Responsive metal complexes: A click-based "allosteric scorpionate" Complex permits the detection of a biological recognition event by EPR/ENDOR spectroscopy

Article abstract of DOI:10.1002/chem.200802425

Chemical sensing is a mature field, and many effective sensors for small anions and cations have been devised. Metal complexes have been used widely for this purpose, but there are fewer reports of their use in the detection of organic and biological analytes. To date metal complexes have been used in sensing via the direct displacement of a pre-existing ligand by an analyte, or by an adventitious complementarity between the complex and analyte. These strategies do not permit a general approach to the sensing of biological molecules with metal complexes because of the demands to engineer molecular recognition into the complex architecture. We describe a fundamentally new approach to this field - the "allosteric scorpionate" metal complex. The binding partner of a biological analyte is attached to a scorpionate ligand on a metal complex, remote from the metal centre. Binding of the analyte causes a change in the primary coordination sphere at the metal, thereby revealing the presence of the biological molecule. We show that azamacrocyclic complexes with a triazole scorpion ligand may be easily assembled with the [3+2] Huisgens 'click' cycloaddition. We demonstrate the synthesis of a biotin-functionalised cyclam derivative using this methodology. This, and our previously communicated zinc sensor, are to the best of our knowledge the first examples of a triazole being employed as a scorpion ligand on an azamacrocycle. Coordination by the triazole to the metal is perturbed by the binding of avidin to the pendant ligand. This event can be sensitively detected with EPR spectroscopy, and the details of the coordination change probed with ENDOR spectroscopy, confirming the loss of the axial triazole nitrogen donor upon binding to avidin. This represents the first metal complex where remote, 'allosteric' coordination of an analyte has been shown to cause a change in the primary coordination sphere of the metal. Since the synthesis is modular and straightforward, other biological ligands may easily be introduced, and the associated binding events may be probed.

Full text of DOI:10.1002/chem.200802425

DOI: 10.1002/chem.200802425
Responsive Metal Complexes: A Click-Based “Allosteric Scorpionate”
Complex Permits the Detection of a Biological Recognition Event by
EPR/ENDOR Spectroscopy
Emiliano Tamanini,[a] Stephen E. J. Rigby,[a] Majid Motevalli,[a] Matthew H. Todd,*[b] and
Michael Watkinson*[a]
Abstract: Chemical sensing is a mature
field, and many effective sensors for
small anions and cations have been de-
vised. Metal complexes have been used
widely for this purpose, but there are
fewer reports of their use in the detec-
tion of organic and biological analytes.
To date metal complexes have been
used in sensing via the direct displace-
ment of a pre-existing ligand by an an-
alyte, or by an adventitious comple-
mentarity between the complex and
analyte. These strategies do not permit
a general approach to the sensing of
biological molecules with metal com-
plexes because of the demands to engi-
neer molecular recognition into the
complex architecture. We describe a
fundamentally new approach to this
a biological analyte is attached to a
scorpionate ligand on a metal complex,
remote from the metal centre. Binding
of the analyte causes a change in the
primary coordination sphere at the
metal, thereby revealing the presence
of the biological molecule. We show
that azamacrocyclic complexes with a
triazole scorpion ligand may be easily
assembled with the [3+2] Huisgens
ꢁclickꢀ cycloaddition. We demonstrate
the synthesis of a biotin-functionalised
cyclam derivative using this methodol-
ogy. This, and our previously communi-
cated zinc sensor, are to the best of our
triazole being employed as a scorpion
ligand on an azamacrocycle. Coordina-
tion by the triazole to the metal is per-
turbed by the binding of avidin to the
pendant ligand. This event can be sen-
sitively detected with EPR spectrosco-
py, and the details of the coordination
change probed with ENDOR spectros-
copy, confirming the loss of the axial
triazole nitrogen donor upon binding
to avidin. This represents the first
metal complex where remote, ꢁalloste-
AHCTUNGTRENGNrUN icꢀ coordination of an analyte has
been shown to cause a change in the
primary coordination sphere of the
metal. Since the synthesis is modular
and straightforward, other biological li-
gands may easily be introduced, and
the associated binding events may be
probed.
knowledge the first examples of
a
Keywords: azamacrocycles click
·
chemistry · copper · EPR spectros-
copy · sensors
field—the
“allosteric
scorpionate”
metal complex. The binding partner of
Introduction
Metal complexes have been used extensively as drugs and
imaging agents but there is an increasing awareness that
their full potential in medicine and biology has not yet been
exploited since metal complexes are typically non-selective
in their interactions with biological molecules.[1] Whilst
direct changes to a metalꢀs coordination environment as a
result of analyte binding has been used as a sensing strategy
for many years,[2] for example through the displacement of a
coordinating dye (Strategy I, Figure 1),[3] the use of metal
complexes in the sensing of organic and biological analytes
has been much less explored.[4] Hamachi and co-workers
used a zinc(II) complex for the sensing of phosphorylated
peptides that detected the phosphate moiety rather than a
[a] Dr. E. Tamanini, Dr. S. E. J. Rigby, M. Motevalli, Dr. M. Watkinson
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road, London E1 4NS (UK)
Fax : (+44)20-7882-7427
E-mail: m.watkinson@qmul.ac.uk
[b] Dr. M. H. Todd
School of Chemistry
The University of Sydney
NSW 2006 (Australia)
Fax : (+61)2-9351-3329
E-mail: m.todd@chem.usyd.edu.au
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200802425.
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A new approach would be the generation of complexes
where selective binding of an analyte to a remote, pendant
site alters the primary coordination sphere and thereby con-
verts the metal centre into an active catalytic site or one
that emits a detectable signal that can be spectroscopically
probed to elicit information on the nature and strength of
the interaction (Strategy V, Figure 1). This may be thought
of as an ꢁallosteric scorpionꢀ approach. Specificity between
ligand and receptor is not reliant on the structure of the
metal complex itself, which acts as a reporter. We present
here the first demonstration of such a sensing strategy.
