Unnatural multidentate metal ligating α-amino acids

Article abstract of DOI:10.1016/j.tetlet.2006.06.128

A family of penta- and hexadentate metal ligating α-amino acids, suitably protected for Fmoc solid-phase chemistry, has been prepared. These residues incorporate the mono-amides of ethanolaminetriacetic acid, ethylenediaminetriacetic acid, and ethylenediaminetetraacetic acid as side chains. Side chains are tethered varying distances (n) from the Cα-carbon to allow metal binding events to occur at distinct distances from the peptide backbone. These residues are designed to allow the facile installation of metal chelates along a peptide backbone.

Full text of DOI:10.1016/j.tetlet.2006.06.128

Tetrahedron Letters 47 (2006) 6277–6280
Unnatural multidentate metal ligating a-amino acids
Christopher M. Micklitsch, Qian Yu and Joel P. Schneider*
Department of Chemistry and Biochemistry, University of Delaware, 115 Brown Lab, Newark, DE 19716, United States
Received 18 May 2006; accepted 23 June 2006
Abstract—A family of penta- and hexadentate metal ligating a-amino acids, suitably protected for Fmoc solid-phase chemistry, has
been prepared. These residues incorporate the mono-amides of ethanolaminetriacetic acid, ethylenediaminetriacetic acid, and eth-
ylenediaminetetraacetic acid as side chains. Side chains are tethered varying distances (n) from the Ca-carbon to allow metal binding
events to occur at distinct distances from the peptide backbone. These residues are designed to allow the facile installation of metal
chelates along a peptide backbone.
Ó 2006 Elsevier Ltd. All rights reserved.
At functional metal sites within proteins, the ligands are
provided, by either the amide backbone or more gener-
ally, the side chains of amino acid residues. Natural
metal binding residues such as histidine, methionine,
cysteine, aspartic, and glutamic acids individually act
as low affinity mono- or bidentate ligands in the absence
of a protein scaffold.1,2 However, evolution of protein
structure has culminated in exquisite protein folds that
offer secondary structural elements and outer sphere
interactions, such as hydrogen bonding, that uniquely
position the side chains of ligating residues.3–5 These
nonlocal structural attributes, contributed from the en-
tire protein scaffold, result in high affinity metal binding
sites.
peptides15–20 and are beginning to explore the possibility
of using this mechanism to prepare metal-containing
materials for use in microfluidics and sensor fabrication.
Successful preparation of these materials relies on our
ability to incorporate metal ions at precise sequential
positions within each peptide comprising the assembly.
Metal binding sites need to be incorporated within a
given peptide reliably, affording metal complexes of sig-
nificant stability. In addition, the ability to transpose a
given metal binding site into any peptide would enable
the preparation of focused libraries of metal-binding
self-assembling peptides. Designing such sites using only
naturally occurring amino acid residues would be a slow
and daunting task.
The de novo design of peptide and protein scaffolds that
bind metal ions in predictable geometries using only nat-
urally occurring amino acids is a field making notable
progress, but is extremely difficult.6–9 In order to design
a high affinity site a priori, not only does one need to be
concerned with the coordination sphere of the metal
binding site, but equally important is the overall three
dimensional structure of the entire protein that will serve
to position the ligating side chain ligands.
Here, we report the synthesis of a family of amino acids
that contain penta- and hexadentate aminocarboxy side
chain ligand (Scheme 1) compounds 11–19. Each amino
acid represents an autonomous metal binding site capa-
ble of binding one metal ion without the aid of other res-
idue side chains elsewhere in the sequence. Three classes
of residues have been prepared. The first, residues 11–
13, and the second class, residues 14–16, display penta-
dentate ligands as side chains, the mono-amides of etha-
nolaminetriacetic acid, and ethylenediaminetriacetic
acid, respectively. The third class, comprising residues
17–19, displays a hexadentate ligand, the mono-amide
of ethylenediaminetetraacetic acid (Scheme 1—ligating
atoms are shown in bold for residues 11–13 for refer-
ence). By varying the ligation number and identity of
ligating atoms, each class should form metal complexes
characterized by distinct stability constants and kinetic
labilities. Within each class of residue, the number of
The use of metal binding peptides and proteins is gaining
prominence in materials science.10–14 We are interested in
fabricating novel biomaterials from self-assembling
Keywords: Metal ligating a-amino acids; Metalloprotein de novo
design.
*
Corresponding author. Tel.: +1 302 831 3024; fax: +1 302 831
6335; e-mail: schneijp@udel.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.06.128

