Introducing a new family of biotinylated Ir(III)-pyridyltriazole lumophores: Synthesis, photophysics, and preliminary study of avidin-binding properties

Article abstract of DOI:10.1021/om5007962

Six new biotinylated reagents derived from monocationic pyridyl-1,2,3-triazole-based Ir(III) complexes of the general formula [(C^∧N)₂Ir(N^∧N-spacer-X-CO-biotin)]⁺, where HC^∧N is 2-phenylpyridine, N^∧N is the neutral chelating (2-pyridyl)-1,2,3-triazole ligand, the term spacer refers to alkyl (-C₁₁H₂₂) or aromatic (p-phenyl- or 4,4′-biphenyl-) chains, and X is NH or O, have been prepared and characterized. Upon photoexcitation, all six complexes in fluid CH₂Cl₂ and aqueous solutions at room temperature displayed intense - with quantum yields as high as 0.60 (complex 4b) - and quite long-lived blue-green luminescence, corresponding in each case to a vibronically structured emission profile peaking at ca. 480 and ca. 508 nm. These features, in combination with the reduced rigidochromic shift that was observed from the same samples frozen at 77 K, suggested the ³LC-type excited states as the prevalent contributors to the emission. The interactions of these new biotinylated complexes with avidin have been studied by 4′-hydroxyazobenzene-2-carboxylic acid (HABA) assays and emission titrations. In general, the amplification of the emission intensity of the Ir(III) complexes occurring upon their interaction with avidin was observed. It is also worth mentioning how a similar or better response was displayed by [(C^∧N)₂Ir(N^∧N-spacer-O-CO-biotin)]⁺-type complexes, in which biotin is appended to the Ir(III) lumophore through an ester moiety.

Full text of DOI:10.1021/om5007962

Article
pubs.acs.org/Organometallics
Introducing a New Family of Biotinylated Ir(III)-Pyridyltriazole
Lumophores: Synthesis, Photophysics, and Preliminary Study
of Avidin-Binding Properties
Andrea Baschieri,† Sara Muzzioli,† Valentina Fiorini,† Elia Matteucci,† Massimiliano Massi,‡
Letizia Sambri,*,† and Stefano Stagni*,†
†Department of Industrial Chemistry “Toso Montanari”, University of Bologna, Viale Risorgimento 4, I-40136 Bologna, Italy
‡Department of Chemistry, Curtin University, GPO Box U 1987, Perth, Australia, 6845
S
* Supporting Information
ABSTRACT: Six new biotinylated reagents derived from
monocationic pyridyl-1,2,3-triazole-based Ir(III) complexes of
the general formula [(C∧N)2Ir(N∧N-spacer-X-CO-biotin)]+,
where HC∧N is 2-phenylpyridine, N∧N is the neutral chelating
(2-pyridyl)-1,2,3-triazole ligand, the term “spacer” refers to
alkyl (−C11H22) or aromatic (p-phenyl- or 4,4′-biphenyl-)
chains, and X is NH or O, have been prepared and
characterized. Upon photoexcitation, all six complexes in
fluid CH2Cl2 and aqueous solutions at room temperature
displayed intensewith quantum yields as high as 0.60
(complex 4b)and quite long-lived blue-green luminescence,
corresponding in each case to a vibronically structured
emission profile peaking at ca. 480 and ca. 508 nm. These
features, in combination with the reduced rigidochromic shift that was observed from the same samples frozen at 77 K, suggested
3
the LC-type excited states as the prevalent contributors to the emission. The interactions of these new biotinylated complexes
with avidin have been studied by 4′-hydroxyazobenzene-2-carboxylic acid (HABA) assays and emission titrations. In general, the
amplification of the emission intensity of the Ir(III) complexes occurring upon their interaction with avidin was observed. It is
also worth mentioning how a similar or better response was displayed by [(C∧N)2Ir(N∧N-spacer-O-CO-biotin)]+-type
complexes, in which biotin is appended to the Ir(III) lumophore through an ester moiety.
of d6 metal complexes as luminescent probes for bio-
imaging applications has been achieved through the peripheral
INTRODUCTION
■
Environment-responsive luminescence is one of the features that
decoration of the ligand set with biologically relevant functional
has driven some classes of luminescent metal complexes to huge
groups.6 Following this approach, Lo and co-workers reported
scientific popularity.1 In this context, Ru(II) polypyridyls,2 Ir(III)
cyclometalates,3 and tris-carbonyl Re(I) diimine complexes4
numerous examples of Re(I)- and Ir(III)-based luminescent
complexes7 in which one or more coordinated ligands were
are probably the most emblematic examples. The emission
output of these metal-containing molecules can be readily
derivatized with a wide variety of end groupssome examples of
which are represented by biotin, indole, estradiol,5a,6−8 and
perturbed following their interactions with a wide range of
sugars5a,7,9in order to enable the optical detection of the cor-
analytes including cations and anions, O2, and biological targets
such as proteins and DNA.5 The reasons for such pronounced
responding bioconjugation products and, in general, to endow
the modified lumophores with increased lipophilicity. Among the
sensitivity are usually found in their displaying radiative processes
multitude of different options, the one dealing with the
that originate from excited states of “pure” metal to ligand
conjugation of Re(I)- and Ir(III)-based lumophores with biotin
charge-transfer (MLCT) nature, as for the Ru(II) complexes, or
has been extensively explored in light of the extremely high
from emissive levels that can be described as admixtures of
MLCT and ligand-centered (LC)-type states, as in the cases of
affinity that is displayed by biotin with respect to the tetra-
meric glycoprotein avidin. It has to be noted that, while the
third-row transition metals such as Re(I) and, to a greater extent,
introduction of biotin in the backbone of such molecules does
Ir(III). Taken together, these aspects have prompted the
scientific community to design and prepare an incredibly vast
number of ligands to be employed for building new families of
not cause any change in the intrinsic photophysical features of
the metal-based fluorophores, the luminescence output of the
luminescent metal complexes with continuously improved
emission performances and remarkable sensing capabilities.1−3
Received: August 2, 2014
In particular, truly decisive progress in the use of such classes
Published: October 7, 2014
© 2014 American Chemical Society
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Scheme 1. Ligands and Complexes Studied in This Work
biotinylated species is avidin-sensitive.5a,6−8 This feature, in con-
junction with the improvement of the cellular uptake perfor-
mance that derives from the presence of biotin itself, has led to
the use of similar biotin−avidin arrays as the core system of many
assays and probes.10 In this context, apart from the examples of
neutral Ir(III) complexes described by Hong and co-workers in
2008,11 the biotinylated Ir(III)-based species that have been
reported in the literature usually deal with cationic Ir(III)
complexes of the general formula [(C∧N)2Ir(N∧N)]+, which
consist of two identical cyclometalating ligands (C∧N) and one
neutral ancillary ligand (N∧N).5a,6,7 The site for biotinylation
may vary from the cyclometalating C∧N units to the N∧N neutral
one, and, in general, the biotin-containing aliphatic or aromatic
pendant arms have been appended to the structure of such
lumophores through the formation of the corresponding amines
or amides. In this regard, Lo and co-workers have demonstrated
how the use of a similar synthetic strategy might also lead to the
production of Ir(III) multibiotinylated complexes as attractive
examples of cross-linkers for avidin.8b,c However, within the
framework of the design of biotinylated Ir(III) complexes, a great
deal of attention has been paid to the modification and/or the
variation of the cyclometalating C∧N ligands from the archetypal
cyclometalated phenylpyridine (ppy), while the structure of the
neutral N∧N ligand usually consists of 2.2′-bipyridine (bpy) or
1,10-phenanthroline (phen) type backbones.5a,6,7 These latter
considerations introduce the scope of this present work. Here,
we report the synthesis, the study of the photophysical features,
and the preliminary account of the avidin-binding properties of a
series of six new biotinylated Ir(III) complexes (Scheme 1) of
the general formula [(C∧N)2Ir(N∧N-spacer-X-CO-biotin)]+
(Scheme 1), where HC∧N is 2-phenylpyridine and X is NH or
O, that introduce some elements of novelty for this class of
Ir(III)-based lumophores. First, the architecture of the N∧N
ligands described herein differs from those that have been
reported so far since, in all of the six new complexes, it is
represented by a metal-chelating pyridyl-1,2,3-triazole unit. Also,
relative to the design of the biotinylated N∧N ligands, the bio-
tin moiety has been appended to aromatic (para phenyl and
4,4′-biphenyl) or aliphatic (C11H22-) pendant chains through the
formation of the “traditional” amide-type linkage as well as the
first reported examples of ester-type ones (Scheme 1).
