Histidine analog amino acids providing metal-binding sites derived from bioinorganic model systems

Article abstract of DOI:10.1002/ejoc.200900472

Metalloproteins are of utmost importance for nearly all biological processes. Valuable information about their functionalities came from mutagenesis studies and led to the de novo design of artificial proteins. Advances in peptide chemistry enable the tot

Full text of DOI:10.1002/ejoc.200900472

FULL PAPER
DOI: 10.1002/ejoc.200900472
Histidine Analog Amino Acids Providing Metal-Binding Sites Derived from
Bioinorganic Model Systems
André Nadler,[a] Christina Hain,[a] and Ulf Diederichsen*[a]
Keywords: Amino acids / Metal binding / Coordination modes / Zinc finger / Click chemistry / Bioinorganic chemistry
Metalloproteins are of utmost importance for nearly all bio-
logical processes. Valuable information about their function-
alities came from mutagenesis studies and led to the de novo
design of artificial proteins. Advances in peptide chemistry
enable the total synthesis of complete proteins and allow the
incorporation of non-proteinogenic amino acids. Chelating
amino acids utilized to introduce artificial metal-binding sites
into proteins should mimic the side chains of the natural resi-
dues in order to minimize structural consequences within the
target proteins. In this paper a synthetic method is described
for the preparation of amino acids bearing a metal ligand
system connected to the β-carbon via triazole formation.
Thereby, a histidine isoster is generated with an additional
metal-binding site in proximity to the peptide backbone. Two
representative building blocks bearing the ligands triaza-
cyclononane (tacn) and bis(picoloyl)-amine (bpa) were syn-
thesized and incorporated into model peptides.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Tandem zinc finger repeats are among the best known
B-DNA-binding protein domains.[6] Every individual ca-
nonical zinc finger domain contains a structural zinc atom
that is coordinated by two cysteine and two histidine resi-
dues.[7] In the case of zinc finger domains, metal binding is
essential for adaptation of the protein fold that is required
for specific DNA-binding within the major groove. Never-
theless, with His4 zinc coordination it is possible to render
the zinc center also functional for DNA cleavage.[8] Metal-
Introduction
Transition metal ions are crucial for the molecular ma-
chinery of living cells. They are essential for correct protein
folding as structural metal ions, act as key constituents of
electron transfer pathways or serve as reactive catalytic spe-
cies in active sites of numerous enzymes.[1] These capabili-
ties require subtle variations of coordination geometry and
donor atom pattern which are guaranteed by the ability of
polypeptide chains to adopt a large number of different
conformations. Unique properties are observed for multinu-
clear metal centers in a number of prominent cases such as
the oxygen evolving complex or the copper A center that
have no analog in inorganic coordination chemistry.[2]
These characteristics of protein metal-binding sites make
them promising targets for chemical modifications, chang-
ing the coordination sphere or introducing additional metal
ions. Artificial binding sites in the proximity to a natural
metal center might serve as spectroscopic probes.[3] On the
other hand, the well-defined secondary structure of pep-
tides is suited to provide a structurally rigid chiral scaffold
for catalytically active metal complexes. So far, this has
been exploited using short peptides of defined secondary
structures functionalized with artificial metal-chelating
amino acids.[4,5] Well-defined protein interaction with bio-
molecules or recognition sites for small molecules is likely
to provide higher selectivity of the catalyzed reactions.
based nucleases often contain two metal ions (Mg2+, Mn2+
,
Zn2+) in spatial proximity that act in concert during the
phosphodiester cleavage.[9] In this context, it would be of
interest to generate binuclear metal centers in specifically
DNA-binding protein domains like zinc fingers. Incorpo-
rating a second metal ion into mononuclear structural
metal-binding sites might be possible by functionalizing the
side chain of a suitable positioned residue with a metal-
chelating moiety derived from bioinorganic coordination
chemistry. Replacing one of the coordinating histidine resi-
dues with an artificial metal-chelating amino acid would
result in a binuclear zinc binding site. As a prerequisite for
DNA recognition it is essential to preserve the three-dimen-
sional structure of the zinc finger motif.[6] Controlling the
spatial positioning of the introduced metal ion is the central
challenge of this approach requiring careful building block
design. The modified amino acids should be able to mimic
histidine as ligand of the structural zinc while providing an
additional metal-binding motif.