6278
C. M. Micklitsch et al. / Tetrahedron Letters 47 (2006) 6277–6280
O
X
1.
pentafluorphenol, DCC, CH2Cl2
HO
N
CO2tBu
CO2tBu
2. Fmoc-Dap-OH (n =1), or Fmoc-Dab-OH (n=2)
or Fmoc-Orn-OH (n=3); DIEA, pH 9, DMF
X = O (2),
X = N(Boc) (5),
X = N(CH2CO2t-Bu) (10)
CO2t-Bu
CO2t-Bu
CO2t-Bu
CO2t-Bu
N
N
O
O
O
N
O
N
N
CO2t-Bu
NH
NH
n
CO2t-Bu
FmocHN
NH Boc
CO2t-Bu
n
n
OH
OH
OH
FmocHN
FmocHN
O
O
O
11 (n=1) 90%
12 (n=2) 89%
13 (n=3) 92%
14 (n=1) 87%
15 (n=2) 68%
16 (n=3) 77%
17 (n=1) 73%
18 (n=2) 79%
19 (n=3) 82%
Scheme 1.
methylene groups separating the ligand from the Ca-
carbon of the amino acid is varied (n = 1–3) so that
the distance between the metal complex and the peptide
backbone can be controlled. The residues have been
Fmoc-protected so that standard solid phase peptide
synthesis can be employed to easily prepare peptides
containing one or multiple copies of any of these desired
metal binding residues.
in 90%, 89% and 92% yield, respectively, from mono-
acid 2. Ligand 2 was prepared by first coupling 2-(2-
chloroethoxy)ethanol to di-t-butyl iminodiacetate in
the presence of sodium iodide affording di-t-butyl pro-
tected 1 in 49% yield (Scheme 2). Platinum oxide-
assisted oxidation of 1 yields the corresponding mono-
acid 2 in 89% yield.
The coupling of 5 to Fmoc-Dap, -Dab or -Orn (Scheme
1) affords residues 14–16 in 87%, 68%, and 77% yield,
respectively. Scheme 3 provides the synthetic route for
the N-Boc, di-t-butyl ester protected ligand 5. Briefly,
treatment of ethanolamine with t-butyl bromoacetate
followed by bromination affords 3 in 87% yield for the
two steps. Next, bromine is displaced by the tosylate salt
of glycine benzyl ester yielding 4 in 76% yield. In concur-
rent reactions, the secondary amine of 4 is N-Boc pro-
tected using t-butoxy carbonyl anhydride and the
benzyl ester is de-protected via hydrogenation to afford
5 in 63% yield.
Historically, polycarboxylate ligands have found wide
applications in industrial, medical, and agricultural set-
tings, due to their ability to form metal chelates with
high binding efficiency.21 We chose three polycarboxy-
lates as side chains that should bind a wide range of
transition metals with differing affinities. For example,
the well-characterized hexadentate ligand ethylene-
diaminetetraacetic acid22,23 (precursor to the side chains
of residues 17–19), binds Fe2+, Cu2+, Ni2+, Zn2+, and
Cr3+ with 1014–1023 MÀ1 affinity, with Cr3+ forming
the most thermodynamically stable chelate. In compari-
son, the pentadentate ligand, ethylenediaminetriacetic
acid (precursor to the side chains of residues 14–16)
binds to Cr3+ with 106 MÀ1 affinity.24 Lastly, replacing
one of the ligating nitrogens with an oxygen donor
affords a side chain that should demonstrate preference
for binding harder metal ions (residues 11–13).
CO2t-Bu
HN
CO2t-Bu
O
HO
Cl
A facile convergent synthesis is reported that couples the
pentafluorophenol active esters of mono-acids 2, 5 or 10
to N-a-Fmoc-diaminopropionic acid (Fmoc-Dap), N-a-
Fmoc-diaminobutyric acid (Fmoc-Dab) or N-a-Fmoc-
ornithine (Fmoc-Orn) (Scheme 1). Active esters of 2,
5, and 10 are prepared via dicyclohexylcarbodiimide
(DCC) activation of the corresponding carboxylic acids
in the presence of pentafluorophenol and are used
directly in the subsequent coupling reaction. Commer-
cially available Fmoc-protected fragments react
quickly (within 2 h) and cleanly with 1.2 equiv of active
ester under basic solution conditions (pH 9) resulting in
the formation of compounds 11–19 in good to excellent
yield (see Supplementary data for detailed experimentals).
For example, compounds 11, 12, and 13 were produced
Na2CO3, NaI
49%
PtO2, O2
CO2t-Bu
CO2t-Bu
O
HO
N
NaHCO3
89%
1
O
O
CO2t-Bu
CO2t-Bu
HO
N
2
Scheme 2.

