Coordination to Imidazole Ring Switches on Phosphorescence of Platinum Cyclometalated Complexes: The Route to Selective Labeling of Peptides and Proteins via Histidine Residues

Article abstract of DOI:10.1021/acs.bioconjchem.6b00598

In this study, we have shown that substitution of chloride ligand for imidazole (Im) ring in the cyclometalated platinum complex Pt(phpy)(PPh₃)Cl (1; phpy, 2-phenylpyridine; PPh₃, triphenylphosphine), which is nonemissive in solution, switches on phosphorescence of the resulting compound. Crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies of the substitution product showed that the luminescence ignition is a result of Im coordination to give the [Pt(phpy)(Im)(PPh₃)]Cl complex. The other imidazole-containing biomolecules, such as histidine and histidine-containing peptides and proteins, also trigger luminescence of the substitution products. The complex 1 proved to be highly selective toward the imidazole ring coordination that allows site-specific labeling of peptides and proteins with 1 using the route, which is orthogonal to the common bioconjugation schemes via lysine, aspartic and glutamic acids, or cysteine and does not require any preliminary modification of a biomolecule. The utility of this approach was demonstrated on (i) site-specific modification of the ubiquitin, a small protein that contains only one His residue in its sequence, and (ii) preparation of nonaggregated HSA-based Pt phosphorescent probe. The latter particles easily internalize into the live HeLa cells and display a high potential for live-cell phosphorescence lifetime imaging (PLIM) as well as for advanced correlation PLIM and FLIM experiments.

Full text of DOI:10.1021/acs.bioconjchem.6b00598

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Article
Coordination to Imidazole Ring Switches On Phosphorescence
of Platinum Cyclometalated Complexes: The Route to Selective
Labeling of Peptides and Proteins via Histidine Residues
Anastasia I. Solomatina, Pavel S Chelushkin, Dmitrii V. Krupenya, Ivan S.
Podkorytov, Tatiana O. Artamonova, Vladimir V. Sizov, Alexei S Melnikov,
Vladislav V. Gurzhiy, Elena I. Koshel, Vladislav Shcheslavskiy, and Sergey P Tunik
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00598 • Publication Date (Web): 15 Nov 2016
Downloaded from http://pubs.acs.org on November 19, 2016
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Coordination to Imidazole Ring Switches On Phosphorescence of
Platinum Cyclometalated Complexes: The Route to Selective La-
beling of Peptides and Proteins via Histidine Residues
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†
†,‡
†
⊥
Anastasia I. Solomatina, Pavel S. Chelushkin, Dmitrii V. Krupenya, Ivan S. Podkorytov,
#
†
§,#
Tatiana O. Artamonova, Vladimir V. Sizov, Alexei S. Melnikov, Vladislav V. Gurzhiy,° Elena I.
¦
•
*,†
Koshel, Vladislav I. Shcheslavskiy, Sergey P. Tunik
†
St. Petersburg State University, Institute of Chemistry, Universitetskii pr. 26, 198504, St. Petersburg, Russia
‡
Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoi pr. V.O., 31, 199004, St. Peters-
burg, Russia
§
St. Petersburg State University, Department of Physics, Ulianovskaya Str., 3, 198504, St. Petersburg, Russia
⊥St. Petersburg State University, Biomolecular NMR Laboratory, Botanicheskaya 17, St. Petersburg 198504, Russia
#
Peter the Great St.Petersburg Polytechnic University, Research center of nanobiotechnologies, Polytechnicheskaya,
29, 195251 St. Petersburg, Russia
¦
°
St. Petersburg State University, Institute of Earth Sciences and Biology Department, University emb. 7/9, 199034,
St. Petersburg, Russia
•
Becker & Hickl GmbH, Nahmitzer Damm 30, 12277, Berlin, Germany
KEYWORDS Platinum complexes, stimuli responsive phosphorescence, histidine, peptides, proteins, selective covalent
conjugation, FLIM/PLIM imaging
ABSTRACT: In this study we have shown that substitution of chloride ligand for imidazole (Im) ring in cyclometalated
platinum complex, Pt(phpy)(PPh )Cl (1; phpy = 2-phenylpyridine; PPh = triphenylphosphine), which is non-emissive in
3
3
solution, switches on phosphorescence of the resulting compound. Crystallographic and NMR spectroscopic studies of
the substitution product showed that the luminescence ignition is a result of Im coordination to give the
[
Pt(phpy)(Im)(PPh )]Cl complex. The other imidazole-containing biomolecules such as histidine and histidine-
3
containing peptides and proteins also trigger luminescence of the substitution products. The complex 1 proved to be
highly selective towards the imidazole ring coordination that allows site-specific labelling of peptides and proteins with 1
using the route, which is orthogonal to the common bioconjugation schemes via lysine, aspartic/glutamic acids or cyste-
ine and does not require any preliminary modification of a biomolecule. The utility of this approach was demonstrated on
i) site-specific modification of the ubiquitin – a small protein that contains only one His residue in its sequence, and ii)
preparation of nonaggregated HSA-based Pt phosphorescent probe. The latter particles easily internalize into the live
HeLa cells and display high potential for live cell phosphorescence lifetime imaging (PLIM) as well as for advanced corre-
lation PLIM/FLIM experiments.