RESULTS AND DISCUSSION
■
Ligand Design and Synthesis. We have designed a set of
neutral ligands, that might be schematized as [(N∧N)-spacer-X-
CO-biotin], in order to provide molecules that can coordinate to
the Ir(III) metal center through the N∧N moiety and, at the same
time, are capable of interaction with the avidin target with the
biotin end unit. In particular, all the ligands reported herein have
been prepared in good yield by following a convergent procedure
that entailed the synthesis of the two different subunits
the Ir(III)-coordinating pyridyl triazole and the biotin arm
which were finally combined through the formation of the de-
sired ester or amide (see Schemes 2, 3, and 4 and Figures S1−S14,
Supporting Information). In detail, all the pyridyl triazole
intermediates 8, 14a,b, and 1612 were obtained by following the
well-known CuI-catalyzed azide−alkyne cycloaddition
(CuAAC)13 involving use of 2-ethynylpyridine and the
appropriate alkyl or aryl azides.14 Then, the synthesis of the
ligand 1a was carried out by a two-step procedure involving
the further transformation of the hydroxylpyridine-triazole 8 into
the amine 9, followed by its reaction with biotin-succinimino
derivative 11. On the other hand, the key intermediate 8 was
employed with no further transformation to give ligand 1b in 60%
yield by reaction with the biotin acyl chloride 12 (Scheme 2).
The syntheses of ligands containing aromatic spacers 2a, 2b, 3a,
and 3b are reported in Schemes 3 and 4. In particular, the amide
2a and the ester 2b were obtained in 63% and 55% yields,
respectively, upon the condensation of the pyridyl triazole
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Scheme 2. Synthetic Route to Ligands 1a and 1b
Scheme 3. Synthetic Route to Ligands 2a and 2b
Scheme 4. Synthetic Route to Ligands 3a and 3b
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Scheme 5. Synthesis of Ir(III) Complexes 4a,b, 5a,b, and 6a,b
intermediates 14a and 14b with biotin 10, in accordance with a
previously reported procedure for similar aromatic substrates.15
Relative to the preparation of the ligands 3a,b (Scheme 4), the
pyridyl triazole 16, following a Suzuki coupling procedure, was
first treated with the appropriate phenylboronic derivative 17a,b
in order to obtain the corresponding biphenyl derivatives 18a
and 18b.16 Then, the latter compounds were finally converted
into the biotin ligands 3a (63% yield) and 3b (63% yield).
The target Ir(III) complexes 4−6 were synthesized through
a well-established procedure16 that involved the reaction of
the dimer 19 with a slight molar excess of the proper ligand in a
3:1 dichloromethane/ethanol (v/v) mixture at room temper-
ature (Scheme 5).
luminescence performances of the various biotinylated Ir(III)
triazole complexes described herein. At room temperature, upon
excitation of the 1MLCT feature (λ = 380 nm) of the
corresponding diluted dichloromethane solution (ca. 10−5 M),
each of the new Ir(III) complexes displays bright blue-green
luminescence originating from excited states of triplet character,
as witnessed by the increase of emission quantum yield and the
concomitant elongation of the emission lifetimes that took place
on passing from air-equilibrated to deoxygenated solutions
(Table 1). The emission profiles were found substantially
coincident for all complexes and always consisted of an intense
band centered at ca. 480 nm followed by an almost equally
intense vibronic progression peaking at ca. 508 nm. The oc-
currence of such similarly structured emission spectra from
Ir(III) cyclometalated complexes is usually representative for the
The formation of the desired cationic Ir(III) complexes was
first confirmed by electrospray ionization mass spectrometry
(ESI-MS) with the observation of the patterns of signals whose
(m/z) values matched those of the expected positive molecular
ions. Despite its being complicated from the presence of at least
25 magnetically inequivalent hydrogens or carbons all resonating
in the aromatic region, the analysis of the NMR spectra (1H; 13C)
was also congruent with the formation of the target Ir(III)
species (Supporting Information, Figures S15−S19).
3
3
interplay of LC/3MLCT type emissive excited states. In our
cases, even though the determination of the radiative rate
constants (kr) for the air-equilibrated dichloromethane solutions
provided values (see Table 1) consistent with the 3MLCT nature
of the emissions,3 the very small rigidochromic blue-shift of
the emission maxima that is observed at 77 K indicates the
3
3
likely prevalence of the LC excited states over the MLCT
ones (Table 1, Figure 2, and Figures S18−S22, Supporting
Information). The minor contribution coming from such polar
CT states could also be deduced from observing how all the
biotinylated complexes 4−6a,b retained their luminescence
features in an aqueous environment, with steady-state emission
spectra that were found almost exactly superimposable to the
ones obtained from dichloromethane solutions (Table 1, Figure 2,
and Figures S26−S30, Supporting Information) and radiative
rate constants (kr) lower than those calculated in organic
medium.3 Conversely, as previously reported by Lo and co-
workers for biotinylated Ir(III) substrates,8e a significant
elongation of the emission lifetimes was observed upon passing
from organic to aqueous solutions, an effect that might likely be
explained by considering the mostly apolar character of the
emissive excited states and the lipophilic environment that is
brought by the spacer arms. However, as these trends are general
for the whole series of complexes 4−6a,b, no obvious cor-
relations of the emissive performances of the new Ir(III)
complexes with respect to the different type of linkers (ester or
amide) and/or the nature (aliphatic or aromatic) of the spacer
arms can be made.
Photophysical Properties. The relevant absorption and
emission features of the Ir(III) complexes are summarized in
Table 1.
In dichloromethane solutions maintained at room temper-
ature, all the Ir(III) complexes show quite typical and similar
absorption profiles. Up to 300 nm, each spectrum is dominated
by intense and spin-allowed ligand-centered (1LC) π−π*
transitions involving both the cyclometalating and the ancillary
1
ligands. Weaker and broader spin-allowed MLCT and spin-
forbidden 3MLCT bands are found at longer wavelength (300 to
380 nm) (Figure 1). Apart from some slight hypsochromic shifts
of the absorption maxima, this picture (see Table 1, Figure 1, and
Figures S21−S25, Supporting Information) does not vary to a
significant extent when the absorption spectra of the same com-
plexes were recorded from the corresponding aqueous (50 mM
potassium phosphate buffer at pH 7.4/DMSO (95:5, v/v))
solutions.
The luminescence properties of the new biotinylated Ir(III)
complexes are rather similar to those of the parent and biotin-free
cationic Ir(III) triazole complexes that have been described by
De Cola and co-workers.17a This similarity provides an initial
indication for the not particularly important influence played
by the different types of biotin pendant arms, as well as by
the presence of the biotin moiety itself, in determining the
Interaction with Avidin. According to the approach re-
ported by Lo and co-workers in their pioneering works,18 the
avidin-binding properties of the biotinylated Ir(III) complexes
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a
Table 1. Photophysical Properties of the Obtained Complexes
bcd
, ,
be
,
absorption
emission, 298 K
emission, 77 K
complex
(solvent)
λabs (nm):
τ (μs)
Φ
kr
knr
(10−4ε) (M−1 cm−1
)
λem (nm)
τ (μs) air
under Ar
Φair
under Ar
(105 s−1
)
(106 s−1
)
λem (nm)
τ (μs)
4.2
4a (CH2Cl2)
4a (buffer)
4b (CH2Cl2)
4b (buffer)
5a (CH2Cl2)
5a (buffer)
255 (4.5)
381 (0.66)
248 (4.0)
374 (3.8)
255 (4.5)
381 (0.55)
268 (4.0)
376 (3.8)
269 (4.6)
380 (0.16)
270 (4.0)
285 (3.8)
385 (0.63)
268 (4.7)
383 (0.37)
255 (2.9)
263 (2.9)
378 (0.27)
270 (6.1)
294 (5.3)
382 (0.69)
266 (4.1)
303 (3.8)
406 (0.63)
272 (5.5)
288 (5.1)
382 (0.39)
272 (3.3)
286 (3.1)
387 (0.49
476, 508
0.14 (90)
0.055 (10)
0.57
0.42
0.048
0.148
0.60
0.55
3.4
2.3
4.3
1.1
9.2
3.2
6.8
1.5
2.7
1.5
6.8
2.9
474, 506
478, 504
476, 508
478, 508
478, 506
478, 506
0.13
0.15
1.1
0.064
0.070
0.12
470, 505
472, 506
2.1 (20)
4.9 (80)
0.62 (60)
0.090 (40)
0.13
0.51
4.7 (90)
1.7 (10)
0.30 (70)
0.10
0.041 (30)
5b (CH2Cl2)
5b (buffer)
476, 508
478, 506
0.13
0.42
0.50
0.10
0.22
0.25
0.34
7.7
5.2
6.9
1.9
470, 506
470, 506
3.9
6a (CH2Cl2)
6a (buffer)
6b (CH2Cl2)
6b (buffer)
476, 508
478, 506
476, 506
478, 504
0.23
0.47
0.13
0.39
1.2 (88)
0.080
0.020
0.12
3.5
4.0
2.0
6.8
2.4
6.0 (84)
1.7 (16)
0.36 (12)
0.42
9.2
0.82
0.41
472, 506
4.9 (67)
2.9 (33)
0.060
1.5
a
“Buffer” means 50 mM potassium phosphate buffer at pH 7.4/DMSO (95:5, v/v); “air” denotes air-equilibrated samples, “under Ar” means
b
degassed (O2-free) samples. For the biexponential excited-state lifetimes (τ), the relative weights of the exponential curve are reported in
parentheses. Quantum yields (Φ) are measured versus air-equilibrated [Ru(bpy)3)]Cl2 aqueous solution (Φ = 0.028). kr = (Φ/τ), knr = (1 − Φ)/τ
with reference to air-equilibrated samples. In frozen CH2Cl2 matrices.
c
d
e
Figure 1. Absorption profiles of complex 4a in CH2Cl2 solution at 298 K (continuous line) and in 50 mM potassium phosphate buffer at pH 7.4/DMSO
(95:5, v/v) (dashed line).