We decided to prepare a 1,2,3-triazole derivative as a
scaffold that closely resembles the imidazole ring of Nε-sub-
stituted histidine[10] (Figure 1) since the CuI-catalyzed [3+2]
cycloaddition of alkynes and azides (click chemistry) offers
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
Tammannstr. 2, 37077 Göttingen, Germany
Fax: +49-551-39-22944
E-mail: udieder@gwdg.de
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to combine preparation of a histidine analog ligand with facilitating chromatographic purification. Furthermore,
functionalization by an additional ligand system for metal with two amino functions being protected as carbamates,
binding.
the tacn ring loses its metal coordination capability and the
click reaction can be performed without removal of residual
copper from the metal-binding pocket. The tacn ligand 3
was synthesized by a standard procedures and bbt 2 was
obtained following a modified procedure from Wieghardt
et al.[18] The second ligand system bpa 4 was synthesized
according to a literature protocol.[19] For connecting the li-
gands 2 and 4 to the triazole moiety a C2 linker was used
to retain the possibility of a potential bridging coordination
mode via N2 and N3 of the triazole moiety. Thereby, for-
mation of thermodynamically favored five-membered che-
late rings in case of Zn2+ binding is facilitated. Therefore,
2-azidoethyl tosylate (5) was synthesized by reaction of eth-
ylene glycol ditosylate (6) with sodium azide. The (2-azido-
ethyl)-substituted tridentate ligands 7 and 8 were obtained
in moderate to good yields by nucleophilic substitution of
the tosylate by the corresponding secondary amino groups
of the precursors bbt 2 and bpa 4. In a final step the Fmoc-
protected amino acid building blocks 1 and 9 were obtained
using CuI-mediated click chemistry. Building block 1 was
synthesized by using standard conditions, whereas in case
of building block 9 higher catalyst loadings were required
and subsequent treatment with Na2EDTA was necessary
due to copper contamination of the product even after
chromatographic purification (Schemes 1 and 2). Boc de-
protection of derivative 1 yielded the free amino acid 10
which was used for metal-coordination experiments.
Figure 1. Comparison of a Nε-substituted histidine derivative with
an alanyl-triazolyl building block.
Amino acids of this type should still be able to coordi-
nate the transition metal of the natural metal-binding site
as 1,2,3-triazoles are known to bind metal ions via N2 or
N3.[11–12] Binding via N2 would be equivalent to Nε-coordi-
nation of histidine and binding via N3 would mimic the
unusual Nδ-coordination mode. Synthetically, a modular
approach can be applied employing commercially available
Fmoc-propargylglycine and azido functionalized derivatives
of ligands from inorganic coordination chemistry as build-
ing block precursors.
The first ligands employed were triazacyclononane (tacn)
and bis(picoloyl)amine (bpa) as they are excellent models
for N3 coordination, a coordination motif that can be
found in many metalloproteins. Within this study two
amino acids were synthesized and incorporated in peptides
with DNA-binding potential. The new amino acids are de-
signed to serve as bridging zinc binders using the triazole
unit for coordination of the structural and covalently at-
tached triazacyclononane (tacn) and bis(picoloyl)amine
(bpa) ligands for binding of the second zinc ion. Incorpora-
tion of these amino acids in DNA-binding peptides by solid
phase peptide synthesis was established and zinc coordina-
tion was observed.
Results and Discussion
Preparation of Amino Acids Functionalized with Metal
Ligands
Side chain functionalized amino acids acting as metal
ligands can be obtained by alkylating the amino group of
lysine,[13] by nucleophilic ring opening of serine lactone with
suitable nitrogen nucleophiles, or by nucleophilic side-chain
substitution.[14] Furthermore, modified tyrosine derivatives
and amino acids substituted with bipyridine or phenanth-
roline ligands[15] have been used, e.g. for the de novo de-
sign of functional metallopeptides.[16] Synthesis of the tacn
functionalized building block 1 required a discrimination of
the three secondary amino functions. Two different possibil-
ities to generate such a molecule are described in literature
leading either to dialkyl compounds,[17] or to 1,4-bis(tert-
butoxycarbonyl)-1,4,7-triazacyclononane (bbt, 2).[18] The
latter option was chosen due to the lower polarity of bbt 2 Scheme 1. Synthesis of the tacn functionalized building block 1.
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Metal-Binding Histidine Analogs
ids 10 and 12 and two of their donor atoms (N2 of the
triazole moiety of 10 and Nε of 12) are isosteric with the
corresponding Nε atoms of histidine residues.
Metal Binding of Functionalized Peptides
Binding of zinc and copper ions by the monomers 9 and
10 was investigated by means of UV/Vis spectroscopy and
ESI-MS.