INTRODUCTION
nescence “switching on” in the presence of a specific bio-
molecule(s). In this context, cyclometalated platinum
complexes are among the most advanced “switch on”
phosphors because of their square-planar configuration
providing high sensitivity to subtle environmental chang-
es. As a result, these compounds are successfully used as
Recent years have seen rapid growth in implementation
1–4
of phosphorescent metal complexes into bioimaging
5
–7
and biosensing. This progress is mainly determined by
the unique photophysical properties of these lumino-
8
phores: they exhibit enhanced photostability, large
Stokes shifts (from 100 to 400 nm) and long excited state
lifetimes (μs to ms), the latter makes possible complete
signal discrimination from short-lived background fluo-
rescence through spectral or temporal resolution. One
of the challenges in this area is development of stimuli-
responsive probes demonstrating highly selective lumi-
7,12–15
2
DNA sensors and protein stains.
2
Nevertheless, currently available cyclometalated plati-
num “switch on” probes lack binding regioselectivity,
which stems from noncovalent mode of their interaction
9,10
11
7,12–15
with biomolecules.
For instance, cyclometalated plat-
inum “switch on” probes for DNA are either intercalators
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Bioconjugate Chemistry
7
or minor groove binders. In these systems, selectivity is
achieved either via selective recognition of some specific
detection and monitoring of their presence in tissues and
biological media of particular interest.
1
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1
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1
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1
6,17
DNA structures such as G-quadruplex,
or via using the
In this communication, we describe the first, to the best
of our knowledge, example of luminescence “switching
on” in cyclometalated platinum complex as a result of
coordination bond formation with imidazole ring or with
biomolecules, which contain this group in their structure,
i.e. histidine (His) and histidine-containing peptides and
proteins. We also show that this binding is highly selec-
tive and dominates over interaction with any other amino
acid residues present in proteins. This unique feature
provides a novel route to site-specific labeling of peptides
and proteins as demonstrated by regioselective probe
insertion into ubiquitin – the protein that contains only
one His residue in its amino acid sequence. As a further
development of this approach, preparation of nonaggre-
gated HSA-based Pt phosphorescent probe is also demon-
strated. This probe easily internalizes into live HeLa cells
and displays high potential for recently emerged simulta-
aptamers that form double helix exclusively in the pres-
ence of the analyte molecule specifically recognized by
the aptamer. In the case of proteins, “switch on” probes
are typically incorporated into their hydrophobic cavities
7,18
(
“pockets”), the way that does not provide substantial
regioselectivity and also makes binding reversible with
1
2–15
the tendency to dissociate upon high dilution.
cent publications
In re-
it was found that Ru(II) diimine and
Ir(III) cyclometalated complexes display luminescence
switch on” upon interaction with histidine with superior
1
9–21
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“
selectivity of the response over other natural amino acids.
However, the authors did not study the chemical details
of the complex-histidine interaction having limited it to
ESI mass-spectroscopic investigation, based on which the
1
9
authors of earliest publication even concluded that there
is no covalent interaction between histidine and Ru ion.
We believe that alternative binding mechanism, namely
formation of covalent/coordination bonds, plays a key
role in emission “switching on” and provide the resulting
high regioselectivity and enhanced stability of the com-
neous fluorescence/phosphorescence lifetime imaging
34,35
(FLIM/PLIM)
of live bio-objects.