Azamacrocycles, such as cyclam, are the perfect scaffold
for this strategy since they exhibit robust coordination
chemistry (including a range of scorpion complexes),[15]
which has resulted in their application in a diverse number
of biological and medicinal areas.[16] It has previously been
reported that the stereochemistry of five-coordinate cop-
per(II) complexes with pentadentate nitrogen donors is very
susceptible to changes in the local environment about the
metal centre.[17] Given this and the weak binding of the axial
ligand in CuII complexes due to the Jahn–Teller effect we se-
lected this basic system for a proof of concept study of the
allosteric scorpion model. The lability of pendant arm scor-
pionate ligands has been known for some time through ob-
servation of changes in the metalꢀs coordination sphere.[15d]
Recently the lability of primary amine scorpionate donors in
aqueous solution has been shown to be essentially independ-
ent of the metal ion in complexes of CoII, NiII and CuII.[18]
Given this wealth of basic work, there are surprisingly
few applications of scorpion complexes in sensing. Those
that have appeared rely on either acid labilisation of the
scorpion ligand resulting in molecular devices capable of
monitoring changes in pH,[19] or on competitive substitution
of the labile ligand by an anion[2b] or nucleobase[10] as de-
scribed above. To the best of our knowledge no reports have
appeared using scorpion complexes in the general sensing of
biological molecules, and there have been no reports of
complexes built to function on an allosteric scorpion model.
We hypothesised that a strong binding interaction with a
biological analyte might be able to perturb the primary co-
ordination sphere of the metal by remote binding of the
scorpion arm. In our design the presence of the biological
analyte is relayed to a change in the metalꢀs primary coordi-
nation by selective binding to the sidearm. This offers signif-
icant potential for the sensing of biological molecules, due
to the selectivity in the interaction with the biological target
of interest, and the generality of the design.
Figure 1. Sensing strategies for organic/biological analytes involving
metal complexes. The mechanism demonstrated here is the “allosteric
scorpion” model.
direct detection of the biological molecule itself.[5] Alterna-
tively, there can be an adventitious recognition event be-
tween complex and analyte (Strategy II, Figure 1): several
(largely N-containing) natural products such as flavins and
amino acids have also been shown to coordinate effectively
to the metal centre of azamacrocyclic complexes[6] and
direct coordination of amino acid side chains to zinc(II) aza-
macrocycles is involved in the anti-HIV activity of a xylyl
bis-cyclam.[7] The detection of nucleobases by this method is
well-known,[8] but Kimura and Aoki have also demonstrated
the detection of nucleobases by the displacement of a metal-
bound scorpion ligand (Strategy III, Figure 1).[9] In the case
of a scorpionate coumarin ligand, sensing of anions was pos-
sible in aqueous solution.[10] It is also possible to detect bio-
logical analytes with metal complexes by photoelectron
transfer between the metal centre and an appropriately deri-
vatised analyte brought together by hydrogen bonding
(Strategy IV, Figure 1),[11] and sensing has also been well-ex-
plored using electron transfer between antenna molecules
attached to macrocyclic lanthanide complexes.[12] Metal
complexes hold great promise for the detection of diverse
organic and biological analytes—they may be assembled
through a metal templating effect,[13] and Hayashi and co-
workers have shown, through the use of reconstituted myo-
globin, that protein binding can alter the photophysical
properties of an encapsulated zinc–porphyrin complex.[14]
However, in all of the chemical sensing strategies de-
scribed above that rely on a change in the coordination en-
vironment of the metal complex for analyte detection there
is a de facto requirement for direct binding of the analyte to
the metal centre. Selectivity in these systems therefore also
requires either the construction of a complex that is comple-
mentary to the analyte (i.e. conversion of the complex to a
host) or adventitious complementarity between the macro-
cycle and the analyte.
We report here a proof of concept study of this allosteric
scorpion design, and use EPR and ENDOR spectroscopy to
detect a biological recognition event, namely the binding of
biotin by the protein avidin at biologically relevant concen-
trations of analyte. We further show the nature of the
change in the coordination chemistry of the metal complex
by ENDOR spectroscopy, which is due to the programmed
change in the coordination mode of a scorpionate ligand in
line with the design.
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M. H. Todd, M. Watkinson et al.
The interaction
between
biotin and avidin represents the
strongest known interaction in
Nature resulting in it finding a
wide array of applications in
biology and chemistry.[20] Only
rarely however, has the system
been employed to probe
changes at a metal centre, al-
though it has been shown that
biotinylated ruthenium and iron
complexes show changes by lu-
minescence spectroscopy[21] and
cyclic voltammetry[22] upon
avidin binding. An obvious
method to probe changes in the
coordination environment of a
metal is UV/Vis spectroscopy;
however, we elected to use
EPR spectroscopy since it is in-
herently sensitive and respon-
sive to subtle changes in the co-
ordination sphere of a metal,
and can operate in the micro-
Scheme 1. Synthesis of cyclam–biotin complex 6. a) OHC
HATU, DIPEA, DMAP; d) TFA (20%) in CH2Cl2; e) CuAHCUTNGTRENNUNG
G
ACHTUGNRTEN(NUGN OAc)3; b) Pd/C, H2; c) biotin,
molar range[23] required in biological samples, precluding the
need for high concentrations of the analyte, in this case
avidin. Further, it was anticipated that fine details of any
changes in the coordination chemistry of the complex upon
avidin binding could be elucidated with ENDOR spectros-
copy.
Significant levels of asymmetric induction have been at-
tained in catalytic reactions as a result of the avidin–biotin
interaction affecting the secondary coordination sphere of a
metal, suggesting that binding of the large proteinaceous an-
alyte might be sufficient to perturb the local environment
about the metal centre to allow detection of the binding
event.[24] It was therefore essential that we prepared a
simple, first-generation biotinylated metal complex and ex-
amined whether any changes in the metal centre could be
observed by EPR spectroscopy upon avidin binding.
Results and Discussion
A copper(II) complex of a cross-bridged cyclam analogue
functionalised with biotin has previously been prepared, but
binding to (strept)avidin was not demonstrated.[25] We pre-
pared the related, novel biotinylated copper(II) complex 6
(Scheme 1) and unequivocally demonstrated its binding to
avidin using standard titration methods (see the Supporting
Information).
Figure 2. X-band EPR spectra at 20 K of a 100 mm aqueous solution of a)
compound 6, b) compound 6 plus one equivalent of avidin c) the differ-
ence spectrum b) minus spectrum a).
While there was clearly binding between 6 and avidin, as
evidenced by the standard titrimetric assay, there were no
changes in the EPR spectrum of the complex upon addition
of avidin. Figure 2a and b are both consistent with the pres-
ence of square-planar CuII. The relatively small g anisotropy,
rise to the ꢁovershootꢀ feature at high field, are typical of
planar N4 complexes. Importantly, the difference spectrum
(Figure 2c) clearly shows the spectra to be essentially identi-
Cu
g? =2.06 and gjj =2.19, and the Ajj value of 198 G, giving
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Responsive Metal Complexes
FULL PAPER
cal. As the unpaired electron of the CuII ion resides princi-
pally in the dx2ꢀy2 orbital, changes in coordination in this
plane will have the greatest influence on its EPR spectrum.