Zinc and copper complexes of the triazole building
blocks 9 and 10 are readily formed in polar solvents such
as MeOH or MeCN or mixtures of organic solvents and
water and were detected by ESI-MS. The free amino acids
could no longer be detected when equimolar amounts of
metal salts were added indicating complete formation of the
respective coordination compounds. Both building blocks
exhibit a strong tendency of dimerization upon metal com-
plexation under ESI-MS conditions even in diluted solu-
tions (100 µ) (Figure 2). This was not observed when ESI-
MS data were collected for the amino acids under metal
free conditions; formation of binuclear dimeric complexes
seems probable. A comparable effect is not observed in the
case of peptides P1–P3 (Figure 3), so it is likely that the
carboxy function of the free amino acids 9 and 10 are in-
volved in metal chelation. Coordination modes that include
a participation of the carboxy group are discussed in litera-
ture for Tc complexes of 1,2,3-triazole alanines.[10] Oligo-
mer P3 contains two different metal-binding sites; accord-
ing to ESI-MS data oligomer P3 forms binuclear zinc com-
plexes (Figure 4).
Scheme 2. Synthesis of the bpa functionalized building block 9.
With respect to the synthesis of bifunctional peptides
containing two different metal-binding sites a third triden-
tate metal-chelating building block was synthesized. We de-
cided to prepare an ornithine derivative bearing two car-
boxyl groups in order to allow metal coordination via Nε
as it is usually the case for histidine residues. The carboxyl-
ates might act as bridging ligands, generating a well-defined
binuclear metal centre. Numerous possibilities for alkylat-
ing the Nζ position of lysine have been described in litera-
ture[13] and can be employed for functionalizing ornithine.
Reductive amination with a suitable aldehyde was chosen
as the most straightforward alkylation method. Glyoxylic
acid tert-butyl ester was prepared from tert-butyl bromo-
acetate according to a literature protocol[13a] and treated
with Nα-Fmoc-ornithine 11 to yield building block 12
(Scheme 3).
Scheme 3. Synthesis of the ornithine derivative 12.
Incorporation of Metal-Binding Amino Acids in Peptides
Building blocks 1, 9 and 12 were incorporated into model
peptides by HOBt/HBTU activation and regular coupling
times.[20] Polar peptide sequences containing multiple lysine
residues resulting in a net positive charge were prepared in
order to mimic the chemical properties of DNA-binding
proteins. Thereby, the metal-binding capability of the artifi-
cial amino acids could be checked under conditions that
Figure 2. ESI-HRMS-of 10Cu. Upper spectrum: experimental
data. Lower spectrum: simulated for the dimer (10Cu)2
[C56H66N14O8Cu2]2+
.
UV spectra of the copper complexes of the tacn and bpa
closely resemble the natural situation. Peptides P1 and P2 functionalized building blocks were recorded to assign
(H2N-KFKGXGKFK-COOH with X = 10 or 9) are mono- characteristic bands that can serve as fingerprint signals
nuclear whereas P3 (H2N-12-IRI-10-GKFK-COOH) is a when investigating larger peptides or small proteins func-
dinuclear model which is derived from the H(X)3H motive tionalized with the metal-chelating amino acids.
that is commonly found in zinc-induced α-helices. The two
>Metal-specific UV absorption was indeed observed for
histidines are substituted by the metal-chelating amino ac- the Boc-deprotected amino acid 10. Upon copper binding
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the Cu(OAc)2 bands at 212 and 240 nm were red-shifted to
232 and 272 nm, respectively. A new band at 358 nm ap-
peared which is probably due to a triazole–Cu2+ LMCT
transition (Figure 5). A similar CT band at 396 nm was ob-
served when amino aicd 10 was incorporated into a peptide
P1 (Figure 6). Therefore, UV spectroscopy is well-suited to
indicate copper complexation at the correct binding site.
Figure 3. ESI-HRMS of P1Zn. Upper spectrum: experimental
data. Lower spectrum: simulated for [C61H101N20O11Zn]3+
.
Figure 6. UV spectra of Cu(OAc)2 (solid line) and the Cu2+ com-
plex of P1 (dashed line, difference spectrum, absorption of the free
peptide subtracted).
