126H); MS (ES+): m/z calcd for C111H159N19O30: 2183.5; found: 1093.4
[M+2H]2+
,
1104.3 [M+Na+H]2+
,
1112.4 [M+K+H]2+
,
1124.5
Procedure B: Peptide 1b was synthesized on Rink amide MBHA
(100 mg, loading 0.72 mmolg)À1, as described for peptide 1a. We used as
first amino acid the Fmoc-LysACTHNUTRGNE(UNG TegN3)-OH 17 and the corresponding
amount of coupling reagents HBTU, HOBt, DIPEA (2:2.2:6 equiv).
After complete attachment to the resin the synthesis was further per-
formed as described for peptide 1a (31 mg, 46%). White powder, Rt =
8.92 min (RP-C18, 25–60% water (0.1% TFA)/acetonitrile); MS (ES+):
m/z calcd for for C43H69N9O14 [M+H]+: 936.06; found 936.4 [M+H]+,
958.4 [M+Na]+.
[M+Na+K]2+, 1131.5 [M+2K]2+, 1084.7 [MÀH2O+2H]2+
.
General procedure for complexation of the terpyridine-based ligands
with metal salts: A solution of the appropriate metal salt in acetonitrile
(0.5 equiv) was added to a solution of the terpyridine-based ligand
(1 equiv) in acetonitrile (5 mL), and the mixture was stirred for 30 min.
The solvent was removed in vacuo and the product was washed with
water and crystallized with diethyl ether before use.
A
ACHTUNGTREN(NUG 20b)2]ACHTUGNTRENN(UNG ClO4)2: The complexation was performed as described in
Solution-phase synthesis of compound 19 through amide coupling be-
tween the carboxy terminated ligand 2a and the amino-terminated affini-
ty label 1a: Carboxy-terminated ligand 2a (1 equiv, 2.53 mg, 6.2 mmol),
DIC (1 equiv, 1.95 mg, 2.5 mL, 12.52 mmol) and HOBt (1 equiv, 1.69 mg,
12.52 mmol) were dissolved in anhydrous DMF (0.3 mL) under argon and
allowed to stir at RT for 5 min. Dried amino-terminated epoxysuccinyl
peptide 2a (1.25 equiv, 10.64 mg, 15.67 mmol) was dissolved in a minimum
amount of anhydrous DMF and added to the reaction mixture. The reac-
tion mixture was allowed to stir until complete conversion of the diacid,
as monitored by HPLC. The solvent was removed in vacuo and the crude
of reaction was analyzed on a C18 reverse phase HPLC column using a
linear gradient of water (0.1% TFA)/acetonitrile and purified on a corre-
sponding preparative C18 reverse phase HPLC column (see General),
using the same gradient. Fractions containing the mono-epoxide product
were pooled, frozen and lyophilized to obtain 19 as white powder
(1.9 mg, 29%). Rt =15.2 min (RP-C18, 5–35% water (0.1% TFA)/aceto-
nitrile); MS (ES+): m/z calcd for C54H69N9O14 1068.5 [M+H]+; found:
1068.7 [M+H]+, 1090.7 [M+Na]+, 1106.5 [M+K]+.
AHCTUNGTRENNUNG
tive HPLC on a C18 reverse phase column using a linear gradient of
water/acetonitrile. Violet powder (94%); MS (ES+): m/z calcd for [FeII-
(20b)2]=[C214H302FeN38O60]: 4422.7; found: 1475.5 [(20b)2 +FeII +H]3+
,
1482.6 [(20b)2 +FeII +Na]3+
,
1488.1 [(20b)2 +FeII +K]3+
,
1488.9
[(20b)2 +FeII +2Na]3+, 1495.4 [(20b)2 +FeII +Na+K]3+, 1499.9 [(20b)2 +
FeII +2 K]3+, 1507.4 [(20b)2 +FeII +2H+ClO4]3+, 1107.3 [(20b)2 +FeII +
2H]4+, 1112.4 [(20b)2 +FeII +H+Na]4+, 1116.0 [(20b)2 +FeII +H+K]4+
,
1116.9 [(20b)2 +FeII +2Na]4+
, , 1125.3
1121.4 [(20b)2 +FeII +Na+K]4+
[(20b)2 +FeII +2K2]4+, 1140.7 [(20b)2 +FeII +2H+K+ClO4]4+
.
