Biomimetic ligands for transition metals: Catechol-containing peptides

Article abstract of DOI:10.1016/S0960-894X(00)00303-6

The tetrapeptide ligands 6a and 6b containing a catechol moiety have been synthesised and their metal binding with Fe(III), Mn(III) and Cu(II) has been studied using fluorescence spectroscopy. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00303-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1617±1620
Biomimetic Ligands for Transition Metals:
Catechol-Containing Peptides
Muhammed A. Ashraf,a,b Keith Jonesa,* and Sheetal Handab,y
aSchool of Applied Chemistry, Kingston University, Penrhyn Road, Kingston-upon-Thames, Surrey, KT1 2EE, UK
bDepartment of Chemistry, King's College London, Strand, London, WC2R 2LS, UK
Received 17 March 2000; accepted 22 May 2000
AbstractÐThe tetrapeptide ligands 6a and 6b containing a catechol moiety have been synthesised and their metal binding with
Fe(III), Mn(III) and Cu(II) has been studied using ¯uorescence spectroscopy. # 2000 Elsevier Science Ltd. All rights reserved.
One of the major challenges in current organic chemistry
is to ®nd ecient catalysts for the chemo- and stereo-
selective insertion of one oxygen atom into various
organic molecules under mild conditions. Transition
metal-containing enzymes capable of achieving such
transformations are well known1,2 but the main e€orts
in this area have followed a non-biomimetic course.
However, there are limited reports illustrating the e€ec-
tiveness of peptides3 5 and a-amino acids6 as ligands of
metallic species in asymmetric reactions. In addition,
simple peptides such as the physiologically active tri-
peptide, Gly±His±Lys have been shown to exhibit
strong chelating ability to metal ions.7 Such peptides all
contain a histidyl residue and chelation of metal ions
has been shown to involve the terminal amino nitrogen
and histidine imidazole nitrogen. This chelating domain
is also a feature of serum albumins (His±Gly±Gly).8
These systems have been shown to produce oxidising
species upon complexation.9 The aim of this project was
to design and synthesize small peptides containing the
Scheme 1.
*Corresponding author. Tel.: +44-208-547-8450; fax: +44-208-547-7562; e-mail: keith.jones@kingston.ac.uk
yPresent address: BP-Amoco Chemical Ltd., Chertsey Road, Sunbury, Middlesex, TW16 7LL, UK.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00303-6

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M. A. Ashraf et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1617±1620
key His±Gly±Gly structural motif along with a metal-co-
ordinating group at the N-terminus. In addition to
being a good ligand for redox-active transition metals,
the N-terminus group should also act as a ¯uorsecent
probe such that metal binding could be followed by
observing ¯uorescence quenching. In this way, potential
new oxidation catalysts could be readily identi®ed. We
now wish to report on the synthesis and metal-binding
of two such peptide-based ligands.
The N-terminus group chosen was the 1,2-dihydroxy-
benzene (catechol) group. This group is found in side-
rophores,10 the natural iron chelators and it also possesses
suitable ¯uorescent properties to act as a probe for bind-
ing.11 The regioisomeric ligands 6a and 6b, di€ering
only in the positioning of the 1,2-diol functionality with
respect to the attaching chain were synthesised as outlined
in Scheme 1. Tritylation of the imidazole nitrogen of
histidine methyl ester was accomplished in 75% yield
Figure 1. Uncorrected ¯uorescence emission of 6a and 6b in response to the addition of Fe(III), Mn(III) and Cu(II) metal cations in methanol: 0,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 molar equivalents. Exicitation was carried out at 305nm for (6a) and 293nm for (6b).