RESULTS AND DISCUSSION
The information concerning interaction of stimuli re-
plexes with histidine and histidine containing biomole-
20,21
19
22–24
sponsive Ir(III)
and Ru(III) probes with histidine and
cules. Indeed, many types of Pt complexes,
anti-tumor drugs,
including
25–27
histidine containing biomolecules indicated that lumi-
nesce switch on of the starting non-emissive complexes is
due to their interaction with imidazole functionality of
histidine to give imidazole substituted compounds, which
display considerable emission enhancement compared to
the parent complexes. However, none of these communi-
cations reported the details of this chemistry and its rela-
tion to the photophysical characteristics of the resulting
are prone to forming coordination
bonds with different biomolecules, particularly, with pro-
teins, but there is no reports, which would describe lumi-
nescence “switching on” as a result of this process. Anoth-
er important issue is regioselectivity of binding: Pt com-
pounds typically form complexes with various side groups
23,28
in proteins that results in nonselective binding.
Con-
sequently, the development of cyclometalated platinum
complexes that could “switch on” their luminescence up-
on selective binding with some specific groups in biomol-
ecules is strongly desired. In this respect it is also worth
noting that histidine-rich proteins play a very important
luminescent complexes. Our nearly accidental findings
II
that well known platinum complex Pt (phpy)(PPh )Cl (1;
3
where phpy
=
2-phenylpyridine;
PPh3
=
tri-
phenylphosphine) demonstrates emission “ignition” upon
interaction with HSA (human serum albumin) prompted
29–33
role in many biological processes
that makes their
Figure 1. Synthesis of complexes 1, 1-Im and 1-His. A. Synthetic scheme. Conditions: (a) PPh , CH Cl /Hexane, 4°C; (b) CH Cl ,
3
2
2
2
2
Imidazole (3 equiv), 25°C, 30 min; (c) DMSO/H O, 25°C. B. Emission spectra of 1 and mixtures of 1 with 5 equivalents of imidaz-
ole (1-Im) or histidine (1-His); [1] = 0.4 mM; solvent - DMSO; λex = 350 nm.
2
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product shows the dominating signal ([1-Cl+Im]
Page 4 of 22
+
us to perform detailed investigation of coordination
chemistry and photophysics of 1 with imidazole (Im) and
imidazole containing derivatives, including such biomol-
ecules as ubiquitin and HSA. This study clearly indicates
that the platinum ion coordination to Im functionality
plays a key role in the phenomena observed and demon-
strated high potential of the suggested approach to regio-
selective labeling of histidine containing biomolecules
and application of 1 in stimuli responsive bioimaging.
=
1
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679.1624 m/z) that corresponds to the substitution of the
chloride ligand with the imidazole (Figure S5). Similarly,
mixing of 1 with histidine results in appearance of the
+
+
related [1-Cl+His] (766.19 m/z) ion in the ESI mass-
spectrum that is indicative of a similar chloride ion sub-
stitution for histidine (Figure S6). Reaction of 1 with both
imidazole and histidine is reversible with rather high
equilibrium concentrations of initial compounds; conse-
quently, 3-5-fold excess of imidazole-containing reagents
is needed to shift the equilibrium to the right (Figure S7).
The imidazole substituted complex 1-Im easily forms
well-defined crystals, while 1-His does not give single
crystals suitable for XRD analysis. The solid state struc-
The complex 1 has been already reported in the article
devoted to investigation of anticancer activity of cy-
clometalated platinum(II) complexes. However, this
study describes rather scanty analysis (limited to IR and
0
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1
H NMR spectroscopies) of its structure and stereochem-
ture of 1-Im ([Pt(phpy)(PPh )(Im)]Cl) determined by sin-
istry and does not contain any information on its reactivi-
ty and photophysical properties. Therefore we prepared 1
by reaction of triphenylphosphine with the [Pt(2-
3
gle crystal X-ray crystallography (Figure 2) revealed that it
retains square-planar geometry completely analogous to
that of 1 with the chloride and phosphine ligands in cis-
and trans-positions to the nitrogen atoms of the metallat-
ed ring, respectively. Imidazole coordinates to the plati-
num ion via the N-center bearing free electron pair to
give cationic species with the chloride ion in the outer
phenylpyridine)dmsoCl]
precursor
in
dichloro-
methane/hexane mixture (Figure 1, A) and performed its
exhaustive structural characterization. Complex 1 demon-
strates polymorphism in solid state: it forms two types of
crystals with different unit cell parameters (Table S1). The
XRD study of the both polymorphs showed that 1 (Figures
1
1
sphere of the complex. The study of 1-Im using the H- H
3
1
COSY (Figure S8) and P NMR (Figure S3) spectroscopy
shows that it retains the same structure in solution.