[FeII (ClO4)2: Violet powder (6 mg, 92%); 1H NMR (400 MHz,
AHCTUNGTRENUNNG ACHTUNRGTEG(NNUN 2a)2]ACHTNUGTRENNUGN
[D3]MeCN): d=8.81 (d, 3J=8.8 Hz, 4H; H3’, H5’), 8.60 (t, 3J=8.8 Hz,
2H; H4’), 8.38 (d, 3J=9.2 Hz, 4H; H3, H3’’), 7.80 (d, 3J=9.2 Hz, 4H; H4,
H4’’), 6.94 (s, 4H; H6, H6’’), 4.19 (s, 8H; CH2O), 3.85 ppm (s, 8H;
CH2COOH); MS (ES+): m/z calcd for [FeII
874.63; found: 437.5 [(2a)2 +FeII]2+
[(2a-2CO2)2 +FeII +ClO4]+, 1073.0 [(2a)2 +FeII +2
[FeII
(2b)2]
ACHTUNGTRENNUNG
,
,
AHCTUNGTRENNUNG
Solution-phase synthesis of compound 20 through click reaction between
the ethynyl terminated ligand 1b and the azide-terminated affinity label
1b: [CuACHTUNGTRENNUNG(CH3CN)4]PF6 (1 equiv, 1.1 mg, 2.8 mmol) dissolved in degassed
acetonitrile (30 mL) was added under argon to a degassed solution of
compound 2b (1 equiv, 1.0 mg, 2.8 mmol) in acetonitrile/water (6:1,
0.5 mL), and the reaction mixture was allowed to stir for 30 min at RT.
Upon addition of the copper(I) salt the solution turned immediately dark
red, characteristic for copper(I) complexes of terpyridine ligands. Peptide
E
N
ACHTUNGTRENNUNG
[D3]MeCN): d=8.86 (d, 3J=8.0 Hz, 4H; H3’, H5’), 8.65 (t, 3J=8.0 Hz,
2H; H4’), 8.41 (d, 3J=8.4 Hz, 4H; H3, H3’’), 7.80 (d, 3J=8.4 Hz, 4H; H4,
H4’’), 6.89 (s, 4H; H6, H6’’), 4.18 (s, 8H; CH2O), 3.92 (d, 4J=2.4 Hz, 8H;
CH2), 2.59 ppm (t, 4J=2.4 Hz, 4H; CH); MS (ES+): m/z calcd for [FeII-
(1b)2]=[C46H38FeN6O4]: 794.2; found: 397.4 [(2b)2 +FeII]2+
,
880.9
357.4 [{2bÀ2-
[(2b)2 +FeII +(BF4)]+, 377.4 [(2bÀCH2CCH)2 +FeII]2+
,
2178
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 2170 – 2180

Protein–Inorganic Arrays
FULL PAPER
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E
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A
[D3]MeCN): d=8.80 (d, 3J=8.0 Hz, 4H; H3’, H5’), 8.56 (t, 3J=8.0 Hz,
2H; H4’), 8.39 (d, 3J=8.4 Hz, 4H; H3, H3’’), 7.77 (d, 3J=8.4 Hz, 4H; H4,
H4’’), 7.67 (s, 4H; CHtriazole), 6.93 (s, 4H; H6, H6’’), 5.08 (s, 8H; N-CH2),
4.33 (s, 8H; CH2O); 4.21 (s, 8H; OCH2), 1.47 ppm (s, 36H; tBu); MS
(ES+): m/z calcd for [FeII(21)2]=[C70H82FeN18O12]: 1423.3; found: 711.5
[(21)2 +FeII]2+
,
627.5 [(21À3tBu)2 +FeII]2+, 599.6 [(21À4tBu)2 +FeII]2+, 1521.2 [(21)2 +
FeII +(ClO4))]+.
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
[NiII(21)2](ClO4)2: Green powder (10.1 mg, 89%); MS (ES+): m/z calcd
for [NiII(21)2]2+ =[C70H82NiN18O12]2+
NiII]2+
,
684.6 [(21ÀtBu)2 +NiII]2+
,
[(21À3tBu)2 +NiII]2+, 600.6 [(21À4tBu)2 +NiII]2+, 1523.3 [(21)2 +NiII +
(ClO4))]+.
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Financial support from European Commission by the EXT Grant 25085,
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ACHTUNGTRENNUNGische Forschungstiftung for scholarship funding.
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