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Metal-Ion-Binding Peptides From Catalysis to Protein Tagging.

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Peptide-Based Ligands
Metal-Ion-Binding Peptides: From Catalysis
to Protein Tagging
Giulia Licini* and Paolo Scrimin*
combinatorial chemistry · homogeneous catalysis ·
metal ions · peptides · sensors
etal ions are among the most important cofactors in proteins. They play
a key role in regulating the activity of
proteins both at allosteric sites and at
catalytic ones. The repertoire of natural
amino acids comprises several functional groups in the side arms for the
purpose of binding metal ions. Appropriate positioning of these groups in the
protein sequence ensures the binding of
a diverse range of metal ions, often with
remarkable selectivity, according to
their nature and their position, as determined by the secondary and tertiary
conformation adopted by the polymer.
It is thus not surprising that chemists are
making more and more use of peptide
sequences in their selection of ligands
for metal ions for diverse applications.[1]
A common approach takes advantage of
the natural sequences known to bind a
specific metal ion. Chemists, however,
have also expanded the supply of coordination motifs provided by natural
amino acids by synthesizing new ones
and by introducing specific capping
units into peptide sequences.[2] The reported applications range from the synthesis of catalysts for reactions not
necessarily finding an equivalent in the
biological world to obtaining probes for
analytical purposes.
One of the important advantages of
a peptide sequence over other building
blocks is the fact that a solid-phase
[*] Prof. Dr. G. Licini, Prof. Dr. P. Scrimin
Department of Organic Chemistry
and ITM-CNR, Padova Section
University of Padova
Via Marzolo, 35131 Padova (Italy)
Fax: (+ 39) 049-827-5239
synthesis, and hence a combinatorial,
approach to the preparation of libraries[3] of molecules can be accomplished
with ease. The challenge in these cases is
to devise methodologies for the screening of the libraries. This approach requires the possibility of estimating the
binding strength of the ligands for the
metal ions and, depending on the final
use of the complexes, to also evaluate
their activity as catalysts or sensing
Functional metal peptides are clearly the most appealing targets. Novel
peptide-based catalysts were obtained
by Francis and Jacobsen from metalbinding combinatorial libraries for the
epoxidation of alkenes.[4] A combinatorial approach to the cleavage of phosphates by hydrolytic metallopeptides
was reported by Berkessel and H-rault.[5] Their strategy allowed the direct
on-beads screening of a 625-member
library of undecapeptides with a chromogenic model phosphate. In this way
they were able to find active compounds
whose efficiency was also tested in
homogeneous solution. Combinatorial
optimization of the tripeptide Xaa-XaaHis for complexation to Ni2+ ions and
deoxyribose-based (oxidative) cleavage
of B-form DNA was performed by Long
and co-workers.[6] The procedure they
have employed is to generate two libraries (by using 18 naturally occurring
amino acids and excluding Cys and
Trp) in which the first and second
position of the ligand were varied. The
optimized metallopeptide Ni2+-Pro-LysHis was found to cleave DNA an order
of magnitude better than Ni2+-Gly-GlyHis, the reference compound used as a
starting point for the selection process.
Interestingly, metal complexation and
the T/A-rich site selectivity of the optimized metallopeptide were not altered,
while the affinity of DNA binding was
only slightly increased. Thus, the observed increased activity is mostly related to the geometry of binding, as
suggested by molecular models.
The stereoselective catalysis of a
series of rather different reactions was
performed by Hoveyda's research group
using metallopeptide complexes. In this
case, the highly crucial identification of
the most effective chiral ligand/metal
salt couple for a specific process has
been performed by preliminary parallel
screening of different metal precursors
with readily modifiable (modular) ligands. In all cases, peptide-based Schiff
bases with different O, N, or P donors
were used in the presence of both early
and late transition metal ions: the development of efficient and highly stereoselective C C bond-forming processes, such as TiIV-catalyzed CN addition to
epoxides[7] and imines,[8] AlIIICN addition to ketones,[9] ZrIV-catalyzed dialkyl
zinc addition to imines,[10] Cu-catalyzed
conjugate addition to enones,[11] and
allylic substitution,[12] have been achieved. As an example, pyridinyl peptidic
derivatives have been found to be effective chiral ligands for the Cu-catalyzed
allylic substitution of di- and trisubstituted alkenes. This catalytic system
allowed the synthesis of (R)-( )-sporochnol with 82 % ee in 82 % overall
The strategy towards the selection of
the optimal reaction conditions was
based on a rational rather than a combinatorial screening approach with the
following steps (Scheme 1): a) The identification of the most reactive substrates
and metal precursors (allylic phosphates
DOI: 10.1002/anie.200301668
Angew. Chem. Int. Ed. 2003, 42, 4572 –4575
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 1. The protocol used by Hoveyda and co-workers for the optimization of a peptide-based ligand/Cu salt combination for the asymmetric
allylic substitution of alkenes.