2
, A and S1) displays square-planar geometry typical for
cyclometalated Pt(II) complexes with the structural pa-
rameters in the range typical for this type of platinum
compounds. The chloride ligand occupies cis-position to
the nitrogen atom of the metallocycle, whereas the phos-
phine ligand was found in trans-position.
More sophisticated behavior is characteristic for the 1-
+
His complex in solution. The ESI mass-spectrum of the
(1:His = 1:5) reaction mixture displays only one signal ([1-
+
Cl+His] ) corresponding to the product of chloride for
histidine substitution (Figure S6) in addition to the sig-
31
nals of the starting compounds. However, the P NMR
spectrum (Figure S3, S7) of the same mixture consists of
several signals with relatively close chemical shifts that
indicates formation of a few species corresponding to dif-
ferent conformers or isomers of coordinated histidine in
1
1
the complexes formed. Detailed analysis of the H- H
1
COSY (Figure S9) and the aliphatic region in the H NMR
spectrum (Figure S10) revealed the presence of four iso-
meric species of 1-His. Figure S10 demonstrates experi-
1
β
α
mental H spectra of histidine C H -C H moiety in 1-His
2
Figure 2. Solid-state structure of 1a (A) and 1-Im (B). A.
Complex 1a. Selected bond distances (Å): Pt1–C11 2.004(3),
Pt1–N1 2.085(3), Pt1–P1 2.2250(1), Pt1–Cl1 2.376(1). Selected
angles (°): C11–Pt1–P1 95.38(9), C11–Pt1–N1 80.4(1), N1–Pt1–Cl1
2.60(8), Cl1–Pt1–P1 91.54(3). B. Complex 1-Im. Selected bond
distances (Å): Pt1–C11 2.013(3), Pt1–N1 2.096(2), Pt1–N2
.101(2), Pt1–P1 2.2377(9). Selected angles (°): C11–Pt1–P1
and simulated spectra of each isomer; the Table S2 shows
the main parameters of the simulated spectra. The exist-
ence of two isomers can be explained by tautomerism of
37
imidazolium ring; thus both stereo-inequivalent nitro-
9
gen atoms may act as donors of electron pair for platinum
atom. Each of these isomers in principle can form some
conformers with different orientation of amino acid chain
with respect to cyclometalated fragment. The influence of
sterically bulky phosphine ligand together with intramo-
lecular non-covalent interactions between coordinated
histidine and other parts of the molecule slow down in-
terconversion of the conformers and allows distinguishing
2
9
9
5.9(1), C11–Pt1–N1 80.5(1), N1–Pt1–N2 91.3(1), N2–Pt1–P1
2.36(7), N1–Pt1–N2–C30 100.4(3). Hydrogen atoms are omit-
ted for clarity.
1
1
31
Combination of the H- H COSY (Figure S2) and
P
+
NMR spectra (Figure S3), as well as ESI mass spectromet-
ric data (Figure S4) indicate that 1 retains the same struc-
ture in solution.
3
1
1
of four different species in the P and H NMR spectra.