Conclusions
Novel metal-chelating amino acids are of significant
interest for the de novo design of metallopeptides and are
well-suited for modification of naturally occurring metal-
binding sites in proteins. Two histidine mimicking Fmoc
amino acid building blocks 1 and 9 bearing tridentate bis-
(picoloyl)amine and triazacyclononane ligands were synthe-
sized by means of CuI-mediated click chemistry. The modu-
lar synthetic approach facilitates the variation of the chelat-
ing moiety. Both building blocks were incorporated into
model peptides by standard SPPS protocol. Binding of
Cu2+ and Zn2+ by amino acid monomers 9 and 10 and pep-
tides P1, P2 and P3 was investigated by means of UV spec-
troscopy and ESI-HRMS. The new ligands were proven to
be efficient zinc and copper binders, both as amino acids
and when incorporated into peptides. They can be used for
the functionalization of peptides and small proteins with
additional metal-binding sites.
Figure 4. ESI-HRMS of P3Zn2. Upper spectrum: experimental
data. Lower spectrum: simulated for [C65H11N22O15Zn2]3+
.
Experimental Section
General Remarks: All reagents were of analytical grade and used
without further purification. Solvents were of the highest grade
available. Dry solvents were stored over molecular sieves (4 Å).
Glass equipment utilized for reactions under inert atmosphere was
flame dried before use. Triazacyclononane and bis(2-picolyl)amine
were prepared as described in literature.[18,19] 1H and 13C NMR
Figure 5. UV spectra of Cu(OAc)2 (solid line) and the Cu2+ com-
plex of amino acid 10 (dashed line, as difference spectrum, absorp-
tion of the free amino acid is subtracted).
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Metal-Binding Histidine Analogs
spectra were recorded with a Varian Unity 300 spectrometer or a
Varian Inova 600 spectrometer. Chemical shifts are quoted in parts
per million (ppm) downfield of TMS. Coupling constants are given
in Hz. UV spectra were recorded with a Jasco V-550 UV/Vis spec-
trophotometer. ESI-MS data were obtained with a Finnigan (type
LGC or TSQ 7000) spectrometer, high resolution spectra were ob-
(220 µL 1.61 mmol) was added and the reaction mixture was stirred
under reflux for 20 h. The solvent was removed in vacuo, the resi-
due was dissolved in aqueous K2CO3 solution (30 mL) and ex-
tracted with DCM (3ϫ20 mL). The combined organic phases were
dried with Na2SO4 and the solvent was removed in vacuo. The
residue was purified by distillation (90 °C, 1 ϫ10–4 bar). Product 8
was obtained as brownish oil; yield 190 mg, 67%. Rf (iPrOH/H2O/
HOAc, 79:20:1) = 0.50. UV/Vis (λmax): ε = 214 nm. 1H NMR
tained with
a Bruker spectrometer (Apex-Q IV 7T). Flash
chromatography was performed using Merck silica gel 60. Thin
layer chromatography (TLC) was carried out using Merck alumi-
num sheets of silica gel 60 F254. Non-fluorescence quenching com-
pounds were detected using a solution of ninhydrine in ethanol.
Fmoc solid phase peptide synthesis was carried out by a standard
HOBt/HBTU activation. HPLC analysis was performed using a
Pharmacia Äkta basic instrument (pump type P-900, variable wave-
length detector) with a linear gradient of A (0.1% TFA in H2O) to
B (0.1% TFA in MeCN/H2O, 8:2). Peptides were analyzed using a
YMC J’spere column ODS-H80, RP-C18, 250ϫ4.6 mm, 4 µm,
80 Å, with a flow rate of 1 mL per min. Preparative purification
was performed using a YMC J’spere column ODS-H80, RP-C18,
250ϫ20 mm, 4 µm, 80 Å, with a flow rate of 10 mL per min.
3
4
5
(300 MHz, CDCl3): δ = 8.49 (ddd, JHH = 4.9, JHH = 1.8, JHH
=
0.9 Hz, 2 H, H6), 7.64 (ddd, 3JHH = 7.8, 3JHH = 7.3, 4JHH = 1.8 Hz,
3
2 H, H4), 7.51 (d, JHH = 7.8 Hz, 2 H, H3), 7.13 (ddd, 3JHH = 7.3,
4
3JHH = 4.9, JHH = 1.1 Hz, 2 H, H5) 3.84 (s, 4 H, Ar-CH2), 3.30
3
3
(t, JHH = 6.0 Hz, CH2), 2.80 (t, JHH = 6.0 Hz, 2 H, 2 H, CH2)
ppm. 13C NMR (75.6 MHz, CDCl3): δ = 158.83 (C2), 148.84 (C6),
136.41 (C4), 122.84 (C3), 122.01 (C5), 60.54 (CH2Ar), 53.19 (CH2),
48.87 (CH2) ppm. ESI-MS: m/z (%) = 269 (57) [M + H]+, 291 (100)
[M + Na]+. HRMS (ESI): calcd. for [C14H17N6]+: 269.15092; found
269.15100.