Given that this plane is occupied by the nitrogen ligands of
the macrocycle, it appears that binding to avidin can only
induce perturbations of the secondary coordination sphere
which is too distant from the dx2ꢀy2 orbital to influence the
EPR spectrum and suggested as anticipated that a change in
the primary coordination sphere was required for transduc-
tion of the binding event.
We were attracted by the broad utility of the Sharpless/
Huisgens CuI-catalyzed [3+2] click cycloaddition of alkynes
and azides to generate the ꢁscorpionꢀ ligand.[26] The reaction
has been recently used in the incorporation of biomolecules
into radiolabelled complexes[27] and in lanthanide-containing
dendrimers in which the resulting triazole acts as a non-co-
ordinating photosensitizer.[28] We hypothesised that the
triazole could be perfectly situated to act as a chelating
ligand at the weakly coordinating axial position and we have
demonstrated this principle by developing a highly selective
sensor for zinc using this approach.[29] This sensor and the
present report are to the best of our knowledge the first ex-
amples of a triazole being used as a scorpionate ligand on
an azamacrocycle. Hence to test the robustness of the coor-
dination of the triazole we prepared model complex 10
(Scheme 2), and were delighted to find that single-crystal X-
ray crystallography revealed that the expected coordination
geometry was adopted with the triazole occupying the apical
site and the trans I (R,S,R,S) (++++) conformation being
adopted (Figure 3).
Figure 3. Single-crystal X-ray structure of 10 (cation) showing the scor-
pion role of the triazole moiety.[30]
NaHCO3, Na2CO3, NaH2PO4, KH2PO4, Na3PO4 and
(CO2H)2). In only two cases did we observe significant
changes in the UV/Vis spectra (for the phosphate and hy-
droxide anions, Figure 4), due to the adversely high pH of
these solutions (also see Supporting Information). These
measurements clearly demonstrate that the interaction be-
tween the triazole and the copper(II) centre is relatively
Bond lengths about the copper(II) centre are typical of
ꢀ
ꢀ
ꢀ
related complexes (Cu1 N1 2.051, Cu1 -N2 2.002, Cu1 N3
ꢀ
ꢀ
2.005, Cu1 N4 2.025, Cu1 N5 2.323 ꢃ). Given that we
wished to use the perturbation of the scorpion-like interac-
tion as a means of detecting the nature of the interaction of
the proteinaceous ligand with the complex we were keen to
investigate whether the addition of large quantities of com-
peting anions affected the coordination sphere of the com-
plex. We therefore monitored the effect of the addition of
large excesses (ca. 200 equiv) of a number of anionic ligands
on 10 (NaF, NaCl, KBr, KI, NaOH, NaOAc, K2SO4,
Figure 4. Visual detection of the perturbation of the coordination sphere
of 10 (left hand vial) in the presence of hydroxide (right hand vial).
robust and that perturbations in
the
primary
coordination
sphere of complexes of this
type are not likely to occur in
biological samples as a result of
simple anion exchange for the
triazole, unlike in closely relat-
ed cyclen systems in which the
pendant arm donors were labile
in the presence of a number of
anions at neutral pH.[10a,11] We
were therefore encouraged that
the ꢁclickꢀ-generated system had
potential in the detection of
protein binding.
Scheme 2. Synthesis of model cyclam-click complex 10. a) Propargyl bromide, Na2CO3; b) benzyl azide,
.
CuSO4, Na ascorbate; c) TFA (20%) in CH2Cl2; d) Cu
N
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M. H. Todd, M. Watkinson et al.
noise ratio is relatively poor as
a consequence of the biological-
ly relevant concentration of 14
used, clear negative features in
the gjj region of the difference
spectrum are observed which
are marked with arrows and are
consistent with a significant re-
duction in the concentration of
the five-coordinate species on
binding to avidin, although the
feature at 3058 G is poorly re-
solved due to line width. In ad-
dition an apparently first deriv-
ative line at 2948 G (marked 1
in the difference spectrum) re-
sults from changes in intensity
of the spectra arising from both
four- and five-coordinate spe-
cies. Even more obvious and
significant deviations are ob-
served in the g? region of the
difference spectrum reflecting
the increased magnitude and
shift in this signal upon avidin
Scheme 3. Synthesis of cyclam-click-biotin conjugate 14. a) 3-Azidopropylamine, CuSO4, Na ascorbate; b)
biotin, HATU, DIPEA, DMAP; c) TFA (20%) in CH2Cl2; d) Cu(ClO4)2·6H2O, pH 8.
AHCTUNGTRENNUNG
To validate our allosteric scorpion concept we prepared
complex 14 (Scheme 3). The click cycloaddition allows the
incorporation of biotin onto azamacrocycles more easily
than traditional synthetic approaches.[31]
binding. This clearly shows that the binding of avidin is able
to perturb the primary coordination sphere of the metal and
that this process can be detected spectroscopically.
Although these EPR measurements provide qualitative
evidence that the coordination sphere of the copper(II)
The binding between 14 and avidin was shown to be es-
sentially identical to that between avidin and 6 (see the Sup-
porting Information). The EPR spectrum of 14 at biological-
ly relevant concentrations in frozen solution (Figure 5a) was
clearly different to that of 6 and now indicated that a mix-
ture of two coordination environments was present, namely
a five-coordinate copper(II) species resulting from coordina-
tion of the triazole and a square-planar copper(II) species
that is analogous to 6. Two overlapping contributions to the
EPR spectrum are evident. One is quite similar to the spec-
trum of 6 having g? =2.06 and gjj =2.19 but with a smaller
Cu
Ajj of 186 G. The second contributing spectrum (the gjj
features of which are marked with * in Figure 5a) exhibits
gjj =2.25 and AjjCu =176 G, while maintaining the g? =2.06.