Though there are several hypothetic variants of different
stabilized conformers to be formed, we would like to
avoid extended speculations on this topic, since it is far
beyond
The complex 1 readily reacts with imidazole and imid-
azole-containing molecules, e.g. histidine, to give substi-
tuted cationic complexes 1-X (X = Im or His), the latter
+
being soluble in water. The ESI mass-spectrum of the
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Bioconjugate Chemistry
Table 1. Photophysical properties of 1 and its complexes with imidazole and different imidazole-containing bi-
omolecules in DMSO and water.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
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5
5
5
5
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5
5
5
5
5
6
a
b
Compound
Abs, nm
ε×10-3, M-1cm-1)
278 (17), 285 (17), 314sh The compound
Em, nm
Ex, nm
τ(aer)*,
Φ(aer)*,
%
τ(deg)*,
μs
Φ(deg)*,
%
a
a,b
a
a,b
µs
(
1
in DMSO
(
(
7.9), 327sh (6.4), 380 is not lumines-
2.9) cent
1-Im
268 (18), 276 (18), 296sh 477, 510, 538sh
8.1), 316 (6.5), 327 (6.3),
62 (2.8)
311sh,
360
340sh, 0.5±0.1
0.17±0.03
0.8±0.2
0.8±0.1
1.4±0.1
0.23±0.0
5
(
3
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2
3
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0
in DMSO
1
-His
in DMSO
-His
in H O
275 (22), 300sh (10.3), 316 478, 511, 540sh
(8.5), 327 (8.0), 362 (3.3)
283, 318sh, 326, 0.8±0.1
360
1.1±0.2
0.5±0.1
2.0±0.4
18±4
1
267 (24), 312 (8.1), 322 477, 509, 538sh 282, 320, 355
7.6), 353 (3.4)
0.8±0.1
0.36±0.08 1.9±0.2
(
2
1-Ubq
272 (10), 296sh (3.8), 316 478, 511, 539sh
3.0), 326 (2.7), 360 (1.1)
275, 317, 326, 0.8±0.1
360
1.1±0.2
15±3
1.4±0.1
20.0±2
(
in H O
2
1-HSA
277 (41), 312sh (4.5), 326sh 477, 510, 538sh
3.8), 360sh (1.8)
275, 316, 327, 15.6±2
360
(
in H O
2
a
b
Excitation wavelength 350 nm; Emission wavelength 510 nm; *aer/deg – aerated/degassed solution.
Figure 3. Electron density difference plots for the lowest energy triplet emission (T → S ) of the complexes 1, 1-Im and 1-His.
1
0
During the electronic transition, the electron density increases in the blue areas and decreases in the red areas. The emission
wavelength was estimated from the total energy difference of the optimized states.
of the scope of the present paper. It is also very probable
that isomerization described above prevents formation of
single crystals suitable for XRD analysis.
center is switching on the complex luminescence. The
complex 1 reveals detectable green luminescence only in
solid state (Table S3) but does not display any emission in
solution. On the contrary, addition of imidazole or histi-
dine to the solution of 1 gives rise to green luminescence
within less than 5 minutes that is necessary to yield ap-
preciable amount of the substituted product (Figure 1, B).
Photophysical properties of 1 and its substitution prod-
ucts with imidazole and histidine are summarized in Ta-
ble 1. All reaction products of 1 with imidazole and imid-
azole containing biomolecules display nearly identical
emission bands (Fig. S11,B) that indicates origin of emis-
sion from the same chromophoric center, evidently from
Interaction of 1 with compounds containing imidazole
ring is not unexpected: binding with histidine via imidaz-
ole function has been documented for different platinum
2
2,38,39
40
complexes,
including those with terpyridine ligand,
24,41
and cyclometalated Pt(C^N) complexes.
However, to
the best of our knowledge, none of the above reports have
mentioned luminescence ignition as a result of imidazole
ring coordination. In the present study, we showed that
the most notable feature accompanying coordination of
imidazole containing ligands to the cyclometalated Pt(II)
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with
4
2
the {Pt(C^N)} moiety. Both excitation and emission
bands fall into expectable wavelength ranges observed for
the other cyclometalated Pt(C^N) complexes. These
observations, together with large Stokes shift, lifetimes in
microsecond domain and clearly resolved vibrational
complex
resulting
from
its
interaction
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(bio)molecules containing imidazole ring was not previ-
ously described.
4
2
From the above data on the interaction of 1 with imid-
azole and histidine, one can anticipate a similar lumines-
cence switch on upon reaction of 1 with the biomolecules
containing imidazole ring or histidine, such as histidine
containing peptides and proteins. This perspective may
open an avenue for application of 1 in regioselective label-
ing of these biomolecules accompanied by luminescence
of the conjugated platinum complex. To date, main bio-
conjugation strategies involved linking of the luminescent
probes via amino or carboxylic functions presented in
peptides or proteins (ε-amino groups of lysine or N-
terminal amino groups, side groups of aspartic or glutam-
-
1
spacing of ca. 1360 cm point to IL/MLCT phosphores-
4
2
cence typical for this class of platinum complexes.