M. A. Ashraf et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1617±1620
1619
under standard conditions. N-Trityl hisitidine methyl ester
1 was then condensed with N-Cbz protected diglycine 2 to
give the protected tripeptide 3 in 84% yield. Consider-
able experimentation was carried out to ®nd the most
suitable protecting groups for the phenolic hydroxyl
groups in the dihydroxybenzoate moiety. The condi-
tions for the removal of ketal-based protecting groups
proved to be too severe for the system and ®nally, benzyl
ether protection was chosen. Removal of the Cbz group
from tripeptide 3 followed by condensation separately
with the protected 2,3-dihdroxybenzoic acid 4a and the
protected 3,4-dihydroxybenzoic acid 4b, gave the pseudo-
tetrapeptides 5a and 5b in yields consistently between
75% and 85%. Catalytic hydrogenation removed the
benzyl ethers in high yield and ®nally treatment with
pyridinium hydrochloride in methanol removed the trityl
group to give 6a and 6b in overall yields of some 90%
from 5a and 5b.
equivalents of metal to ligand. On titration with Mn(III)
(spectrum 5), virtually no quenching is observed in
marked contrast to the corresponding case with 6a (spec-
trum 2). Finally, titration with Cu(II) (spectrum 6) shows
a similar result to that obtained with ligand 6a (spectrum
3), namely progressive quenching but at a notably slower
`rate' and leaving a small but noticeable residual ¯uo-
rescence. We interpret these results as showing that with
Fe(III) and particularly Mn(III), peptide 6b shows little
or no co-ordination via the catechol unit. This is explic-
able in terms of the twist required in the system to incor-
porate binding to both the imidazole and the catechol in
6b owing to the disposition of the diol functionality in this
ligand. However, it appears that 6b can form a complex
with Cu(II) involving the catechol group although a com-
parison of the quenching with that observed for 6a with
Cu(II) would indicate a more weakly bound complex.
In summary, we have prepared two peptide-based cate-
chol-containing ligands and have explored their metal
binding using the ¯uorescent properties of the catechol
unit. These studies have shown that the 2,3-dihydroxy-
benzamide unit in 6a is a better system for metal chela-
tion than the 3,4-dihydroxybenzamide unit in 6b. With a
rapid screening method available and reliable chemistry
which can be transfered to the solid phase, we are now
in a position to investigate related ligands and explore
their chemical reactivity.
Although ¯uorescence has been widely used in peptide
chemistry, it has usually been employed as a means of
detection and quanti®cation of small amounts of peptides
either through the ¯uorscence properties of tryptophan,
derivatisation with the dansyl12 group or incorporation
of non-natural amino acids13 with speci®c ¯uorescent
properties. The use of ¯uorescence quenching to explore
metal binding has previously been accomplished by
Imperiali who derivatised the peptides studied using a
dansyl group.12c,d In our case, the dihydroxybenzoate
group, in addition to metal chelation shows excellent
¯uorescence properties with excitation of 6a at 305 nm
leading to strong emission at 385 nm and excitation of
6b at 293 nm leading to strong emission at 360 nm. The
results of metal binding studies with Fe(III) chloride,
Mn(III) acetate and Cu(II) acetate are shown in Figure
1. Spectrum 1 shows that a progressive quenching of
¯uorescence occurs up to 0.5 molar equivalents of Fe(III)
to 6a. Spectrum 2 shows a similar progressive quenching
of ¯uorescence emission of 6a on addition of Mn(III).
However, in this case complete quenching requires 1
molar equivalent of Mn(III). Spectrum 3 shows the
results obtained for 6a and Cu(II). This latter combina-
tion closely mirrors the results for 6a and Mn(III). From
this series of results, we conclude that Fe(III) forms a
preferential 2:1 complex with 6a involving 2 molecules of
6a to each Fe(III) ion whilst Mn(III) and Cu(II) form
1:1 complexes. The ¯uorescence quenching provides
evidence for binding of metal ion to the catechol unit but
we have no direct evidence of the site(s) of further co-
ordination. Clearly, the histidine imidazole would be the
most favoured option for the next co-ordination site in
6a. This would then produce a 3-point attachment of 6a
to metal ions. The 1:1 complexes formed with Mn(III)
and Cu(II) could also involve metal binding via the
amide groups.
Acknowledgements
We thank the E.P.S.R.C. for a studentship under the
programme in Catalysis and Catalytic Processes (MAA),
the ULIRS NMR and mass spectrometry services and
the B.B.S.R.C. ¯uorescence spectroscopy facility at
King's College London.
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The behaviour of peptide 6b under similar conditions
with Fe(III), Mn(III) and Cu(II) is shown in spectra 4, 5
and 6 respectively. In spectrum 4, ¯uorescence quenching
with Fe(III) is again observed. However, this time it is
much less complete than in the case of 6a and Fe(III) as
judged by the degree of quenching (c.f. spectrum 1).
Additionally, it does not plateau out at 0.5 molar

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