4−6 have been investigated by the standard HABA (4,4′-
hydroxyazobenzene-2-carboxylic acid) assay followed by lumi-
nescence titrations.
In the former instance, the HABA assay relies on the higher
affinity for avidin that is displayed by biotin (Kd = ca. 6 × 10−6 M)
with respect to that of HABA (Kd = ca. 10−15 M),10 the binding of
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Figure 2. Emission profiles of complex 4a obtained from a CH2Cl2 solution at 298 K (red trace) and 77 K (black trace) and from a 50 mM potassium
phosphate buffer at pH 7.4/DMSO (95:5, v/v) (blue trace).
HABA to avidin being witnessed by an intense absorption feature
centered at 500 nm. As a consequence, the displacement of
HABA from the corresponding (HABA)4-avidin adducts, as the
result of addition of unmodified biotin or biotinylated molecules,
results in the decrease of the absorbance at 500 nm. This effect is
also clearly evident from the addition of the biotinylated Ir(III)
complexes 4−6 to solutions of avidin presaturated with HABA.
In each case, the results of the HABA assay indicate that all of
the new Ir(III) complexes bind avidin with the same stoichio-
metry ([Ir]/[avidin] = 4:1) as that deduced by carrying out
the same experiments with unmodified biotin. The results of
such spectrophotometric titrations are reported in Supporting
Information Figures S31 to S33. The behavior of our new bio-
tinylated Ir(III) complexes 4−6 was studied by performing
luminescence titrations in which avidin, as the analyte, was
titrated with the appropriate Ir(III) complex (Figure 3). In
particular, avidin (3.8 μM) was dissolved in a phosphate buffer
solution (pH = 7.4), and titrations were carried out by adding
aliquots of 5 μL of a solution of the appropriate Ir(III) complex
(0.55 mM). Each avidin-complex titration (“main experiment”)
was compared to those obtained from two different control
experiments, the first one consisting in the titration of avidin-free
buffer solution and the second being represented by the titration
of a buffer solution containing avidin presaturated with an excess
of unmodified biotin. In all cases the comparative analysis of
the three emission titrations unequivocally indicated how the
emission intensitywhich was always monitored by considering
the higher energy emission feature (λmax = ca. 480 nm)and
the corresponding emission lifetime of the Ir(III) complexes
were significantly enhanced upon interaction with avidin.
The occurrence of this effect is likely due to the ensuing in-
crease of rigidity and lipophilicity in the local environment of
the biotinylated ligand. In particular, as can be observed in the
emission titration curves reported in Figure 3, the enhancement
of the emission intensity reached its maximum in correspond-
ence with what can be considered as a proper equivalence point
that occurred, in all cases, at [Ir]:[avidin] ca. 4:1. After this point,
the slope of the curves became almost identical to that displayed
by each of the control experiments, the only exceptions being
complexes 4a and, to a lower extent, 6a, where the intervention of
nonspecific interaction between the free Ir(III) probes and the
saturated protein cannot be confidently excluded.
In general, at the equivalence point, each of the Ir(III) com-
plexes showed a different response in terms of increase of
emission intensity and elongation of lifetimes. In particular,
among the amide-linked derivatives 4a, 5a, and 6a, the highest
emission amplification factor (I/I0) (see Table 2) was displayed
a
Table 2. Relative Emission Intensities and Emission
b
Lifetimes of the Biotin Complexes 4−6 in the Absence and in
c
the Presence of Avidin and in the Presence of Avidin and
d
Excess Biotin in Aerated 50 mM Potassium Phosphate Buffer
pH 7.4/DMSO (9:1, v/v) at 298 K
b
c
d
complex
I0 (τ/μs)
I/I0 (τ/μs)
I/I0 (τ/μs)
4a
5a
6a
4b
5b
6b
1.0 (0.61)
1.0 (0.30)
1.0 (0.47)
1.0 (0.62)
1.0 (0.42)
1.0 (0.39)
1.4 (0.81)
6.0 (1.0)
1.0 (0.64)
1.0 (0.30)
1.5 (0.42)
1.4 (0.64)
1.0 (0.42)
0.9 (0.41)
13 (0.80)
8.1 (1.1)
2.6 (0.94)
5.3 (0.66)
a
Emission intensities were measured in correspondence with the
maximum at higher energy (λ = 480 nm); emission lifetimes (τ) are
b
referred to air-equilibrated samples. [Ir] = 15.5 μM; [avidin] = 0 M;
c
[biotin] = 0 M. [Ir] = 15.5 μM; [avidin] = 3.8 μM; [biotin] = 0 M.
d
[Ir] = 15.5 μM; [avidin] = 3.8 μM; [biotin] = 380 μM.
by complex 6a, while the most relevant elongation of the
emission lifetimes was observed for the complex 5a. Relative to
the ester-linked complexes 4b, 5b, and 6b, the most pronounced
amplification of emission intensity was exhibited by complex 4b
(I/I0 = 8.1), whereas the extent of the lifetime elongation was found
essentially constant along the series. In general, the magnitude of
such amplication factors is quite in line with those reported by
Lo and co-workers.5a,7b However, from the comparison of the
results obtained for the amide-containing complexes [(C∧N)2Ir-
(N∧N-C11H22-NH-CO-biotin)]+ 4a, 5a, and 6a with those relative
to ester-based ones [(C∧N)2Ir(N∧N-C11H22-O-CO-biotin)]+ 4b,
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●
▼
Figure 3. Emission titration curves for the titrations of (i) 3.80 μM avidin ( )), (ii) 3.80 μM avidin and 3.80 μM unmodified biotin ( ), and (iii) a blank
■
phosphate buffer solution ( ) with complexes 4a, 4b (top), 5a, 5b (middle), and 6a, 6b (bottom).
5b, and 6b, it is possible to observe how the replacement of the
amide group with an ester moiety did not affect the avidin-binding
properties of the Ir(III) biotinylated complexes. On the contrary,
in some cases, such modification seemed to improve the avidin
affinity of the amide-based complexes. As can be deduced from
Figure 3, the ester complex 4b displays much higher emission
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Mercury 400 MHz or a Varian Inova 600 MHz spectrometer with
tetramethylsilane as the internal standard. ESI-MS analyses were
performed by direct injection of acetonitrile solutions of the compounds
using a Waters ZQ 4000 mass spectrometer. Elemental analyses were
performed on a ThermoQuest Flash 1112 series EA instrument. HABA
assays and emission titrations have been performed according to the
procedures reported by Lo and co-workers.18b
amplification than that exhibited by the amide analogue 4a.
Also, the interaction of avidin with complex 6b, [(C∧N)2Ir-
(4,4′-biphenyl)-O-CO-biotin)]+, is characterized by a more
evident turning point than that displayed by the amide complex
6a, [(C∧N)2Ir-(4,4′-biphenyl)-NH-CO-biotin)]+. Finally, on the
basis of the analysis of these data, no clear rationalization can be
done inconsideration of the aliphatic (as for complexes4a and 4b)
or aromatic (such as the species 5a, 5b, 6a, and 6b) nature of the
spacer arm.