and CuCN) as well as the best Schiff
base for the addition of Et2Zn in THF.
The two best ligands under these conditions afforded ee values of 34 % and
26 %, which were unimpressive starting
points. b) Identification of the best ligand/Cu salt combination using the
above-selected ligands and six copper
salts. c) The determination of the optimal peptide (number of amino acids in
the sequence, nature of the C-terminal
group). d) The tuning of the Schiff base
and the first amino acid. With this
selection process the ee values went up
to 87 % with aryl-disubstituted alkenes
and to 90 % with the aryl-trisubstituted
ones. Similar enantiomeric excesses
were obtained by using a nonrational
approach based on the screening of
parallel libraries of substrates, solvents,
and metal ions.[8a, 10] This observation
may indicate that small, focused libraries may provide results that are as good
as a rational, but more time-consuming,
approach in the selection of a catalytic
An important aspect was raised by
Gilbertson et al. in selecting catalysts
comprised of peptide-based phosphane
Angew. Chem. Int. Ed. 2003, 42, 4572 –4575
ligands and late transition metal
ions:[13, 14] that of tuning the best chiral
ligand/substrate couple. This point underlines the quest for catalysts which are
rather specific for a substrate, a property
typical of enzymes. By studying the PdIIcatalyzed addition of dimethylmalonate
to cyclopentadienyl acetate with catalysts selected from two series of 96- and
40-member peptide libraries, this research group has also addressed the
relevance of the secondary conformation of the peptide on the stereoselectivity of the catalytic process. They
found that b-turns are better than helical
conformations and that the direct
screening of the catalysts anchored on
the solid support where they had been
synthesized is possible in some cases,
with results comparable to those obtained in homogeneous solution.
While catalysis has been proven to
be an excellent proving ground for testing the potentiality of metallopeptides, a
newly emerging application of metallopeptides as fluorescent markers has
been reported by Imperiali and coworkers. Previously, they had focused
their research on peptide-based
cent chemosensors for ZnII ions.[15] The
scope of their research has now widened
to the discovery of a Ln3+-binding peptide for the rapid detection of tagged
proteins. The development of fluorescent proteins as molecular tags may
allow complex biochemical processes
to be correlated with the function of
living cells.[16] The work by Imperiali's
group is based on previous studies
carried out by other research groups
which had focused on the similarities in
ionic radii and coordination preferences
between Ca2+ and Ln3+ ions of sequences based on specific loops of calciumbinding proteins.[17] This previous
work[18] had pinpointed hot positions
that were relevant both for binding and
fluorescence enhancing in 14-mer peptides. The screening[19] by Imperiali and
co-workers of a large (up to 500 000
member) library of peptides of the
general sequence Ac-Gly-Xaa-ZaaXaa-Zaa-Xaa-Gly-Trp-Zaa-Glu-ZaaZaa-Glu-Leu (where Xaa were potential metal-binding residues Asp, Asn,
Ser, and Glu and Zaa were hydrophobic
amino acids) led to the discovery of a
peptide with a KD value for Tb3+ ions as
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
low as 0.22 mm. The authors have suggested that these lanthanide-binding
tags (LBTs) may constitute a new alternative for expressing fluorescent fusion
proteins by routine molecular biological
techniques. To test this idea they have
appended the most promising sequences
to ubiquitin.