C. M. Micklitsch et al. / Tetrahedron Letters 47 (2006) 6277–6280
6279
lates suitably protected. Diamine 6 is then reacted with
excess t-butyl bromoacetate to yield tri-functionalized
7 in 83% yield. Following this step, the orthogonal ben-
zyl group was selectively removed by hydrogenation
using 10% palladium on carbon. The resulting second-
ary amine 8 was reacted with benzyl bromoacetate to
afford 9 in 90% yield, which was subsequently hydro-
genated to finally yield mono-acid 10 in 96% yield.
1. BrCH2CO2t-Bu,
KHCO3
CO2t-Bu
N
NH2
Br
HO
2. NBS, PPh3
87%
CO2t-Bu
CO2t-Bu
3
BnO2CCH2NH3+ -OTs
N
BnO2C
N
NaI, KHCO3
76%
H
CO2t-Bu
In summary, a family of metal chelating amino acids has
been prepared by reacting the active esters of suitably
protected polycarboxylate ligands with commercially
available Fmoc-protected Dap, Dab, and Orn residues.
The synthesis of these unnatural residues, as well as their
synthetic precursors, proceeds with good to excellent
yields. We are currently utilizing these penta- and hexa-
dentate residues in the fabrication of metal-containing,
self-assembled peptide-based materials.
4
O
Boc
1. Boc2O
N
HO
N
CO2t-Bu
2. 10% Pd-C, H2
63%
CO2t-Bu
5
Scheme 3.
Finally, coupling the active ester of 10 to Fmoc-Dap,
-Dab, or -Orn afforded 17, 18, and 19 in 73%, 79%,
and 82% yield, respectively. Precursor 10 was prepared
via an optimized procedure originally reported by
Johannes et al.25 (Scheme 4). Here, careful reductive
amination of ethylenediamine affords mono-amine 6 in
80% yield. The benzyl protecting group of 6 allows an
orthogonal protection strategy to be used to install the
mono-free acid of 10 with the remaining three carboxy-
Acknowledgement
This work was supported by the National Science Foun-
dation (CHE 0348323).
Supplementary data
Experiments and characterization spectra (MS, 1H
NMR and 13C NMR) for all prepared compounds are
available in the Supplementary data. Supplementary
data associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2006.06.128.
PhCHO,
NaBH4
H
Ph
N
H2N
NH2
NH2
80%
6
References and notes
t-BuO2C
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BrCH2CO2tBu,
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