Nα-Fmoc-β-[1-(2-bpa-ethyl)-1,2,3-triazol-4-yl]-L-alanine (9): Amine
8 (110 mg, 370 µmol) and Fmoc--propargyl-glycine (150 mg,
450 µmol) were dissolved in tBuOH (12 mL) under inert atmo-
sphere. Solutions of Cu(OAc)2 (164 mg, 820 µmol) in H2O (6 mL)
and of sodium ascorbate in H2O (6 mL) were prepared. All three
solutions were degassed for 10 min by bubbling argon through the
solvent. The two aqueous solutions were transferred to the tBuOH
solution via transfer cannula. The reaction mixture was stirred for
48 h at room temp. The solvent was removed in vacuo, the residue
was dissolved in aqueous Na2EDTA solution (100 mL, 6.71 mm)
and extracted five times with EtOAc (50 mL). The combined or-
ganic layers were dried with Na2SO4 and the solvent removed in
vacuo. The residue was purified by flash chromatography (eluent
DCM/MeOH, 7:3) and amino acid 9 obtained as a yellow solid;
yield 112 mg, 50%. Rf (iPrOH/H2O/HOAc, 5:2:0.1) = 0.30. UV/Vis
(λmax): ε = 298, 262, 207 nm. 1H NMR (300 MHz, CDCl3): δ =
8.37–8.28 (m, 2 H, Ar-CH), 7.61–7.54 (m, 2 H, Ar-CH), 7.42–7.30
(m, 4 H, Ar-CH), 7.24–7.16 (m, 3 H, Ar-CH), 7.12–6.85 (m, 6 H,
Ar-CH), 4.42–4.35 (m, 1 H, Hα), 4.24–4.08 (m, 3 H, Fmoc-CH,
Fmoc-CH2), 4.03–3.91 (m, 2 H, CH2), 3.59 (s, 4 H, NCH2), 3.37–
3.18 (m, 2 H, Hβ), 2.84–2.72 (m, 2 H, CH2) ppm. 13C NMR
(75.6 MHz, CD3OD): δ = 158.17 (COOH), 156.20 (C=O), 148.95
(Ar-C), 143.99 (Ar-C), 143.88 (Ar-C), 141.00 (Ar-C), 136.56 (Ar-
C), 127.43 (Ar-C), 126.94 (Ar-C), 125.19 (Ar-C), 123.41 (Ar-C),
123.02 (Ar-C), 122.13 (Ar-C), 119.66 (Ar-C), 66.44 (Cα), 59.74
(NCH2), 55.50 (Fmoc-CH), 53.69 (CH2), 50.53 (Cβ), 47.88 (CH2),
46.97 (Fmoc-CH2) ppm. ESI-MS: m/z (%) = 626.3 (100). HRMS
(ESI): calcd. for [C34H34N7O4]+: 604.2667; found 604.2670.
1,4-Bis(tert-butoxycarbonyl)-1,4,7-triazacyclononane (2): Et3N
(2.68 mL, 19.3 mmol) was added to a solution of triazacyclononane
3 (1.00 g, 7.75 mmol) in dry DCM (20 mL) under inert atmosphere.
The solution was cooled to 0 °C and stirred for 10 min followed by
addition of Boc2O (3.27 g, 15.5 mmol). Stirring was continued at
room temp. for additional 6 h and the solvent removed in vacuo.
The residue was purified by flash chromatography with the eluent
EtOAc and 2 was obtained as a colorless viscous oil; yield 1.61 g,
63%. NMR spectroscopic data were identically equal to those re-
ported in literature.[18]
(2-Azidoethyl) Tosylate (5): Ethylene glycol ditosylate (1.90 g,
5.13 mmol) and sodium azide (333 mg, 5.13 mmol) were dissolved
in DMSO (10 mL) and the solution was stirred for 16 h at 100 °C.
The reaction mixture was diluted with water (30 mL) and extracted
five times with Et2O (20 mL). The combined organic layers were
dried with Na2SO4 and the solvent removed in vacuo. The residue
was purified by flash chromatography with the eluent DCM and
the title compound obtained as colorless oil; yield 690 mg, 56%.