To shed light onto the reasons of the observed lumines-
cence ignition as well as on the nature of electronic tran-
sitions in 1, 1-Im and 1-His, we performed comparative
DFT and TD DFT calculations for these complexes. Re-
sults of calculations (Figure 3 and Figures S12, S13) show
that for all these complexes the nature/composition of
LUMO and HSOMO (Highest Singly Occupied Molecular
Orbital in triplet state) as well as HOMO and LSOMO
0
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43
(
Lowest Singly Occupied Molecular Orbital in triplet
ic acids or C-terminal carboxylic groups). The probes
state) are essentially similar. Comparative analysis of the
boundary orbitals in 1, 1-Im and 1-His, however, shows
that LUMO (HSOMO) in these complexes are similar in
nature, but there is a substantial difference between
HOMO (LSOMO). In the case of 1, the Cl and Pt orbitals
significantly contribute to HOMO (LSOMO) and the con-
tribution of Pt seems to dominate over that of cyclomet-
alated ligand, resulting in a mixed interligand and metal-
to-ligand transition in the case of low energy excitation.
On the contrary, in 1-Im and 1-His both HOMO
can be involved in one-step conjugation with the above
residues (e.g. via carbodiimide chemistry, using active
esters, iso(thio)cyanates, aldehydes, etc.); these strategies
seem to be the most used ones. Alternatively, the same
residues can be modified to introduce some reactive func-
tionalities enabling further modification by the probe via
43
various “bioorthogonal” chemistries (for example, alkynyl
44,45
or azide groups for “click” chemistry
keto groups for chemical ligation
or aldehyde or
46,47
); these approaches
typically provide very fast conjugation with high yields
but require preliminary modification of biomolecules.
Ultimately, all the above conjugation strategies involve
modification of Lys, Glu and Asp residues; however, be-
cause these residues are the most abundant ones in the
biomolecules, these approaches are not regioselective.
Examples of regioselective conjugations are very rare and
usually limited to peptides rather than to proteins; in the
latter case, only few examples are reported: i) modifica-
tion of human serum albumin (HSA) via free thiol group
(
LSOMO) and LUMO (HSOMO) are localized at the cy-
clometalated ligand, with slightly smaller contribution of
Pt centered orbitals and without any significant contribu-
tion of imidazole ligands into the orbitals responsible for
emission. This means that emissive transitions in the
complexes containing coordinated imidazole moiety are
∗
of almost pure π−π intraligand nature perturbed by the
1–4
heavy atom effect. Moreover, it was also shown that
substitution of chloride with strong field ligands (like
imidazole) results in the growth of the dd excited states
energy, which plays a key role in non-radiative deactiva-
tion of excited states in the Pt(II) complexes of this type,
thus eliminating an important channel of emission
quenching. We believe that both these reasons signifi-
cantly change photophysical behavior of 1-Im and 1-His
compared to that of 1 and result in luminescence ignition
in the imidazole containing complexes.
4
8
of cysteine-34 using thiol-maleimide click chemistry ii)
selective oxidation of N-terminal serine leading to genera-
tion of aldehyde group suitable for oxime and hydrazone
4
7
49
ligations; iii) selective modification of tryptophan.
Note that at least two cases (free thiol groups of cysteine
and N-terminal serine residues) are not typical for pro-
teins; therefore, there is a strong need in alternative bio-
conjugation approaches.
The only examples of closely related behavior are Ru(II)
In this respect, coordination of 1 to imidazole function
in histidine seems to be an alternative to standard biocon-
jugation routes and, contrary to click chemistry or chemi-
cal ligation approaches, does not require any pre-
modification of a protein. Moreover, in some cases this
reaction can provide true regioselective labeling. To eval-
uate selectivity of 1 towards different amino acids, we
added its solution in DMSO to the water solutions of all
20 natural amino acids and run the mass spectra of the
mixtures obtained along with monitoring of luminescence
appearance. We have found that formation of conjugates
and luminescence ignition were observed in the case of
histidine, arginine and cysteine (Figure 4, A). However,
19
20,21
diimine and Ir(III) cyclometalated
complexes, but in
the Pt(II) chemistry there are no direct analogs of the
complexes presented in this study. Somewhat similar ex-
amples of luminescence “ignition” upon interaction with
12–15
biomolecules are: i) a number of “switch-on” Pt probes,
which nonselectively stain proteins; such complexes react
with proteins via non-covalent (presumably, van-der-
Waals) interactions and thus demonstrate preferable
binding to plasma transport proteins (e.g. serum albu-
mins), which possess well-developed hydrophobic cavi-
ties; ii) cyclometalated Pt complex binding to two histi-
24,41
dine residues in an amyloid peptide.