Photophysics. Absorption spectra were recorded at room tem-
perature using a PerkinElmer Lambda 35 UV/vis spectrometer. The
buffer solutions (50 mM potassium phosphate buffer at pH 7.4/DMSO
(95:5, v/v)) containing the Ir(III)-based species were prepared at
first by dissolving the solid samples in DMSO, then diluting with the
aqueous buffer. The obtained solutions were then submitted to sonication
(ca. 5 min). In some cases, as for complexes 5a and, to a lesser extent,
6a, the solutions became a bit opaque within 2 min, therefore determining
a not optimal background subtraction. Uncorrected steady-state emis-
sion and excitation spectra were recorded on an Edinburgh FLSP920
spectrometer equipped with a 450 W xenon arc lamp, double excitation
and single emission monochromators, and a Peltier-cooled Hamamatsu
R928P photomultiplier tube (185−850 nm). Emission and excitation
spectra were corrected for source intensity (lamp and grating) and
emission spectral response (detector and grating) by a calibration curve
supplied with the instrument. The wavelengths for the emission and
excitation spectra were determined using the absorption maxima of the
MLCT transition bands (emission spectra) and at the maxima of the
emission bands (excitation spectra). According to the approach
described by Demas and Crosby,19 luminescence quantum yields
(Φem) were measured in optically dilute solutions (OD < 0.1 at exci-
tation wavelength) obtained from absorption spectra on a wavelength
scale [nm] and compared to the reference emitter by the following
equation:20
CONCLUSIONS
■
In this work, we have reported the synthesis of a series of six new
Ir(III) complexes containing a biotin-appended pyridyl-1,2,3-
triazole derivative as the neutral ligand, their photophysical
characterization, and the preliminary study of their avidin-
binding features. These molecules have been designed in order to
provide luminescent Ir(III) complexes capable of binding avidin
through a biotinylated arm that is carried by a Ir(III)-coordinated
ligand different from the “traditionally” employed bpy- or phen-
type derivatives. In this regard, the presence of the biotin moiety
did not affect the photoemissive performances of the Ir(III)
complexes, since all the new species displayed intense
phosphorescent emission with quantum yield values up to 0.60
in dichloromethane solutions. It is important to note how the
emissive features of the biotinylated Ir(III) complexes were
substantially retained in an aqueous environment. In addition, a
significant elongation of the phosphorescence lifetimes was
observed upon passing from dichloromethane to water solvent,
as the result of the hydrophobicity of the spacer arms that
connect the biotin unit to the Ir(III) phosphors. Taken together,
these features constitute the basis for the study of the avidin-
binding properties of the new complexes. Indeed, the emission
titrations of avidin with the biotinylated Ir(III) complexes as the
titrants suggested the occurrence of the interaction between the
complexes and the protein. In particular, in almost all cases, the
new iridium(III) complexes displayed a relevant emission
intensity enhancement accompanied by emission lifetime
elongation upon their binding to avidin. This effect was observed
both for the “traditional” amide-linked biotin complexes and, in
particular, for the newly reported examples of biotinylated
compounds in which the biotin group was appended to the
Ir(III)-complex structure through esterification. However, even
though these interactions need to be further studied by many
aspects such as cytotoxicity and cellular uptake ability of the new
complexes, the analysis of the results reported herein provided an
important indication about the feasibility of the replacement of
bpy-based biotin carriers with another type of diimine ligand,
such as pyridyl-1,2,3-triazole derivatives, in the structure of
biotinylated Ir(III) phosphors. Finally, it is important to note
how the design strategy we have adopted in this work might
be applied for developing new libraries of luminescent Ir(III)
complexes decorated with a variety of biologically relevant
groups or, more in general, with moieties that might endow the
Ir(III) complexes with the very delicate balance of factors
(lipophilicity, cellular uptake ability, and cellular localization)
that might determine their use as imaging reagents.
2
⎡
⎤
⎥
⎦
⎢
⎥⎢
⎥
⎡
⎤
Ar(λr) Ir(λr) nx
D
D
r
x
⎢
Φ = Φ⎢
⎥⎢
A (λ ) I (λ )
⎥
⎢
⎣
⎥
⎦
x
r
2
⎣
⎦⎣
⎦ n
⎣
x
x
x
x
r
where A is the absorbance at the excitation wavelength (λ), I is the
intensity of the excitation light at the excitation wavelength (λ), n is the
refractive index of the solvent, D is the integrated intensity of the
luminescence, and Φ is the quantum yield. The subscripts r and x refer to
the reference and the sample, respectively. The quantum yield
determinations were performed at identical excitation wavelengths for
the sample and the reference, therefore canceling the I(λr)/I(λx) term in
the equation. All the Ir(III) complexes were measured against an
aqueous solution of [Ru(bpy)3]Cl2 (Φr = 0.028).21 Emission lifetimes
(τ) were determined with the single photon counting technique
(TCSPC) with the same Edinburgh FLSP920 spectrometer using
pulsed picosecond LEDs (EPLED 295 or EPLED 360, fhwm < 800 ps)
as the excitation source, with repetition rates between 1 kHz and 1 MHz,
and the above-mentioned R928P PMT as detector. The goodness of fit
was assessed by minimizing the reduced χ2 function and by visual
inspection of the weighted residuals. To record the 77 K luminescence
spectra, the samples were put in quartz tubes (2 mm diameter) and
inserted in a special quartz dewar filled with liquid nitrogen. The solvent
(dichloromethane) used in the preparation of the solutions for the
photophysical investigations was of spectrometric grade. Degassed
solutions were prepared by gently bubbling argon gas into the prepared
sample for 15 min before measurement. Experimental uncertainties are
estimated to be 8% for lifetime determinations, 20% for quantum
yields, and 2 nm and 5 nm for absorption and emission peaks,
respectively.
Synthesis of Ligands. Synthesis of Ligand 1a. Compounds 9
(208 mg, 0.61 mmol) and 11 (250 mg, 0.79 mmol) were dissolved in
anhydrous DMF (10 mL). Et3N (250 μL, 1.84 mmol) was added to
the solution, and the reaction mixture was stirred at room temperature
for 24 h. Then the solvent was removed by vacuum, and the crude
was washed with Et2O (3 × 15 mL). The solid was dissolved in CH2Cl2
(2 mL) and then precipitated with Et2O to give pure 1a (306 mg) in 92%
yield. 1H NMR (CDCl3, 400 MHz): δ 8.58 (bs, 1H, JHH = 5.1), 8.18 (dt,
1H, JHH = 8.0, JHH = 1.0), 8.16 (s, 1H), 7.78 (td, 1H, JHH = 7.6, JHH = 1.8),
7.23 (ddd, 1H, JHH = 7.6, JHH = 5.0, JHH = 1.3), 6.0 (bs, 1H), 5.9 (bs, 1H),
EXPERIMENTAL SECTION
■
The solvents and chemicals were used as received from sellers, unless
otherwise mentioned. The synthetic procedures for the preparation of
the precursor compounds 8, 9, 11, 14a,b, 16, and 18a,b and the
corresponding characterization are reported in the Supporting
Information, pp S3−S7. NMR spectra were recorded by using a Varian
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Organometallics
Article
5.3 (bs, 1H), 4.51 (dd, 1H, JHH = 7.6, JHH = 5.1), 4.28 (t, 2H, JHH = 7.0),
4.32 (dd, 1H, JHH = 7.6, JHH = 5.1), 3.21 (q, 2H, JHH = 6.9), 3.10−3.15
(m, 1H), 2.91 (dd, 1H, JHH = 12.7, JHH = 5.0), 2.73 (d, 1H, JHH = 12.7),
2.25−2.15 (m, 2H), 2.00−1.90 (m, 2H), 1.80−1.20 (m, 22H) ppm. 13C
NMR (CDCl3, 100 MHz): δ 173.1 (C), 163.4 (C), 150.6 (C), 149.6
(CH), 148.5 (C), 137.2 (CH), 123.0 (CH), 122.1 (CH), 120.5 (CH),
62.0 (CH), 60.4 (CH), 55.6 (CH), 50.7 (CH2), 40.8 (CH2), 39.7
(CH2), 36.3 (CH2), 30.4 (CH2), 29.9 (CH2), 29.8 (CH2), 29.6 (CH2),
29.5 (CH2), 29.47 (CH2), 29.4 (CH2), 29.1 (CH2), 28.3 (CH2), 27.1
(CH2), 26.6 (CH2), 25.9 (CH2) ppm. ESI-MS (m/z): 564 [M + Na+].
Anal. Calcd for C28H43N7O2S (541.76): C, 62.08; H, 8.00; N, 18.10.
Found: C, 61.87; H, 8.10; N, 17.93.
55% yield. 1H NMR (DMSO, 400 MHz): δ 9.34 (s, 1H), 8.67 (d, 1H,
JHH =4.4), 8.13 (d, 1H, JHH = 7.4), 8.08 (d, 2H, JHH = 8.3), 7.96 (dt, 1H,
JHH = 1.9, JHH =7.7), 7.45−7.35 (m, 3H), 6.5 (bs, 1H), 6.4 (bs, 1H),
4.35−4.30 (m, 1H), 4.20−4.15 (m, 1H), 3.20−3.10 (m, 1H), 2.85 (dd,
1H, JHH = 4.8, JHH = 12.8), 2.70−2.55 (m, 3H), 1.75−1.40 (m, 6H) ppm.
13C NMR (DMSO-d6, 101 MHz): δ 172.1 (C), 163.2 (C), 150.9 (C),
150.1 (CH), 150.0 (C), 148.7 (C), 137.8 (CH), 134.5 (C), 123.8 (CH),
123.7 (CH), 122.0 (CH), 121.9 (CH), 120.3 (CH), 61.5 (CH), 59.7
(CH), 55.8 (CH), 40.3 (CH2), 33.7 (CH2), 28.5 (CH2), 28.4 (CH2),
24.8 (CH2) ppm. ESI-MS (m/z): 487 [M + Na+]. Anal. Calcd for
C23H24N6O3S (464.54): C, 59.47; H, 5.21; N, 18.09. Found: C, 59.68;
H, 5.04; N, 18.35.
Synthesis of Ligand 1b. Biotin 10 (185 mg, 0.76 mmol) was
dissolved in SOCl2 (6 mL), and the reaction mixture was stirred at room
temperature for 2 h under a nitrogen atmosphere. Then the excess of
SOCl2 was removed by distillation and the crude was dissolved in
anhydrous acetonitrile (10 mL). A solution of 8 (120 mg, 0.38 mmol) in
anhydrous acetonitrile (5 mL) was then added dropwise. The mixture
was stirred at room temperature for 24 h under a nitrogen atmosphere.
Then the solvent was removed by vacuum and the crude was dissolved
with a 1:1 mixture of EtOAc and aqueous NaHCO3. The aqueous phase
was extracted with ethyl acetate (3 × 10 mL) and DCM (2 × 10 mL).