NMR spectroscopic data were identically equal to those reported
in the literature.[21]
7-(2-Azidoethyl)-1,4-bis(tert-butoxycarbonyl)-1,4,7-triazacyclonon-
ane (7): Tosylate 5 (1.21 g, 5.02 mmol), 1,4-bis(tert-butoxycarb-
onyl)-1,4,7-triazacyclononane (2) (1.50 g, 4.56 mmol) and K2CO3
(503 mg, 5.47 mmol) were suspended in dry MeCN under inert at-
mosphere. The reaction mixture was refluxed for 48 h, cooled to
room temp., filtered and the solvent was removed in vacuo. The
residue was purified by flash chromatography (10 column volumes
DCM, then EtOAc/pentane, 5:1). Compound 7 was obtained as
colorless oil; yield 1.13 g, 62%. Rf (EtOAc/pentane, 3:1) = 0.85. 1H
NMR (300 MHz, [D6]DMSO, 35 °C): δ = 3.43–3.29 (m, 6 H, CH2),
3.23–3.17 (m, 4 H, CH2), 2.77–2.64 (m, 6 H, CH2), 1.41 (s, 18 H,
tBu-H) ppm. 13C NMR (75.6 MHz, [D6]DMSO, 35 °C): δ = 154.55
(C=O), 154.47 (CO), 78.49 (Ctert-O), 78.41 (Ctert-O), 54.48 (CH2),
54.35 (CH2), 53.39 (CH2), 53.14 (CH2), 52.97 (CH2), 50.17 (CH2),
49.50 (CH2), 49.37 (CH2), 49.15 (CH2), 48.96 (CH2), 48.54 (CH2),
48.32 (CH2), 28.03 (CH3) ppm, broad ring 13C signals, two con-
formers. ESI-MS: m/z (%) = 421 (70) [M + Na]+, 819 (100) [2M
+ Na]+. HRMS (ESI): calcd. for [C18H34N6O4]+: 399.2714; found
399.2710.
Nα-Fmoc-β-[1-(2-bbt-ethyl)-1,2,3-triazol-4-yl]-L-alanine (1): A solu-
tion of 7 (750 mg, 1.88 mmol) and Fmoc--propargyl-glycine
(947 mg, 2.82 mmol) in dry DMF (10 mL) was degassed for 10 min
by bubbling argon through the solvent. The solution was transfer-
red to a Schlenk flask equipped with CuI (71.4 mg, 376 µmol) and
sodium ascorbate (372 mg, 1.88 mmol) under inert atmosphere via
transfer cannula. The reaction mixture was stirred at room temp.
for 12 h and the solvent was removed in vacuo. The residue was
purified by flash chromatography with the eluent DCM/MeOH,
3:1 and amino acid 1 was obtained as a yellow solid; yield 1.58 g,
86%. Rf (DCM/MeOH, 3:1) = 0.25. UV/Vis (λmax): ε = 300, 288,
1
263, 203 nm. H NMR (300 MHz, [D6]DMSO, 100 °C): δ = 7.88–
7.79 (m, 3 H, Ar-CH, triazol-H), 7.73–7.65 (m, 2 H, Ar-CH), 7.42–
7.26 (m, 4 H, Ar-CH), 7.63 (sbr, NH), 4.33 (mz, 2 H, NArCH2),
N,N-Bis(2-picolyl)(2-azidoethyl)amine (8): Amine
4
(210 mg, 4.23–4.15 (m, 3 H, Fmoc-CH, Fmoc-CH2), 4.09–4.00 (m, 1 H, Hα),
1.05 mmol) and tosylate 5 (255 mg, 1.05 mmol) were dissolved in
dry MeCN (3.0 mL) under inert atmosphere. To this solution Et3N
3.36 (sbr, 4 H, tacn-CH2), 3.20–3.09 (m, 5 H, tacn-CH2, β-CH2),
3.04–2.93 (m, 3 H, NCH2, β-CH2), 2.65–2.60 (m, 4 H, tacn-CH2),
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1.42, 1.40 (2ϫs, 18 H, tBu-H) ppm. 13C NMR (75.6 MHz, [D6]-
DMSO, 35 °C): δ = 155.38 (COOH), 154.43 (C=O), 154.39 (C=O),
154.36 (C=O), 143.60 (Ar-C), 143.57 (Ar-C), 140.43 (Ar-C), 127.36
(Ar-C), 126.82 (Ar-C), 125.03 (Ar-C), 124.98 (Ar-C), 119.84 (Ar-
C), 78.42 [O-C(CH3)3], 78.29 [O-C(CH3)3], 65.52 (Fmoc-CH2),
53.82, 53.44, 53.29, 52.97, 49.71, 49.26, 49.00, 48.47, 47.94, 47.88,
46.61 (Fmoc-CH), 28.05 [C(CH3)3], 28.00 [C(CH3)3] ppm. ESI-MS:
1359.77967; found 1359.78006. TR (5% B Ǟ 30% B in 30 min) =
3.82 min.