These parameters are indicative of a five-coordinate CuII
ion in a square geometry (effectively a ꢁsquare-based
pyramidꢀ).[32] Therefore in frozen solution (and presumably
in liquid solution) 14 exists as an equilibrium mixture of
four- and five-coordinate species. Upon addition of avidin,
subtle but significant changes to the EPR spectrum are ob-
served, (Figure 5b) and the gjj =2.25 contribution is greatly
reduced in intensity, while the g? =2.19 contribution shows
a relative increase in intensity. This suggests an interconver-
sion between the two forms of 14 observed on binding to
avidin and is consistent with the conversion of the five-coor-
dinate copper(II) to square-planar copper(II) as envisioned
in the allosteric scorpion design. This is clearly displayed in
the difference spectrum (Figure 5c). Although the signal to
Figure 5. X-band EPR spectra of a 100 mm aqueous solution at 20 K of a)
compound 14, b) compound 14 plus one equivalent of avidin, c) differ-
ence spectrum bꢀa.
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Responsive Metal Complexes
FULL PAPER
centre in 14 changes upon binding of avidin which is entirely
consistent with the labilisation of the scorpionate triazole,
we sought to probe changes to the primary coordination
sphere of the metal ion in frozen solution in more detail
using ENDOR (electron nuclear double resonance) spec-
troscopy. Figure 6 shows the X-band Davies FID-detected
the metal centre through a change in the metalꢀs primary co-
ordination environment. We have developed a macrocycle
with a scorpionate triazole ligand for this purpose, derived
from a click cycloaddition. The ease of this synthesis, and
the wide range of macrocycles and biological moieties that
may be coupled, make the approach appealingly modular.
The allosteric scorpion approach is also general, in that the
binding event is not dependent on the structure of the metal
complex, and complementarity can exploit inherent binding
capabilities of any receptor and ligand. Since our design has
been shown to sense a biological molecule at biologically
relevant concentrations we suggest this design could have
wide application to target diverse biomedical applications
that would benefit from selective, responsive metal com-
plexes. We assume that a steric interaction between the bio-
tinylated ligand and the bulky analyte avidin forces dissocia-
tion of the triazole ligand from the metal centre, but the
exact mechanism of this key change is currently being inves-
tigated in our laboratories.
Experimental Section
NMR spectra for all novel compounds, titration curves for avidin–HABA
with complexes 6 and 14, and UV/Vis spectroscopic changes for complex
10 in response to addition of anions may be found in the Supporting In-
formation.
Figure 6. X-band Davies FID-detected ENDOR spectra of frozen solu-
tions of 300 mm aqueous solutions of a) compound 14, b) compound 14
plus avidin and c) the difference spectrum aꢀb.
11-(3-Benzyloxycarbonylaminopropyl)-1,4,8,11-tetraaza-cyclotetradecane-
1,4,8-tricarboxylic acid tri-tert-butyl ester (2): N-Cbz-3-Aminopropanal
(benzyl 3-oxopropylcarbamate)[34] (305 mg, 1.47 mmol) in THF (5 mL)
was added to a solution of tri-Boc cyclam (1)[19a] (882 mg, 1.76 mmol) in
ENDOR spectra of 14 recorded in frozen aqueous solutions
at 20 K in the absence and presence of avidin together with
the difference spectrum.[33] The spectrum in the absence of
avidin (Figure 6a) clearly shows the presence of strong 14N
hyperfine coupling of 37.2 MHz, shown as 1, which is signifi-
cantly enhanced when compared to that of the avidin-bound
THF (15 mL). NaBHACTHNUTRGNEUNG(OAc)3 (934 mg, 4.41 mmol) was added and the
mixture was stirred at room temperature for 16 h. The solvent was re-
moved in vacuo, the residue was dissolved in CH2Cl2 (100 mL) and
washed with aqueous NaHCO3 (5%) and water. The organic phase was
dried over MgSO4, filtered, concentrated and purified by flash chroma-
tography on silica gel (EtOAc 100%) to give 2 as a white solid (815 mg,
80% yield). m.p. 44–478C; 1H NMR (270 MHz, CDCl3): d=7.36–7.28
(m, 5H), 5.56 (bs, 1H), 5.07 (s, 2H), 3.38–3.14 (m, 14H), 2.57–2.47 (m,
2H), 2.46–2.32 (m, 4H), 1.88–1.54 (m, 6H), 1.44 (s, 1H), 1.42 ppm (s,
9H); 13C NMR (67.5 MHz, CDCl3): d=156.4 (C), 155.6 (C), 155.3 (C),
136.8 (C), 128.3 (CH), 127.9 (CH), 127.8 (CH), 79.3 (3 ꢄ C), 66.1 (CH2),
53.4 (CH2), 52.7 (CH2), 51.5 (CH2), 47.7 (CH2), 47.3 (3 ꢄ CH2), 46.7
(CH2), 45.9 (CH2), 39.6 (CH2), 28.4 (9 ꢄ CH3), 26.6 ppm (2 ꢄ CH2); IR
(CH2Cl2): n˜ =3055, 2985, 1710, 1685 cmꢀ1; HRMS (ES) calcd for [M+H]+,
(C36H62N5O8) 692.4593, found 692.4599.
1
compound (Figure 6b). There are also additional H hyper-
fine couplings of 13.3 MHz and 11.2 MHz marked as 2 and
3. The magnitude of the 14N coupling is typical of nitrogen
directly coordinated to the paramagnetic metal ion, while
the 1H hyperfine couplings are suggestive of protons at-
tached to a metal ion ligating group, presumably the methy-
ACHTUNGTRENNUNGlene protons linking the triazole to the cyclam framework
which will be inequivalent when the triazole is coordinated
to the copper centre. While the additional features in the
spectrum are not quantitative, they unequivocally show that
in a portion of 14 the copper(II) ion is five-coordinate and
that this is converted to a four-coordinate species, through
the loss of a nitrogen ligand, on binding to avidin. This is en-
tirely consistent with our proposed allosteric scorpion
scheme shown in Figure 1.
11-(3-Aminopropyl)-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-tricarboxylic
acid tri-tert-butyl ester (3): Pd/C (10 mol%, 121 mg) was added to 2
(780 mg, 1.13 mmol) dissolved in MeOH (30 mL), and the resulting mix-
ture was stirred under H2 (1 atm.) for 16 h at room temperature. The
crude product was filtered through a short plug of Celite and the solvent
was removed in vacuo to give 3 as a white solid in quantitative yield. The
product was used without further purification: m.p. 47–498C; 1H NMR
(270 MHz, CDCl3): d=3.46–3.12 (m, 12H), 2.82–2.30 (m, 10H), 1.94–1.54
(m, 6H), 1.44 (s, 18H), 1.43 ppm (s, 9H); 13C NMR (67.5 MHz, CDCl3):
d=155.7 (2 ꢄ C), 155.5 (C), 79.5 (3 ꢄ C), 53.4 (CH2), 52.9 (CH2), 51.4
(CH2), 48.2 (2 ꢄ CH2), 47.4 (m, 4 ꢄ CH2), 45.8 (CH2), 40.2 (CH2), 28.5
(9
ꢄ CH3), 26.6 ppm (m, 2 ꢄ CH2); IR (CH2Cl2): n˜ =3055, 2985,
Conclusions
1686 cmꢀ1; HRMS (ES) calcd for [M+H]+, (C28H56N5O6) 558.4225, found
558.4228.