The combined organic layers were dried over MgSO4 and filtered, and
the solvent was evaporated. The solid was dissolved in CHCl3 (1.5 mL)
and precipitated with hexane to give pure 1b (124 mg) in 60% yield. 1H
Synthesis of Ligand 3a. In a 50 mL two-necked round-bottom flask
equipped with a stirring bar, biotin 10 (220 mg, 0.90 mmol), EDCI·HCl
(207 mg, 1.1 mmol), HOBt (146 mg, 1.1 mmol), and Et3N (173 mg,
238 μL, 1.7 mmol) were dissolved in anhydrous DMF (6 mL) under a
N2 atmosphere. After 10 min 18a (232 mg, 0.9 mmol) was added, and
the reaction was left to stir 24 h at room temperature. Then MeOH was
added to the solution, and the precipitate was filtered and washed with
MeOH (3 × 5 mL) to give pure 3a (305 mg) in 63% yield. 1H NMR
(DMSO, 300 MHz): δ 10.0 (bs, 1H), 9.38 (s, 1H), 8.67 (ddd, 1H, JHH
=
1.0, JHH = 1.5, JHH = 4.8), 8.15−8.05 (m, 3H), 7.96 (td, 1H, JHH =2.0,
JHH = 7.8), 7.95−7.85 (m, 2H), 7.65 (s, 4H), 7.42 (ddd, 1H, JHH = 1.2, JHH
= 4.9, JHH = 7.6), 6.5 (s, 1H), 6.4 (s, 1H), 4.35−4.30 (m, 1H), 4.15−4.10
(m, 1H), 3.20−3.10 (m, 1H), 2.84 (dd, 1H, JHH = 5.0, JHH = 12.2), 2.59
(d, 1H, JHH = 12.2), 2.35 (t, 2H, JHH = 7.3), 1.70−1.40 (m, 6H) ppm. 13
C
NMR (CDCl3, 400 MHz): δ 8.58 (d, 1H, JHH = 4.3), 8.19 (d, 1H, JHH
8.6), 8.16 (s, 1H), 7.79 (t, 1H, JHH = 8.0), 7.25−7.20 (m, 1H), 5.3 (bs,
1H), 5.1 (bs, 1H), 4.52 (dd, 1H, JHH = 7.2, JHH = 5.4), 4.42 (t, 2H, JHH
7.2), 4.32 (dd, 1H, JHH = 8.0, JHH = 5.4), 4.05 (t, 2H, JHH = 6.7), 3.20−
3.15 (m, 1H), 2.92 (dd, 1H, JHH = 12.7, JHH = 5.0), 2.74 (d, 1H, JHH
=
NMR (DMSO-d6, 101 MHz): δ 171.8 (C), 163.2 (C), 150.1 (CH),
150.0 (C), 148.7 (C), 140.5 (C), 139.8 (C), 137.8 (CH), 135.8 (C),
133.5 (C), 127.9 (CH), 127.5 (CH), 123.8 (CH), 121.6 (CH), 121.0
(CH), 120.3 (CH), 119.9 (CH), 61.5 (CH), 59.7 (CH), 55.9 (CH),
40.3 (CH2), 36.7 (CH2), 28.7 (CH2), 28.6 (CH2), 25.6 (CH2) ppm.
ESI-MS (m/z): 562 [M + Na+]. Anal. Calcd for C29H29N7O2S (539.66):
C, 64.54; H, 5.42; N, 18.17. Found: C, 64.28; H, 5.64; N, 18.49.
Synthesis of Ligand 3b. In a 50 mL two-necked round-bottom flask
equipped with a stirring bar, biotin (37 mg, 0.15 mmol), EDCI·HCl
(74 mg, 0.39 mmol), HOBt (42 mg, 0.31 mmol), and Et3N (84 μL,
0.60 mmol) were dissolved in anhydrous DMF (6 mL) under a nitrogen
atmosphere. After 10 min, 18b (32 mg, 0.10 mmol) was added, and the
reaction was left to stir 24 h at room temperature. Then further EDCI·
HCl (43 mg, 0.23 mmol) was added, and the reaction mixture was
stirred under N2 at room temperature overnight. Then the solvent was
removed by vacuum, and the crude was washed with MeOH to give 3b
in 55% yield. 1H NMR (DMSO, 400 MHz): δ 9.40 (s, 1H), 8.68 (d, 1H,
JHH = 4.2), 8.20−8.10 (m, 3H), 8.00−7.90 (m, 3H), 7.85−7.80 (m, 2H),
7.42 (dd, 1H, JHH = 7.1, JHH = 5.1), 7.30−7.20 (m, 2H), 6.5 (bs, 1H), 6.4
(bs, 1H), 4.35−4.30 (m, 1H), 4.20−4.15 (m, 1H), 3.20−3.10 (m, 1H),
2.85 (dd, 1H, JHH = 12.4, JHH = 5.0), 2.65−2.55 (m, 3H), 1.75−1.40 (m,
6H) ppm. 13C NMR (DMSO-d6, 101 MHz): δ 172.2 (C), 163.2 (C),
150.9 (C), 150.1 (CH), 150.0 (C), 148.7 (C), 140.1 (C), 137.8 (CH),
136.8 (C), 136.2 (C), 128.5 (CH), 128.4 (CH), 128.8 (CH), 122.9
(CH), 121.7 (CH), 121.1 (CH), 120.3 (CH), 61.5 (CH), 59.7 (CH),
55.8 (CH), 40.3 (CH2), 33.8 (CH2), 28.5 (CH2), 28.4 (CH2), 24.9
(CH2) ppm. ESI-MS (m/z): 563 [M + Na+]. Anal. Calcd for
C29H28N6O3S (540.64): C, 64.43; H, 5.22; N, 15.54. Found: C, 64.65;
H, 5.50; N, 15.79.
=
=
12.7), 2.32 (t, 2H, JHH = 7.3), 2.00−1.90 (m, 2H), 1.75−1.55 (m, 6H),
1.50−1.20 (m, 16H) ppm. 13C NMR (CDCl3, 101 MHz): δ 173.6 (C),
163.1 (C), 150.4 (C), 149.3 (CH), 148.2 (C), 137.0 (CH), 122.8 (CH),
121.9 (CH), 120.3 (CH), 64.5 (CH2), 61.9 (CH), 60.1 (CH), 55.3
(CH), 50.5 (CH2), 40.5 (CH2), 33.9 (CH2), 30.2 (CH2), 29.4 (CH2),
29.36 (CH2), 29.3 (CH2), 29.2 (CH2), 29.0 (CH2), 28.6 (CH2), 28.31
(CH2), 28.26 (CH2), 26.4 (CH2), 25.9 (CH2), 24.8 (CH2) ppm. ESI−
MS (m/z): 565 [M + Na+]. Anal. Calcd for C28H42N6O3S (542.74): C,
61.96; H, 7.80; N, 15.48. Found: C, 62.17; H, 7.68; N, 15.73.
Synthesis of Ligand 2a. In a 50 mL two-necked round-bottom flask
equipped with a stirring bar, biotin 10 (220 mg, 0.90 mmol), EDCI·HCl
(207 mg, 1.1 mmol), HOBt (146 mg, 1.1 mmol), and Et3N (173 mg,
238 μL, 1.7 mmol) were dissolved in anhydrous DMF (6 mL) under a
nitrogen atmosphere. After 10 min, 14a (214 mg, 0.9 mmol) was added
and the reaction was left to stir 72 h at room temperature. Then the
solvent was removed by vacuum, and the crude was purified by column
chromatography on neutral alumina (DCM/MeOH = 94:6) to give 2a
(263 mg, 0.57 mmol) in 63% yield. 1H NMR (DMSO-d6, 400 MHz): δ
10.30 (s, 1H), 9.28 (s, 1H), 8.72 (bd, 1H, JHH = 4.6), 8.17 (d, 1H, JHH
=
8.1), 8.05−7.95 (m, 3H), 7.95−7.85 (m, 2H), 7.45 (ddd, 1H, JHH = 1.1,
JHH = 4.8, JHH = 7.4), 6.5 (bs, 1H), 6.4 (bs, 1H), 4.40−4.35 (m, 1H),
4.25−4.15 (m, 1H), 3.25−3.15 (m,1H), 2.89 (dd, 1H,, JHH = 4.8, JHH
=
12.0), 2.64 (d, 1H, JHH = 12.0), 2.42 (t, 2H, JHH = 7.5), 1.80−1.40 (m,
6H) ppm. 13C NMR (DMSO-d6, 101 MHz): δ 172.0 (C), 163.2 (C),
150.1 (CH), 150.0 (C), 148.5 (C), 140.2 (C), 137.8 (CH), 131.9 (C),
123.7 (CH), 121.4 (CH), 121.2 (CH), 120.2 (CH), 120.1 (CH), 61.5
(CH), 60.0 (CH), 55.8 (CH), 40.3 (CH2), 36.7 (CH2), 28.7 (CH2),
28.6 (CH2), 25.5 (CH2) ppm. ESI-MS (m/z): 488 [M + Na+]. Anal.
Calcd for C23H25N7O2S (463.56): C, 59.59; H, 5.44; N, 21.15. Found:
C, 59.27; H, 5.58; N, 20.93.