Peptide P3: Peptide synthesis was done according to a standard
Fmoc protocol.[20] Building blocks 1 and 12 were used in twofold
excess. Purification was carried out via reverse-phase HPLC (see
above); yield 5.40 mg, 12%. HRMS (ESI): calcd. for
[C65H112N22O15]2+: 721.4412; found 721.4400. TR (5% B Ǟ 60%
B in 30 min) = 13.65 min.
m/z (%)
= 732 (100) [M –
H]–. HRMS (ESI): calcd. for
[C38H52N7O8]+: 734.38719; found 734.38686.
Metal Coordination Experiments: All UV measurements were car-
ried out in MeCN or MeCN/H2O, 9:1. Ligand concentration was
adjusted to 50 µ. Cu(OAc)2 and Zn(OAc)2 were used as metal
sources. Complexes were prepared in situ by adding the equimolar
amounts of stock solution and diluting to the desired concentra-
tion. A 100 µ solution of copper and zinc complexes was prepared
for mass spectrometry. UV and ESI-HRMS data of in situ prepared
complexes.
Nα-Fmoc-β-[1-(2-tacn-ethyl)-1,2,3-triazol-4-yl]-L-alanine·3 TFA (10):
Fmoc-β-[1-(2-bbt-ethyl)-1,2,3-triazol-4-yl]--alanine (1) (100 mg,
136 µmol) was dissolved in TFA (1 mL) and stirred for 5 min at
room temp. The solvent was removed in vacuo and the residue
suspended in Et2O. The precipitate was collected via centrifugation
and washed twice with cold Et2O. Amino acid 1 was obtained as a
colorless solid; yield 108 mg, 95%. Rf (DCM/MeOH, 3:1) = 0.05.
UV/Vis (λmax): ε = 299, 288, 264, 208 nm. 1H NMR (300 MHz,
CD3OD): δ = 7.83–7.76 (m, 3 H, Ar-H), 7.68–7.58 (m, 2 H, Ar-
H), 7.40–7.26 (m, 4 H, Ar-H), 4.60–4.52 (m, 2 H, CH2), 4.40–4.18
(m, 4 H, Hα, Fmoc-CH, Fmoc-CH2), 3.48–3.42 (m, 4 H, CH2),
3.22–2.80 (m, 10 H, CH2, β-CH2) ppm. 13C NMR (75.6 MHz, [D6]-
DMSO, 100 °C): δ = 143.43 (Ar-C), 140.32 (Ar-C), 128.39 (Ar-C),
127.12 (Ar-C), 126.73 (Ar-C), 126.56 (Ar-C), 124.67 (Ar-C), 120.47
(Ar-C), 65.61 (Fmoc-CH2), 55.88, 54.15 (Cα), 48.69, 46.47, 46.09,
43.84, 43.84, 43.04, 42.58, 42.52 ppm. ESI-MS: m/z (%) = 534 (100)
[M + H]+, 556 (75) [M + Na]+. HRMS (ESI): calcd. for
[C28H35N7O4]+: 534.2823; found 534.2814.
9Cu: RMS (ESI): calcd. for [C68H64N14O8Cu2]2+ (dimeric species):
665.1806, 665.6823, 666.1800, 666.6814, 667.1829, 667.6806,
668.1822; found 665.1807, 665.6821, 666.1822, 666.6812, 667.1806,
667.6812, 668.1818. UV/Vis (λmax): ε = 287, 236, (difference spec-
trum, absorption of the free amino acid subtracted), (λmax): ε =
299, 260, 207 nm (absolute values).
9Zn: RMS (ESI): calcd. for: [C68H64N14O8Zn2]2+
: 666.1802,
666.6818, 667.1796, 667.6803, 668.1781, 668.6796, 669.1767,
669.6782, 670.1760, 670.6776, 671.1792; found 666.17987,
666.68158, 667.18318, 667.67994, 668.17690, 668.67928, 669.17960,
670.17531, 671.17918.
Nα-Fmoc-Nε,Nε-Bis(tert-butoxycarbonylmethyl)-
L-ornithine
(12):
[10Zn]: HRMS (ESI): calcd. for [C56H68N14O8Zn2]2+ (dimeric spe-
cies): 596.1958, 596.6975, 597.1950, 597.6959, 598.1938, 598.6953,
599.1923, 599.6938, 600.1916, 600.6932; found 596.1960, 596.6960,
597.1960, 597.6947, 598.1937, 598.6938, 599.1932, 599.6927,
600.1917, 600.6920.