We have shown it is possible to detect the binding of a bio-
logical molecule to a metal complex that operates on an al-
losteric scorpion model, where remote binding is relayed to
11-{3-[5-(2-OxohexahydrothienoACHTUNTRGNEU[GN 3,4-d]imidazol-4-yl)pentanoylamino]-
propyl}-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylic acid tri-tert-
butyl ester (4): Biotin (374 mg, 1.67 mmol), HATU (634 mg, 1.67 mmol),
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M. H. Todd, M. Watkinson et al.
DIPEA (580 mL, 3.33 mmol) and DMAP (13 mg, 10 mol%) were pre-ac-
tivated in DMF (10 mL) for 10 min at room temperature. Then
gel (EtOAc/n-hexane 6:4 to 8:2) to give 8 as a white solid (552 mg, 89%
yield): m.p. 52–548C; 1H MNR (CDCl3, 270 MHz): d=7.36–7.12 (m,
6H), 5.44 (s, 2H), 3.68 (s, 2H), 3.36–3.06 (m, 12H), 2.58–2.42 (m, 2H),
2.42–2.22 (m, 2H), 1.88–1.70 (m, 2H), 1.70–1.52 (m, 2H), 1.38 (s, 18H),
1.34 ppm (s, 9H); 13C NMR (CDCl3, 67.5 MHz): d=155.5, 155.3, 143.9,
134.6, 128.9, 128.5, 127.8, 122.2, 79.3, 53.8, 52.4, 50.7, 48.6, 47.3, 46.6, 45.2,
28.3, 28.2, 26.5 ppm; IR (CH2Cl2): n˜ =3055, 2985, 1685 cmꢀ1; HRMS (ES)
calcd for [M+H]+, (C35H58N7O6) 672.4443, found 672.4449.
3
(620 mg, 1.11 mmol) was added and the yellow solution stirred for 24 h at
room temperature. The solvent was evaporated in vacuo and the crude
was purified by flash column chromatography on silica gel (CHCl3/
MeOH, 90:10 rising to 85:15) to give 4 as a pale yellow solid (450 mg,
52% yield): m.p. 109–1118C; 1H NMR (270 MHz, CDCl3): d=6.24 (br,
1H, NH), 5.49 (br, 1H, NH), 4.53–4.42 (m, 1H), 4.34–4.23 (m, 1H), 3.46–
3.04 (m, 15H), 2.96–2.82 (m, 1H), 2.71 (d, J=12.8 Hz, 1H), 2.58–2.47 (m,
2H), 2.46–2.24 (m, 4H), 2.24–1.98 (m, 4H), 1.92–1.48 (m, 10H), 1.43 ppm
(s, 27H); 13C NMR (67.5 MHz, CDCl3): d=173.5 (C), 164.1 (C), 155.6
(m, 3 ꢄ C), 79.8 (m, 3 ꢄ C), 61.9 (CH), 60.3 (CH), 55.8 (CH), 51.8 (m, 3 ꢄ
CH2), 48.1–45.5 (m, 7 ꢄ CH2), 40.6 (CH2), 37.5 (CH2), 36.0 (CH2), 28.6
(9 ꢄ CH3), 28.4 (CH2), 28.1 (CH2), 26.5 (m, 2 ꢄ CH2), 25.8 ppm (CH2);
IR (CH2Cl2): n˜ =1685, 1465, 1265 cmꢀ1; HRMS (ES) calcd for [M+H]+,
(C38H70N7O8S) 784.5001, found 784.5000.
11-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11-tetraazacyclotetradec-
AHCTUNGERTGaNNUN netrihydrotrifluoroacetate (9): Compound 8 (200 mg, 0.298 mmol) was
deprotected with 20% TFA in DCM (5 mL) for 3 h at room temperature.
The solvent was removed in vacuo to give 9 as a colourless glue (209 mg,
98% yield); 1H MNR (D2O; 270 MHz): d=8.08 (s, 1H), 7.46–7.28 (m,
5H), 5.56 (s, 2H), 4.14 (s, 2H), 3.54–3.32 (m, 6H), 3.32–3.12 (m, 8H),
2.99 (pseudo t, J=6.8 Hz, 2H), 2.18–1.96 ppm (m, 4H); 13C NMR (D2O;
67.5 MHz): d=162.5 (q, TFA), 138.1, 134.4, 129.2, 128.9, 128.2, 126.8,
116.3 (q, TFA), 54.2, 48.9, 47.9, 45.9, 42.1, 41.8, 41.6, 39.2, 39.1, 38.4, 19.4,
19.2 ppm; IR (CH2Cl2): n˜ =3500–2100 (br), 3433, 2854, 2530, 1774,
1670 cmꢀ1; HRMS (ES) calcd for [M+H]+, (C20H34N7) 372.2870, found
372.2870.
11-{3-[5-(2-Oxohexahydrothieno
ACHTUNGTRENNUNG
propyl}-1,4,8,11-tetraazacyclotetradecanetrihydrotrifluoroacetate
Compound 4 (219 mg, 0.28 mmol) was dissolved in TFA (20% in CH2Cl2,
10 mL) and the resulting solution was stirred at room temperature over-
night. The solvent was removed in vacuo to give 5 as a pale yellow solid
Complex 10: Compound 9 (212 mg, 0.298 mmol) was dissolved in water
(3 mL) and 2 N NaOH was added dropwise to adjust the pH to 8. A so-
lution of CuSO4 6H2O (110 mg, 0.298 mmol) in MeOH (3 mL) was
added dropwise and the dark blue solution was stirred overnight at room
temperature. The solvent was evaporated, the residue dissolved in
MeOH (10 mL) and filtered through a plug of Celite. MeOH was evapo-
rated in vacuo to give 10 as a dark purple powder (159 mg, 84% yield).