Synthesis of Complexes. Synthesis of Complex 4a. In a 50 mL
round-bottom flask equipped with a stirring bar, the ligand 1a (54 mg,
0.10 mmol) was dissolved in a 3:1 mixture of CH2Cl2/EtOH (8 mL),
and the dimer 19 (54 mg, 0.05 mmol) was added. The mixture was left
to stir at rt for 24 h. The solvent was removed, and the crude was
dissolved in DCM (1 mL) and precipitated with Et2O to give 4a (63 mg)
in 60% yield. 1H NMR (CDCl3, 300 MHz): δ 10.66 (s, 1H), 9.13 (d, 1H,
JHH = 8.2), 8.04 (t, 1H, JHH = 7.7), 7.95−7.85 (m, 2H), 7.80−7.70 (m,
3H), 7.70−7.60 (m, 3H), 7.47 (d, 1H, JHH = 5.7), 7.25−7.20 (m, 1H),
7.05−6.85 (m, 6H), 6.45−6.35 (m, 1H), 6.35−6.25 (m, 2H), 5.8 (bs,
1H), 5.0 (bs, 1H), 4.55−4.40 (m, 3H), 4.35−4.30 (m, 1H), 3.30−3.10
(m, 3H), 2.92 (dd, 1H, JHH = 5.6, JHH = 12.9), 2.75 (d, 1H, JHH = 12.9),
2.30−2.20 (m, 2H), 2.00−1.15 (m, 24H) ppm. 13C NMR (CDCl3,
150 MHz): δ 173.4 (C), 168.3 (C), 167.6 (C), 163.8 (C), 150.1 (C),
149.5 (CH), 149.3 (CH), 148.5 (C), 148.4 (CH), 146.6 (C), 144.6 (C),
Synthesis of Ligand 2b. In a 50 mL two-necked round-bottom flask
equipped with a stirring bar, biotin 10 (37 mg, 0.15 mmol), 14b (71 mg,
0.30 mmol), and HOBt (41 mg, 0.30 mmol) were added under N2 to
anhydrous DMF (3.5 mL). After bubbling N2 in the solution for 15 min,
Et3N (84 μL, 0.60 mmol) and EDCI·HCl (43 mg, 0.23 mmol) were
added, and the reaction mixture was stirred under N2 at room
temperature for 24 h. Then further EDCI·HCl (43 mg, 0.23 mmol) was
added, and the reaction mixture was stirred under N2 at room
temperature overnight. Then the solvent was removed by vacuum, and
the crude was washed with MeOH (3 × 5 mL) to give 2b (38 mg) in
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143.7 (C), 143.6 (C), 139.9 (CH), 137.9 (CH), 137.8 (CH), 131.8 (CH),
131.7 (CH), 130.6 (CH), 130.0 (CH), 128.5 (CH), 125.9 (CH), 124.6
(2CH), 124.3 (CH), 123.2 (CH), 122.8 (CH), 122.6 (CH), 122.1
(CH), 119.4 (CH), 119.3 (CH), 61.8 (CH), 60.2 (CH), 55.6 (CH),
52.3 (CH2), 40.6 (CH2), 39.4 (CH2), 35.8 (CH2), 29.7 (CH2), 29.3
(CH2), 29.02 (2CH2), 28.99 (2CH2), 28.5 (CH2), 28.04 (CH2), 27.99
(CH2), 26.7 (CH2), 26.1 (CH2), 25.7 (CH2) ppm. ESI-MS (m/z): 1042
[M+]. Anal. Calcd for C50H59ClIrN9O2S (1077.81): C, 55.72; H, 5.52;
N, 11.70. Found: C, 55.49; H, 5.73; N, 12.07.
(m, 1H), 3.15−3.05 (m, 1H), 2.82 (dd, 1H, JHH = 4.7, JHH = 12.6), 2.56
(d, 1H, JHH = 12.6), 2.49 (d, 2H, JHH = 7.5 Hz), 1.75−1.35 (m, 6H) ppm.
13C NMR (CDCl3, 100 MHz): δ 170.6 (C), 169.3 (C), 167.4 (C), 166.5
(C), 162.6 (C), 150.4 (C), 149.9 (C), 148.9 (C), 148.51 (CH), 148.47
(CH), 148.45 (CH), 147.4 (CH), 145.2 (C), 142.7 (C), 142.6 (C),
139.0 (CH), 137.04 (CH), 137.00 (CH), 132.5 (C), 130.9 (CH), 130.7
(CH), 129.7 (CH), 129.0 (CH), 125.8 (CH), 125.2 (CH), 124.3 (CH),
123.7 (CH), 123.3 (CH), 122.3 (CH), 122.2 (CH), 121.8 (CH), 121.7
(CH), 121.2 (CH), 120.6 (CH), 118.6 (CH), 118.4 (CH), 60.9 (CH),
59.2 (CH2), 54.4 (CH), 39.6 (CH2), 32.9 (CH2), 27.30 (CH2), 27.27
(CH2), 23.6 (CH2) ppm. ESI-MS (m/z): 965 [M+]. Anal. Calcd for
C45H40ClIrN8O3S (1000.60): C, 54.02; H, 4.03; N, 11.20. Found: C,
53.79; H, 4.25; N, 10.89.
Synthesis of Complex 4b. In a 10 mL round-bottom flask equipped
with a stirring bar, the ligand 1b (54 mg, 0.10 mmol) was dissolved
in a 3:1 mixture of CH2Cl2/EtOH (4 mL), and the dimer 19 (54 mg,
0.05 mmol) was added. The mixture was left to stir at rt for 24 h. The
solvent was removed, and the crude was dissolved in DCM (1.0 mL) and
Synthesis of Complex 6a. In a 10 mL round-bottom flask equipped
with a stirring bar, the ligand 3a (47 mg, 0.09 mmol) was dissolved
in a 3:1 mixture of CH2Cl2/EtOH (10 mL), and the iridium dimer 19
(48 mg, 0.045 mmol) was added. The mixture was left to stir at rt for 48
h. The solvent was removed, and the resulting residue was dissolved in
MeOH (0.5 mL) and precipitated with Et2O. The solid was filtered
1
precipitated with Et2O to give 4b (83 mg) in 80% yield. H NMR
(CDCl3, 400 MHz): δ 10.66 (s, 1H), 9.15 (d, 1H, JHH = 7.7), 8.03 (t, 1H,
JHH = 7.2), 7.95−7.85 (m, 2H), 7.80−7.60 (m, 6H), 7.47 (d, 1H, JHH
=
5.2), 7.20 (dd, 1H, JHH = 7.5, JHH = 6.0), 7.05−6.85 (m, 6H), 6.31 (t, 2H,
JHH = 7.5), 5.5 (bs, 1H), 5.4 (bs, 1H), 4.55−4.40 (m, 3H), 4.35−4.25
(m, 1H), 4.06 (t, 2H, JHH = 6.7), 3.20−3.10 (m, 1H), 2.88 (dd, 1H, JHH
12.8, JHH = 5.3), 2.78 (dd, 1H, JHH = 12.8, JHH = 3.0), 2.31 (t, 2H, JHH
1
=
=
to give pure complex 6a (56 mg) in 60% yield. H NMR (CD3OD,
400 MHz): δ 9.62 (s, 1H), 8.27 (d, 1H, JHH = 8.0), 8.10−8.00 (m, 3H),
7.85−7.65 (m, 10H), 7.65−7.50 (m, 5H), 7.40−7.35 (m, 1H), 7.05−
6.90 (m, 3H), 6.88 (dt, 1H, JHH = 1.1, JHH = 7.5), 6.81 (dt, 1H, JHH = 1.1,
JHH = 7.5), 6.73 (dt, 1H, JHH = 1.1, JHH = 7.5), 6.21 (dd, 2H, JHH = 7.4 Hz,
JHH = 2.6), 4.60−4.55 (m, 1H), 4.55−4.45 (m, 1H), 3.45−3.35 (m, 1H),
3.10−3.00 (m, 1H), 3.00−2.95 (m, 1H), 2.36 (t, 2H, JHH = 7.2), 1.90−
1.50 (m, 6H) ppm. 13C NMR (DMSO-d6, 101 MHz): δ 171.8 (C),
167.4 (C), 167.0 (C), 161.7 (C), 150.4 (CH), 150.1 (CH), 150.0 (C),
149.5 (CH), 149.34 (C), 149.27 (C), 146.8 (C), 144.52 (C), 144.47
(C), 141.7 (C), 140.7 (CH), 140.1 (C), 139.3 (CH), 139.2 (CH), 135.0
(C), 133.0 (C), 131.9 (CH), 131.3 (CH), 130.7 (CH), 129.9 (CH),
127.9 (CH), 127.7 (CH), 127.5 (CH), 125.7 (CH), 125.5 (CH), 125.5
(CH), 124.4 (CH), 124.2 (CH), 123.4 (CH), 122.9 (CH), 122.3 (CH),
121.7 (CH), 120.3 (2CH), 119.9 (CH), 70.4 (CH), 59.1 (CH2), 56.3
(CH), 53.2 (CH), 36.6 (CH2), 25.6 (CH2), 25.4 (CH2) ppm. ESI-MS
(m/z): 1040 [M+]. Anal. Calcd for C51H45ClIrN9O2S (1075.71): C,
56.94; H, 4.22; N, 11.72. Found: C, 56.67; H, 4.50; N, 12.00.