As shown in Figure 1, an intervening
Gly-Pro-Gly sequence between the Nterminal His tag and LBT tag was
introduced as a stop site to allow
subsequent removal of the His tag. Very
rewardingly, they discovered that protein expression was not diminished by
the presence of the LBT. Metal-ion
competition experiments established
that the LBTs demonstrate good selectivity for Tb3+ ions. They have also
devised a powerful combinatorial
screening[20] on beads to find sequences
with an even better binding ability for
this lanthanide ion. The methodology
utilizes solid-phase split-and-pool combinatorial peptide synthesis where orthogonally cleavable linkers provide the
possibility for an efficient two-step
screening procedure (Figure 2). The initial screen avoids the interference
caused in on-bead screening by photochemically releasing a portion of the
peptides into an agarose matrix for
evaluation. The secondary screening
further characterizes each hit in a defined aqueous solution. It is worth
mentioning that this procedure avoids
Figure 2. Protocol used by Imperiali and co-workers for the synthesis and screening of Tb3+binding peptides. 1) Coupling of N-a-Fmoc-4-nitrophenylalanine; 2) coupling of ANP:HMBA
(1:10); 3) introduction of the spacer sequence; 4) synthesis of a “split-and-pool” library with a
mass spectral ladder; 5,6) deprotection of the amino acid side chains followed by casting into
agarose with 50 mm Tb3+ ions and photolysis. After selection of the beads with luminescent
halos and work-up, they are titrated with Tb3+ ions and the sequence deconvoluted by MALDIMS. Fmoc = 9-fluorenylmethoxycarbonyl.
any interference from the solid support,
as found in the on-bead screening recently reported by Miller and co-workers[21] and Davis and co-workers[22] in
which they looked for products of a
reaction produced by a solid-supported
catalyst. With this approach, and by
further focusing the library, the dissociation constant for Tb3+ ions was decreased to 57 nm for a linear peptide
and 2 nm when it was cyclized through
formation of a disulfide. As a proof of
principle, these peptide sequences were
Figure 1. The principle which is behind lanthanide binding tags (LBT, left) employed by Imperiali
and co-workers and their general strategy towards LBT-tagged proteins (right). Removal of the
His tag can be performed with the TAGZyme protocol (using dipeptidyl aminopeptidase
(DAPase) enzyme).
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
used for the determination of the protein concentration and expression
profiling. The fluorescence of the lanthanide ion can be exploited for this
purpose only if the binding strength is
such that it is possible to operate at
metal-ion concentrations well below
that required by nonspecific binders.
Although ligand peptides[23] have
only recently been exploited, nevertheless an impressive amount of useful
applications of peptide–metal complexes have already emerged. Functional peptides can not only be screened
with a combinatorial approach, but they
can also be incorporated into proteins
expressed in vivo (with the possibility to
encode even unnatural amino acids[24])
and offer the possibility of providing
new tools to study proteins and cellular
functions and to obtain generations of
new synthetic proteins with unusual
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[2] H. Ishida, Y. Inoue, Rev. Heteroat.
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[3] B. E. Turk, L. C. Cantley, Curr. Opin.
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[4] M. B. Francis, E. N. Jacobsen, Angew.
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[5] A. Berkessel, D. A. H-rault, Angew.
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Biochemistry 1999, 38, 2160 – 2166.
[7] K. D. Shimizu, B. M. Cole, C. A. Krueger, K. W. Kuntz, M. L. Snapper, A. H.
Hoveyda, Angew. Chem. 1997, 109,
1781 – 1785; Angew. Chem. Int. Ed.
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[8] C. A. Krueger, K. W. Kuntz, C. D. Dzierba, W. G. Wirschum, J. D. Gleason,
M. L. Snapper, A. H. Hoveyda, J. Am.
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[13] S. R. Gilbertson, S. E. Collibee, A. Agarkov, J. Am. Chem. Soc. 2000, 122, 6522 –
[14] S. R. Gilbertson, X. Wang, Tetrahedron
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[15] G. K. Walkup, B. Imperiali, J. Am.
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[16] J. Lippincott-Schwartz, G. H. Patterson,
Science 2003, 300, 87 – 91.
[17] For the use of EF-hand sequences (a Cabinding motif of calmodulin) as LnIIIbased metallonucleases, see J. T. Welch,
W. R. Kearney, S. J. Franklin, Proc. Natl.
Acad. Sci. USA 2003, 100, 3725 – 3730,
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2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ion, metali, catalysing, protein, tagging, binding, peptide
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