Dark purple crystals, suitable for X-ray crystallography, were obtained by
slow diffusion of toluene into a solution of complex 10 in acetonitrile: IR
(CH3CN): n˜ =1685, 1635, 1103, 1037, 748 cmꢀ1; HRMS (ES) calcd for
[M+ClO4]+, (C20H33O4N7ClCu) 533.1573, found 533.1579.
1
(229 mg, quantitative): m.p. 41–438C; H NMR (270 MHz; D2O): d=4.61
(dd, J=7.9, 4.7 Hz, 1H), 4.41 (dd, J=7.9, 4.4 Hz, 1H), 3.66–3.16 (m,
20H), 3.10 (m, 1H), 3.00 (dd, J=12.8, 4.7 Hz, 1H), 2.77 (d, J=12.8 Hz,
1H), 2.28 (t, J=6.9 Hz, 2H), 2.18–1.98 (m, 4H), 1.97–1.79 (m, 2H), 1.78–
1.28 ppm (m, 6H); 13C NMR (100 MHz, D2O): d=177.6 (C), 165.7 (C),
163.3 (q, C, TFA), 116.7 (q, C, TFA), 62.4 (CH), 60.6 (CH), 55.8 (CH),
51.9 (CH2), 49.8 (CH2), 47.4 (CH2), 45.2–42.0 (m, 4 ꢄ CH2), 40.7 (2 ꢄ
CH2), 40.0 (CH2), 36.6 (CH2), 35.7 (CH2), 28.3 (CH2), 28.0 (CH2), 25.4
(CH2), 23.9 (CH2), 20.9 (CH2), 19.7 ppm (CH2); IR (CH2Cl2): n˜ = 3294
(br), 3055, 2985, 1681 (br) cmꢀ1 HRMS (ES) calcd for [M+H]+,
;
(C23H46N7O2S) 484.3428, found 484.3430.
3-Azido-propylamine (15):[35] Sodium azide (221 mg, 3.4 mmol) was
added to a solution of 1-bromo-3-aminopropane hydrobromide (438 mg,
2.0 mmol) in water (10 mL), and the mixture was stirred at 808C over-
night. The reaction mixture was cooled in an ice bath and diethyl ether
(20 mL) and NaOH (60 mg) were added, keeping the temperature below
108C. After separation of the organic phase, the aqueous phase was ex-
tracted with more Et2O (2ꢄ20 mL). The organic phases were collected,
dried over MgSO4 and filtered. The solvent was carefully removed under
reduced pressure (water bath temperature ca. 308C). The product (15)
was obtained as a colourless liquid (130 mg, ca. 55% yield). Because of
the low boiling point of 15 (ca. 48–508C at 15 mm Hg) the solvent was
not completely removed and a small amount of Et2O was still present,
Complex 6: Compound 5 (200 mg, 0.097 mmol) was dissolved in MeOH/
H2O (1:1, 6 mL) and the pH of the solution was adjusted to 8 by drop-
.
wise addition of 2n NaOH.
A
solution of CuSO4 6H2O (36 mg,
0.097 mmol) in MeOH (1 mL) was added dropwise and the dark purple
solution was stirred overnight at room temperature. The solvent was re-
moved in vacuo, the residue dissolved in MeOH (10 mL) and filtered
through a plug of Celite. MeOH was removed in vacuo to give a dark
purple powder which was recrystallized from hot EtOH/Et2O to give
complex 6 as dark purple crystals (55 mg, 75% yield). IR (CH2Cl2): n˜ =
1681, 1107, 735 cmꢀ1
;
HRMS (ES) calcd for [M+TFA]+,
(C25H45O4N7CuF3S) 659.2496, found 659.2492.
1
1
observed in the H NMR spectrum. H MNR (CDCl3; 270 MHz): d=3.34
(t, J=6.7 Hz, 2H), 2.77 (t, J=6.8 Hz, 2H), 1.70 (q, J=6.7 Hz, 2H),
1.47 ppm (bs, 2H, NH2).
11-Prop-2-ynyl-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylic acid
tri-tert-butyl ester (7): To a solution of 1 (250 mg, 0.50 mmol) in CH3CN
(15 mL) were added Na2CO3 (212 mg, 1 mmol) and propargyl bromide
(67 mL, 0.60 mmol). The mixture was heated at reflux (858C) overnight.
The insoluble salts were removed by filtration and the solvent removed
in vacuo. The crude material was purified by flash column chromatogra-
phy on silica gel to give 7 as a white solid (211 mg, 78% yield): m.p. 47–
11-[1-(3-Amino-propyl)-1H-[1,2,3]triazol-4-ylmethyl]-1,4,8,11-tetraazacy-
clotetradecane-1,4,8-tricarboxylic acid tri-tert-butyl ester (11): To a solu-
tion of 7 (200 mg, 0.372 mmol) and 15 (55 mg, 0.557 mmol) in H2O/
tBuOH (1:1, 3 mL), CuSO4·5H2O (5 mol%, 4.6 mg dissolved in H2O
(0.5 mL)) and sodium ascorbate (10 mol%, 7.4 mg, dissolved in H2O
(0.5 mL)) were added under nitrogen. The yellow solution was stirred at
room temperature overnight. Water (5 mL) was added and the mixture
was extracted with CH2Cl2 (3ꢄ20 mL). The organic phase was washed
with 5% aqueous NaHCO3, dried over MgSO4, filtered and concentrated
to give 11 as a pale yellow solid (214 mg, 90% yield) which was used
without further purification in the next step. An analytical sample was
purified through a short silica column (CHCl3/MeOH, 8:2). 1H NMR
(CDCl3; 400 MHz): d=7.48 (s, 1H), 4.54–4.38 (m, 2H), 3.74–3.60 (m,
2H), 3.40–3.14 (m, 14H), 3.85 (bs, 2H, NH2), 2.58–2.46 (m, 2H), 2.38–
2.26 (m, 2H), 2.12–1.92 (m, 2H), 1.88–1.72 (m, 2H), 1.68–1.56 (m, 2H),
1.36 ppm (s, 27H); 13C NMR (CDCl3; 67.5 MHz): d=155.5, 155.3, 143.1,
122.4, 79.2, 52.7, 50.9, 48.4, 47.3, 46.6, 45.1, 38.0, 33.0, 28.2, 26.4 ppm; IR
(CH2Cl2): n˜ =3455, 2337, 1685 cmꢀ1; HRMS (ES) calcd for [M+H]+,
(C31H59N8O6) 639.4552, found. 639.4553.