Synthesis of Complex 6b. In a 10 mL round-bottom flask equipped
with a stirring bar, the ligand 3b (32 mg, 0.06 mmol) was dissolved in a
3:1 mixture of CH2Cl2/EtOH (8 mL), and the iridium dimer 19 (32 mg,
0.03 mmol) was added. The mixture was left to stir at rt for 48 h. The
solvent was removed, and the crude was purified by column chro-
matography on silica gel (CH2Cl2/ MeOH = 9:1) to give 6b (19 mg) in
30% yield. 1H NMR (CDCl3, 400 MHz): δ 11.79 (s, 1H), 9.38 (d, 1H,
JHH = 8.0), 8.15 (d, 2H, JHH = 7.8), 8.06 (t, 1H, JHH = 7.8), 7.92 (dd, 2H,
JHH = 7.8, JHH = 3.5), 7.80−7.75 (m, 4H), 7.70−7.65 (m, 4H), 7.55−
7.50 (m, 3H), 7.25−7.20 (m, 1H), 7.15 (d, 2H, JHH = 8.5), 7.05−6.95
(m, 4H), 6.95−6.85 (m, 2H), 6.34 (dd, 2H, JHH = 2.5, JHH = 7.4), 5.55 (s,
1H), 5.18 (s, 1H), 4.55−4.45 (m, 1H), 4.35−4.30 (m, 1H), 3.20−3.15
(m, 1H), 2.92 (dd, 1H, JHH = 5.2, JHH = 13.2), 2.75 (d, 1H, JHH = 13.2),
2.60 (, t, 2H, JHH = 7.2), 1.90−1.40 (m, 6H) ppm. 13C NMR (CDCl3,
101 MHz): δ 171.1 (C), 167.5 (C), 166.6 (C), 162.3 (C), 149.6 (C),
149.0 (C),148.9 (C), 148.50 (C), 148.47 (CH), 148.4 (CH), 147.4
(CH), 145.3 (C), 142.7 (C), 142.6 (C), 140.6 (C), 139.0 (CH), 137.0
(CH), 136.9 (CH), 136.1 (C), 134.3 (C), 130.9 (CH), 130.7 (CH),
129.7 (CH), 129.0 (CH), 127.4 (CH), 127.1 (CH), 125.9 (CH), 125.0
(CH), 125.4 (CH), 123.7 (CH), 123.3 (CH), 122.3 (CH), 121.8 (CH),
121.7 (CH), 121.2 (CH), 121.1 (CH), 119.7 (CH), 118.5 (CH), 118.4
(CH), 60.9 (CH), 59.1 (CH), 54.3 (CH), 39.6 (CH2), 33.0 (CH2), 29.7
(CH2), 27.3 (CH2), 23.7 (CH2) ppm. ESI-MS (m/z): 1041 [M+]. Anal.
Calcd for C51H44ClIrN8O3S (1076.69): C, 56.89; H, 4.12; N, 10.41.
Found: C, 57.12; H, 4.35; N, 10.07.
7.1), 1.95−1.90 (m, 2H), 1.75−1.55 (m, 6H), 1.45−1.15 (m, 16H)
ppm. 13C NMR (CDCl3, 150 MHz): δ 173.8 (C), 167.2 (C), 166.7 (C),
162.6 (C), 149.4 (C), 149.1 (C), 149.0 (CH), 148.8 (CH), 148.0 (CH),
146.0 (2C), 143.3 (C), 143.2 (C), 139.2 (CH), 137.3 (CH), 137.2
(CH), 131.2 (CH), 130.9 (CH), 129.7 (CH), 129.0 (CH), 127.85
(CH), 125.3 (CH), 124.0 (CH), 123.8 (CH), 123.6 (CH), 122.6 (CH),
122.3 (CH), 121.8 (CH), 121.3 (CH), 118.8 (CH), 118.7 (CH), 63.7
(CH2), 61.1 (CH), 59.5 (CH), 54.7 (CH), 51.7 (CH2), 39.9 (CH2),
33.2 (CH2), 29.3 (CH2), 28.9 (CH2), 28.63 (CH2), 28.57 (CH2), 28.4
(CH2), 28.1 (CH2), 27.9 (CH2), 27.6 (CH2), 27.5 (CH2), 25.6 (CH2),
25.2 (CH2), 24.1 (CH2) ppm. ESI-MS (m/z): 1043 [M+] Anal. Calcd
for C50H58ClIrN8O3S (1078.79): C, 55.67; H, 5.42; N, 10.39. Found: C,
55.89; H, 5.19; N, 10.67.
Synthesis of Complex 5a. In a 10 mL round-bottom flask equipped
with a stirring bar, the ligand 2a (28 mg, 0.06 mmol) was dissolved
in a 3:1 mixture of CH2Cl2/EtOH (8 mL), and the dimer 19 (32 mg,
0.03 mmol) was added. The mixture was left to stir at rt for 48 h. The
solvent was removed, and the crude was purified by column chro-
matography on silica gel (CH2Cl2/MeOH = 9:1) to give 5a (41 mg)
in 70% yield. 1H NMR (DMSO-d6, 400 MHz): δ 10.40 (s, 1H), 10.15
(s, 1H), 8.47 (d, 1H, JHH = 7.9), 8.30−8.20 (m, 3H), 8.00−7.95 (m, 3H),
7.90 (d, 1H, JHH = 7.2), 7.90−7.80 (m, 3H), 7.75−7.70 (m, 3H), 7.69 (d,
1H, JHH = 5.7), 7.59 (t, 1H, JHH = 6.6), 7.25−7.15 (m, 2H), 7.03 (t, 1H,
JHH = 7.5), 6.95−6.90 (m, 2H), 6.89 (t, 1H, JHH = 7.5), 6.43 (s, 1H), 6.37
(s, 1H), 6.20 (d, 2H, JHH = 7.6), 6.17 (d, 2H, JHH = 7.6), 4.35−4.30 (m,
1H), 4.15−4.10 (m, 1H), 3.15−3.10 (m, 1H), 2.83 (dd, 1H, JHH = 5.5,
JHH = 12.6), 2.59 (d, 1H, JHH = 12.6), 2.40−2.30 (m, 2H), 1.65−1.30 (m,
6H) ppm. 13C NMR (DMSO-d6, 150 MHz): δ 172.2 (C), 167.7 (C),
166.9 (C), 163.2 (C), 152.8 (C), 150.4 (CH), 150.0 (CH), 149.9 (C),
149.5 (CH), 149.4 (C), 149.1 (C), 146.8 (C), 144.5 (C), 144.4 (C),
141.4 (C), 140.6 (CH), 139.3 (CH), 139.2 (CH), 131.9 (CH), 131.3
(CH), 130.7 (CH), 129.9 (CH), 127.6 (CH), 125.7 (CH), 125.4 (CH),
125.0 (CH), 124.4 (CH), 124.2 (CH), 123.4 (CH), 122.9 (CH), 122.3
(CH), 122.1 (CH), 120.3 (CH), 120.1 (CH), 61.5 (CH), 59.7 (CH),
55.8 (CH), 40.3 (CH2), 36.6 (CH2), 28.7 (CH2), 28.5 (CH2), 25.4
(CH2) ppm. ESI-MS (m/z): 964 [M+]. Anal. Calcd for
C45H41ClIrN9O2S (999.61): C, 54.07; H, 4.13; N, 12.61. Found: C,
54.32; H, 4.30; N, 12.42.
Synthesis of Complex 5b. In a 50 mL round-bottom flask equipped
with a stirring bar, the ligand 2b (28 mg, 0.06 mmol) was dissolved
in a 3:1 mixture of CH2Cl2/EtOH (8 mL), and the dimer 19 (32 mg,
0.03 mmol) was added. The mixture was left to stir at rt for 48 h. The
solvent was removed and the crude was purified by column
chromatography on silica gel (CH2Cl2/MeOH = 9:1) to give pure
complex 5b (29 mg) in 50% yield. 1H NMR (CDCl3, 400 MHz): δ 11.6
(s, 1H), 9.24 (d, 1H, JHH = 7.6), 8.04 (d, 1H, JHH = 8.0), 7.98 (t, 1H,
JHH = 8.0), 7.84 (d, 2H, JHH = 8.4), 7.75−7.65 (m, 4H), 7.65−7.55 (m, 2H),
7.43 (d, 1H, JHH = 5.8), 7.20−7.10 (m, 3H), 7.00−6.70 (m, 6H), 6.30−
6.25 (m, 2H), 5.8 (bs, 1H), 5.5 (bs, 1H), 4.45−4.40 (m, 1H), 4.25−4.20
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, chatacterization, and 1H and 13C NMR spectra of the
precursor compounds 8, 9, 11, 14a,b, 16, and 18a,b. 1H and 13C
NMR spectra of all ligands (1−3a,b) and complexes (4−6a,b).