Nα-Fmoc--ornithine hydrochloride (11) (1.00 g, 2.56 mmol), gly-
oxylic acid tert-butyl ester (666 mg, 5.12 mmol), and sodium triace-
toxyborohydride (1.63 g, 7.68 mmol) were dissolved in dry DCE
(30 mL) under inert atmosphere and the reaction mixture was
stirred for 1 h at 0 °C. The reaction mixture was extracted twice
with aqueous NH4Cl, the organic layer was dried with Na2SO4 and
the solvent was removed in vacuo. The residue was purified by flash
chromatography (eluent DCM/MeOH, 95:5) and amino acid 12
was obtained as a yellow solid; yield 895 mg, 60%. Rf (DCM/
10Cu: HRMS (ESI): calcd. for [C56H68N14O8Cu2]2+ (dimeric spe-
cies): 595.1963, 595.6979, 596.1956, 596.6971, 597.1947, 597.6962,
598.1978; found 595.1967, 595.6980, 596.1970, 596.6970, 597.1965,
597.1965, 597.6960, 598.1982. UV/Vis (λmax): ε = 358, 272, 232 nm
(difference spectrum, absorption of the free amino acid subtracted),
(λmax): ε = 358, 298, 264, 208 nm (absolute values).
1
MeOH, 9:1) = 0.30. UV/Vis (λmax): ε = 300, 288, 264, 206 nm. H
NMR (300 MHz, [D6]DMSO, 35 °C): δ = 7.81–7.90 (m, 4 H, Ar-
3
H), 7.68 (d, JHH = 7.4 Hz, 1 H, NH), 7.29–7.44 (m, 4 H, Ar-H),
P1Zn: HRMS (ESI): calcd. for [C61H101N20O11Zn]3+: 451.2411,
451.5755, 451.9069, 452.2412, 452.5731, 452.9075, 453.2491,
453.5764; found 451.2412, 451.5757, 451.9079, 452.2415, 452.4738,
452.9078, 453.2411, 453.5745.
4.16–4.30 (m, 3 H, Fmoc-CH, Fmoc-CH2), 3.70–3.80 (m, 1 H, Hα),
3.33 (s, 2 H, CH2C=O), 3.30 (s, 2 H, CH2C=O), 2.53–2.61 (m, 2
H, δ-CH2), 1.45–1.78 (m, 4 H, β-CH2, γ-CH2), 1.40 (s, 9 H, tBu-
CH3), 1.38 (s, 9 H, tBu-CH3) ppm. 13C NMR (75.6 MHz, [D6]-
DMSO, 35 °C): δ = 170.7 [(CO)OtBu], 169.97 (COOH), 155.65
(C=O), 143.94 (Ar-C), 140.71 (Ar-C), 127.55 (Ar-C), 127.03 (Ar-
C), 125.17 (Ar-C), 120.05 (Ar-C), 80.03 [O-C(CH3)3], 65.40 (Fmoc-
CH2), 55.19 [N-(CH2)2], 54.91 (Cα), 53.41 (Cδ), 46.79 (Fmoc-CH),
29.93 (Cβ), 27.77 [C(CH3)3], 27.51 (Cγ) ppm. ESI-MS: m/z (%) =
583 (70) [M + H]+, 605 (100) [M + Na]+. HRMS (ESI): calcd. for
[C32H42N2O8]–: 581.2868; found 581.2869.
P3Zn: HRMS (ESI): calcd. for [C65H111N22O15Zn2]3+: 522.5722,
522.9066, 523.2385, 523.5723, 523.9042, 524.2385, 524.5699,
524.9042, 525.2361, 525.5705, 525.9049; found 522.5722, 522.9061,
523.2383, 523.5717, 523.9043, 524.2390, 524.5707, 524.9036,
525.2392, 525.5702, 525.9025.
Acknowledgments
Peptide P1: Peptide synthesis was done by a standard Fmoc proto-
col.[20] Building block 1 was used in twofold excess. Purification
was carried out via reverse-phase HPLC (see above); yield 32.0 mg,
79%.
Generous support of the Deutsche Forschungsgemeinschaft (DFG)
(IRTG 1422) is gratefully acknowledged.
HRMS (ESI): calcd. for [C61H103N20O11]3+: 430.60329; found
430.60330. TR (12% B Ǟ 20% B in 30 min) = 14.22 min.
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4598
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Received: May 1, 2009
Published Online: August 4, 2009
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