1
498C; H MNR (CDCl3, 270 MHz): d=3.48–3.15 (m, 14H), 2.71–2.58 (m,
2H), 2.49 (t, J=5.4 Hz, 2H), 2.14 (s, 1H), 1.98–1.77 (m, 2H), 1.76–1.60
(m, 2H), 1.44 ppm (s, 27H); 13C NMR (CDCl3; 67.5 MHz): d=155.8 (C),
155.5 (2 ꢄ C), 79.6 (2 ꢄ C), 79.5 (C), 77.9 (C), 73.2 (CH), 53.0 (CH2),
51.9 (CH2), 50.7 (CH2), 48.0 (CH2), 47.5 (CH2), 46.9 (CH2), 46.7 (CH2),
44.8 (CH2), 41.9 (CH2), 28.5 (9
ꢄ CH3), 25.5 ppm (2 ꢄ CH2); IR
(CH2Cl2): n˜ =3301, 2135, 1685 cmꢀ1; HRMS (ES) calcd for [M+H]+,
(C28H51N4O6) 539.3803, found. 539.3800.
11-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11-tetraazacyclotetradec-
ACHTUNGTRENNUNGane-1,4,8-tricarboxylic acid tri-tert-butyl ester (8): Benzyl azide (130 mL,
1.02 mmol) was added to a solution of 7 (500 mg, 0.929 mmol) in H2O/
tBuOH (1:1, 20 mL) under nitrogen. CuSO4·5H2O (5 mol%, 12 mg dis-
solved in H2O (1 mL)) and sodium ascorbate (10 mol%, 18 mg dissolved
in H2O (1 mL)) were added. The cloudy solution was stirred under nitro-
gen overnight at room temperature. 5% NaHCO3 (5 mL) was added to
the solution and the product was extracted with CH2Cl2 (3ꢄ40 mL). The
organic phase was dried (MgSO4), filtered and concentrated in vacuo.
The crude material was purified by passage through a short plug of silica
11-(1-{2-[5-(2-OxohexahydrothienoACTHNUTRGNE[UNG 3,4-d]imidazol-4-yl)pentanoylamino]-
propyl}-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11-tetraazacyclotetradecane-
1,4,8-tricarboxylic acid tri-tert-butyl ester (12): Biotin (57 mg,
3726
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 3720 – 3728

Responsive Metal Complexes
FULL PAPER
0.235 mmol), HATU (72 mg, 0.235 mmol), DIPEA (54 mL, 0.312 mmol)
[1] T. W. Hambley, Science 2007, 318, 1392–1393.
and DMAP (10 mol%) were added to
a solution of 11 (100 mg,
[2] a) P. D. Beer, S. R. Bayly, Top. Curr. Chem. 2005, 255, 125–162;
b) R. Martꢅnez-MꢆÇez, F. Sancenꢇn, Chem. Rev. 2003, 103, 4419–
4476; c) P. D. Beer, P. A. Gale, Angew. Chem. 2001, 113, 502–532;
Angew. Chem. Int. Ed. 2001, 40, 486–516; d) P. V. Bernhardt, E. G.
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3156–3159; Angew. Chem. Int. Ed. 2001, 40, 3066–3069; b) S. L.
Tobey, E. V. Anslyn, Org. Lett. 2003, 5, 2029–2031; c) M. S. Han,
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Ed. 2002, 41, 3809–3811; d) H. Aꢈt-Haddou, S. L. Wiskur, V. M.
Lynch, E. V. Anslyn, J. Am. Chem. Soc. 2001, 123, 11296–11297.
[4] For example, Lanthanide–cyclodextrin conjugates for the sensing of
small aromatic molecules: C. M. Rudzinski, W. K. Hartmann, D. G.
Nocera, Coord. Chem. Rev. 1998, 171, 115–123.
0.156 mmol) in DMF (3 mL). The solution was stirred overnight at room
temperature. The solvent was then removed in vacuo and the crude was
purified by flash column chromatography on silica gel (CHCl3/MeOH,
8:2) to give 11 as a pale yellow solid (94 mg, 70% yield): m.p. 95–978C;
1H NMR (CDCl3; 270 MHz): d=7.53 (s, 1H), 6.89 (bs, 1H, NH), 6.39
(bs, 1H, NH), 5.56 (bs, 1H, NH), 4.58–4.56 (m, 2H), 4.38 (t, J=6.4 Hz,
2H), 3.44–3.24 (m, 1H), 3.82–3.62 (m, 2H), 3.44–3.02 (m, 15H), 2.90 (dd,
J=12.8, 4.5 Hz, 1H), 2.72 (d, J=12.8 Hz, 1H), 2.68–2.52 (m, 2H), 2.50–
2.34 (m, 2H), 2.26–2.02 (m, 4H), 1.94–1.54 (m, 10H), 1.44 ppm (s, 27H);
13C NMR (CDCl3; 67.5 MHz): d=173.9 (C), 164.4 (C), 155.9 (2 ꢄ C),
155.6 (2 ꢄ C), 143.4 (C), 123.1 (CH), 79.7 (3 ꢄ C), 61.8 (CH), 60.3 (CH),
55.9 (CH), 51.9 (CH2), 51.1 (CH2), 50.0–46.0 (m, 7 ꢄ CH2), 45.5 (CH2),
40.7 (CH2), 36.3 (CH2), 35.9 (CH2), 30.1 (CH2), 28.5 (9 ꢄ CH3), 28.3
(CH2), 28.0 (CH2), 26.5 (CH2), 25.7 ppm (CH2); IR (CH2Cl2): n˜ =3305,
2098, 1678 cmꢀ1
;
HRMS (ES) calcd for [M+H]+, (C41H73N10O8S)
887.5148, found. 887.5142.
11-(1-{2-[5-(2-OxohexahydrothienoACTHNUTRGNE[UNG 3,4-d]imidazol-4-yl)pentanoylamino]-
[5] a) A. Ojida, Y. Mito-oka, K. Sada, I. Hamachi, J. Am. Chem. Soc.
2004, 126, 2454–2463; b) A. Ojida, Y. Miyahara, T. Kohira, I. Hama-
chi, Biopolymers 2004, 76, 177–184; c) a hybrid biosensor was re-
cently reported that employs a mixture of phosphoprotein binding
domains and metal complex-based chemosensor: T. Anai, E.
Nakata, Y. Koshi, A. Ojida, I. Hamachi, J. Am. Chem. Soc. 2007,
129, 6232–6239; for a similar principle applied to the detection of
phosphorylated natural products: d) H.-W. Rhee, C.-R. Lee, S.-H.