Absorption spectra of all Ir(III) complexes. Emission spectra in
6163
dx.doi.org/10.1021/om5007962 | Organometallics 2014, 33, 6154−6164

Organometallics
Article
(11) Kwon, T.-H.; Kwon, J.; Hong, J.-I. J. Am. Chem. Soc. 2008, 130,
3726−3727.
dicholoromethane (rt and 77 K) and aqueous solutions (rt) and
HABA assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
(12) The pyridyl-triazoles 8, 14a,b, and 16 were prepared by slight
modifications of reported procedures: Fletcher, J. T.; Bumgarner, B. J.;
Engels, N. D.; Skoglund, D. A. Organometallics 2008, 27, 5430−5433
(used for 8 and 14a,b). Barral, K.; Moorhouse, A. D.; Moses, J. E. Org.
Lett. 2007, 9, 1809−1811 (for 16).
AUTHOR INFORMATION
Corresponding Authors
*E-mail: letizia.sambri@unibo.it.
*E-mail: stefano.stagni@unibo.it.
■
(13) (a) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org.
Chem. 2006, 51−68. (b) Kolb, H. C.; Finn, M. G.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2001, 40, 2004−2021.
Notes
(14) Andersen, J.; Madsen, U.; Bjorkling, F.; Liang, X. F. Synlett 2005,
̈
The authors declare no competing financial interest.
2209−2213.
(15) For ligand 2a: El Alaoui, A.; Schmidt, F.; Amessou, M.; Sarr, M.;
Decaudin, D.; Florent, J.-C.; Johannes, L. Angew. Chem., Int. Ed. 2007,
46, 6469−6472. For ligand 2b: Davies, S. G.; Mortimer, D. A. B.;
Mulvaney, A. W.; Russell, A. J.; Skarphedinsson, H.; Smith, A. D.;
Vickers, R. J. Org. Biomol. Chem. 2008, 6, 1625−1634.
ACKNOWLEDGMENTS
The authors wish to thank the Toso Montanari Foundation for
financial support.
■
(16) Coppo, P.; Plummer, E. A.; De Cola, L. Chem. Commun. 2004,
1774−1775.
REFERENCES
■
(1) Balzani, V.; Bergamini, G.; Campagna, S.; Puntoriero, F. Top. Curr.
Chem. 2007, 280, 1−36.
(17) (a) Zanarini, S.; Felici, M.; Valenti, G.; Marcaccio, M.; Prodi, L.;
Bonacchi, S.; Contreras-Carballada, P.; Williams, R. M.; Feiters, M. C.;
Nolte, R. J. M.; De Cola, L.; Paolucci, F. Chem.Eur. J. 2011, 17, 4640−
4647. See also: (b) Ladouceur, S.; Zysman-Colman, E. Eur. J. Inorg.
Chem. 2013, 2895−3007. (c) Welby, C. E.; Gilmartin, L.; Marriott, R.
R.; Zahid, A.; Rice, C. R.; Gibson, E. A.; Elliott, P. I. P. Dalton Trans.
2013, 42, 13527−13536. (d) Mydlak, M.; Bizzarri, C.; Hartmann, D.;
Sarfert, W.; Schmid, G.; De Cola, L. Adv. Funct. Mater. 2010, 20, 1812−
1820. (e) Felici, M.; Contreras-Carballada, P.; Vida, Y.; Smits, J. M. M.;
Nolte, R. J. M.; De Cola, L.; Williams, R. M.; Feiters, M. C. Chem.Eur.
J. 2009, 15, 13124−13134.
(18) (a) Lo, K. K.-W.; Hui, W.-K. Inorg. Chem. 2005, 44, 1992−2002.
(b) Lo, K. K.-W.; Chan, J. S.-W.; Lui, L.-H.; Chung, C.-K.
Organometallics 2004, 23, 3108−3116.
(19) Crosby, G. A.; Demas, J. N. J. Phys. Chem. 1971, 75, 991−1024.
(20) Eaton, D. F. Pure Appl. Chem. 1988, 60, 1107−1114.
(21) Nakamura, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697−2705.
(2) Campagna, S.; Puntoriero, F.; Nastasi, F.; Bergamini, G.; Balzani, V.
Top. Curr. Chem. 2007, 280, 117−214.
(3) Flamigni, L.; Barbieri, A.; Sabatini, C.; Ventura, B.; Barigelletti, F.
Top. Curr. Chem. 2007, 281, 143−203.
(4) Kirgan, R. A.; Sullivan, B. P.; Rillema, D. P. Top. Curr. Chem. 2007,
281, 45−100.
(5) (a) Zhang, K. Y.; Lo, K. K.-W. Chemosensing and Diagnostics. In
Coordination and Organometallic Chemistry (Vol. 8) of Comprehensive
Inorganic Chemistry II; Yam, V. W.-W., Ed.; Elsevier: Amsterdam, 2013;
pp 657−732. (b) Lo, K. K.-W.; Li, S. P.-Y. RSC Adv. 2014, 4, 10560−
10585. (c) Shiu, H.-Y.; Chong, H.-C.; Leung, Y.-C.; Zou, T.; Che, C.-M.
Chem. Commun. 2014, 50, 4375−4378. (d) Lo, K. K.-W. Struct. Bonding
(Berlin) 2007, 123, 205−245.
(6) Fernandez-Moreira, V.; Thorp-Greenwood, F. L.; Coogan, M. P.
Chem. Commun. 2010, 46, 186−202 and references therein.
(7) For reviews see: (a) Lo, K. K.-W.; Choi, A. W.-T.; Law, W. H.-T.
Dalton Trans. 2012, 41, 6021−6047. (b) Lo, K. K.-W.; Zhang, K. Y. RSC
Adv. 2012, 2, 12069−12083. (c) Lo, K. K.-W.; Li, S. P.-Y.; Zhang, K. Y.
New J. Chem. 2011, 35, 265−287. (d) Lo, K. K.-W.; Zhang, K. Y.; Li, S.
P.-Y. Eur. J. Inorg. Chem. 2011, 3551−3568. (e) Lo, K. K.-W.; Zhang, K.
Y.; Li, S. P.-Y. Pure Appl. Chem. 2011, 83, 823−840. (f) Lo, K.K.-W.;
Louie, M.-W.; Zhang, K. Y. Coord. Chem. Rev. 2010, 254, 2603−2622.
(g) Lo, K. K.-W. Top. Organomet. Chem. 2010, 29, 115−158. (h) Lo, K.
K.-W.; Tsang, K. H.-K.; Sze, K.-S.; Chung, C.-K.; Lee, T. K.-M.; Zhang,
K. Y.; Hui, W.-K.; Li, C.-K.; Lau, J. S.-Y.; Ng, D. C.-M.; Zhu, N. Coord.
Chem. Rev. 2007, 251, 2297−2310 and references therein.
(8) (a) Lo, K. K.-W.; Tsang, K. H.-K.; Sze, K.-S.; Chung, C.-K.; Lee, T.
K.-M.; Zhang, K. Y.; Hui, W.-K.; Li, C.-K.; Lau, J. S.-Y.; Ng, D. C.-M.;
Zhu, N. Coord. Chem. Rev. 2007, 251, 2297−2310. For some recent
examples of biotinylated Ir(III) complexes see: (b) Leung, S.-K.; Liu, H.-
W.; Lo, K. K.-W. Chem. Commun. 2011, 47, 10548−10550. (c) Leung,
S.-K.; Kwok, K. Y.; Zhang, K. Y.; Lo, K. K.-W. Inorg. Chem. 2010, 49,
4984−4995. (d) Zhang, K. Y.; Lo, K. K.-W. Inorg. Chem. 2009, 48,
6011−6025. (e) Lo, K. K.-W.; Lau, J. S.-Y. Inorg. Chem. 2007, 46, 700−
709. For Ir(III)-indole complexes see: (f) Lau, J. S.-Y.; Lee, P.-K.;
Tsang, K. H.-K.; Ng, C. H.-C.; Lam, Y.-W.; Cheng, S.-H.; Lo, K. K.-W.
Inorg. Chem. 2009, 48, 708−718. For Ir(III) estradiol conjugates see:
(g) Lo, K.K.-W.; Zhang, K. Y.; Chung, C.-K.; Kwok, K. Y. Chem.Eur. J.
2007, 13, 7110−7120.
(9) (a) Law, W. H.-T.; Lee, L. C.−C.; Louie, M.-W.; Liu, H.-W.; Ang,
T. W.-H.; Lo, K. K.-W. Inorg. Chem. 2013, 52, 13029−13041. (b) Louie,
M.-W.; Liu, H.-W.; Lam, M. H.-C.; Lam, Y.-W.; Lo, K. K.-W. Chem.−
Eur. J. 2011, 17, 8304−8308.
(10) (a) Hermanson, G. T. Bioconjugate Techniques; Academic Press:
San Diego, CA, 1996. (b) Green, N. M. Adv. Protein Chem. 1975, 29,
85−133. (c) Wilchek, M.; Bayer, E. A. Methods in Enzymology; Academic
Press: San Diego, CA, 1990; Vol. 184. (d) Wilchek, M.; Bayer, E. A.
Methods Enzymol. 1990, 184, 123−138. (e) Wilchek, M.; Bayer, E. A.
Anal. Biochem. 1988, 171, 1−32.
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