Cho, M.-R. Song, M. Cashel, H. E. Choy, Y.-J. Seok, J.-I. Hong, J.
Am. Chem. Soc. 2008, 130, 784–785; e) S. Aoki, M. Zulkefeli, M.
Shiro, M. Kohsako, K. Takeda, E. Kimura, J. Am. Chem. Soc. 2005,
127, 9129–9139; sensing of phosphorylated membranes: f) K. M.
DiVittorio, W. M. Leevy, E. J. OꢀNeil, J. R. Johnson, S. Vakulenko,
J. D. Morris, K. D. Rosek, N. Serazin, S. Hilkert, S. Hurley, M. Mar-
quez, B. D. Smith, ChemBioChem 2008, 9, 286–293.
propyl}-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11-tetraazacyclotetradecanetri-
hydrotrifluoroacetate (13): Compound 12 (81 mg, 0.0937 mmol) was dis-
solved in TFA (20% in DCM, 5 mL). The resulting solution was stirred
at room temperature overnight and the solvent was removed in vacuo to
give 13 as a pale yellow semi-solid (84 mg, quantitative yield): 1H NMR
(D2O; 270 MHz): d=8.09 (s, 1H), 4.60–4.36 (3 m, total 4H), 4.19 (s, 2H),
3.70–3.14 (m, 18H), 3.13–2.88 (m, 2H), 2.73 (d, J=12.8 Hz, 1H), 2.34–
2.00 (m, 8H), 1.82–1.28 ppm (m, 6H); 13C NMR (CDCl3; 67.5 MHz): d=
176.9, 165.3, 162.4 (q, TFA), 137.1, 127.4, 116.0 (q, TFA), 62.2, 60.4, 55.4,
48.5, 48.4, 45.3, 41.6, 41.3, 39.7, 38.5, 38.4, 38.3, 38.0, 37.8, 36.1, 35.5, 28.8,
28.0, 27.7, 25.2, 18.8 ppm; IR (CH2Cl2): n˜ =1766, 1666 cmꢀ1; HRMS (ES)
calcd for [M+H]+, (C26H49N10O2S) 565.3755, found. 565.3753.
Complex 14: Compound 13 (87 mg, 0.096 mmol) was dissolved in
MeOH/H2O (1:1, 6 mL) and the pH of the solution was adjusted to 8 by
.
dropwise addition of 2n NaOH. A solution of CuSO4 6H2O (35 mg,
[6] a) J. Geduhn, T. Walenzyk, B. Kçnig, Curr. Org. Synth. 2007, 4, 390–
412; b) R. Reichenbach-Klinke, M. Kruppa, B. Kçnig, J. Am. Chem.
Soc. 2002, 124, 12999–13007; c) C. Bazzicalupi, A. Bencini, E.
Berni, A. Bianchi, P. Fornasari, C. Giorgi, B. Valtancoli, Eur. J.
Inorg. Chem. 2003, 1974–1983; d) R. S. Dickins, S. Aime, A. S. Bat-
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0.096 mmol) in MeOH (1 mL) was added dropwise and the dark blue so-
lution was stirred overnight at room temperature. The solvent was evapo-
rated, the residue dissolved in MeOH (10 mL) and filtered through a
plug of Celite. MeOH was evaporated in vacuo to give 14 as dark blue
crystals (63 mg, 79% yield). IR (CH2Cl2): n˜ =1693, 1103, 1037, 752,
725 cmꢀ1
;
HRMS (ES) calcd for [M+ClO4]+, (C26H48O6N10ClCuS)
726.2458, found 726.2465.
HABA assays of complexes 6 and 14:[21] The binding of the copper(II)–
biotin complexes was assessed by HABA assays. Typically, to a mixture
of HABA (300.0 mm) and avidin (7.6 mm) in 50 mm HEPES buffer pH 7.0
(3 mL) were added 10 mL aliquots of the copper(II)–biotin complex
(1 mm). The formation of the copper–avidin adduct was indicated by a
decrease of the absorbance at 500 nm due to the displacement of HABA
from the avidin. By plotting ꢀDA500
versus [Cu]:ACTHNGUTERN[UNG avidin], the binding
of the copper(II)–biotin complex to avidin was determined.
nm
[9] a) S. Aoki, D. Kagata, M. Shiro, K. Takeda, E. Kimura, J. Am.
Chem. Soc. 2004, 126, 13377–13390; b) E. Kimura, H. Kitamura, K.
Ohtani, T. Koike, J. Am. Chem. Soc. 2000, 122, 4668–4677; c) E.
Kimura, T. Koike, Chem. Commun. 1998, 1495–1500; d) E. Kikuta,
M. Murata, N. Katsube, T. Koike, E. Kimura, J. Am. Chem. Soc.
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M. Pelka, M. Subat, I. Dix, P. G. Jones, Eur. J. Org. Chem. 2001,
1943–1949.
Experimental details of EPR spectral analysis: EPR spectra were ob-
tained at X-band using
a Bruker ELEXSYS E500 spectrometer,
equipped with an Oxford Instruments ESR900 liquid helium cryostat.
EPR spectra for blanks (complexes alone) were recorded by using
250 mL of 100 mm solutions of complexes in HEPES buffer. For 1:1 mix-
tures (complexes-avidin) samples were again 250 mL, 100 mm in com-
plexes and 100 mm in avidin.
[10] S. Mizukami, T. Nagano, Y. Urano, A. Odani, K. Kikuchi, J. Am.
Chem. Soc. 2002, 124, 3920–3925.
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388–390; b) C. Turro, C. K. Chang, G. E. Leroi, R. I. Cukier, D. G.
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61, 13–41; f) M. D. Ward, Chem. Soc. Rev. 1997, 26, 365–375.
[12] T. Gunnlaugsson, J. P. Leonard, Chem. Commun. 2005, 3114–3131.
[13] B. Linton, A. D. Hamilton, Chem. Rev. 1997, 97, 1669–1680.
Acknowledgements
We thank the EPSRC (GR/T17014/01) for funding through the Life Sci-
ences Initiative. We are also grateful to the EPSRC for the provision of
the National Mass Spectrometry Service (University of Wales, Swansea)
and the X-ray crystallography service (University of Southampton). We
thank Professor Lisa Hall (Cambridge) for stimulating discussions.
Chem. Eur. J. 2009, 15, 3720 – 3728
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: November 21, 2008
Published online: February 16, 2009
3728
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