Nevertheless, to
the best of our knowledge, luminescence ignition of Pt(II)
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Bioconjugate Chemistry
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Figure 4. A. Emission spectra of 1 (50 μM) in water: DMSO mixture (20 : 1) with 20 natural amino acids (150 μM). Excitation
wavelength 365 nm. B. Dependence of luminescence ignition of 1 on time in the presence of histidine, arginine and cysteine in
water: DMSO mixture (1 : 21). [1] : [amino acid] = 1: 3; [Pt] = 43.5 μM.
emission wavelength of the conjugates are expectedly
different due to coordination of the platinum chromo-
phore to aromatic nitrogen, aliphatic nitrogen and sulfur
coordination functions, respectively. Kinetic studies re-
vealed that interaction with histidine proceeds quite fast
1 and Figure S11). These observations imply, as in the case
of imidazole and histidine, direct coordination of Ubq to
Pt ion through imidazole ring of histidine. Moreover, this
result also suggests that, despite the presence of four ar-
ginine residues in the Ubq sequence, interaction of 1 with
Ubq is highly regioselective towards histidine.
(
quantitative yield is achieved within 5 minutes) while
reaction with two amino acids mentioned above occurs
much slower (Figure 4, B). Moreover, addition of (3+3)
equivalents of histidine/arginine mixture to 1 results in
generation of luminescence with the maximum at 510 nm,
Figure S14 (same as in the case of 1-Im and 1-His), which
differs significantly from that of 1-Arg (broad peak at 520-
550 nm). These observations suggest that kinetic control
of the complex 1 reactions with histidine, arginine and
cysteine results in selective binding of the probe to histi-
dine function in biomolecules containing these amino
acids.
To further prove regioselectivity of this interaction, we
performed a comparative 2D NMR study of 1-Ubq vs Ubq
15
using N enriched samples. Figure 5, A shows relative
1
15
signal loss in the H- N HSQC NMR spectrum of 1-Ubq
compared to that of Ubq. The values of relative signal
1
15
losses were calculated from H- N HSQC spectra, Figure
S16, and assigned to each amino acid residue. These ex-
periments made possible to determine the domains,
which underwent substantial conformational changes
associated with the strongest signal losses. These domains
are painted by red color on the amino acid sequence of
Ubq (upper row in the Figure 5, A) and on the ribbon
structures of the protein (Figure 5, B and C). The data
obtained indicate that four domains of amino acid se-
quence show strongest conformational changes: 2-9
(QIFVKTLT), 13-15 (ITL), 44-49 (IFAGKQ), and 65-68
(STLH); these sequences form β-sheets in native form of
Ubq and are spatially close to each other, the latter one
containing histidine residue (Figure 5). Consequently,
their simultaneous conformational perturbation detected
in the NMR comparative study is most probably caused
by coordination of 1 to His-68. As an independent proof
of coordination site involved into reaction with 1, we per-
formed trypsinolysis of 1-Ubq followed by fingerprint
mass-spectral analysis of the reaction mixture (Table S4).
Both mass spectroscopic signals associated with the pres-
ence of platinum complex were associated with the 64-72
(ESTLHLVLR) sequence ([ESTLHLVLR+1-Cl] = 1676.705
m/z; [ESTLHLVLR+1-Cl-TPP] = 1414.644 m/z). To further
To demonstrate the potential of 1 in regioselective label-
50
ing of proteins, we have chosen ubiquitin (Ubq) as a
model. This small protein (M.W. 8565 Da) contains only
one histidine residue in its sequence (His-68) and thus
can be used to monitor site selectivity in labeling with 1.
In the case of ubiquitin, binding of a probe to histidine is
of particular importance, since carboxylic and amino
groups of the protein cannot be used for labeling because
C-terminal carboxylic group of glycine-76 is the primary
group involved in ubiquitination while all the lysine resi-
dues are the active functionalities in the polyubiquitina-
51
tion process. In this respect, labeling via His-68 is an
extremely useful alternative. We have found that addition
of 1 solution in DMSO to the aqueous Ubq solution re-
sults in appearance of a new signal in the MALDI-TOF
mass-spectrum ([Ubq+1-Cl] = 9176 m/z; Figure S15) that
corresponds to the substitution of chloride ion in 1 with
Ubq molecule. This reaction is accompanied by emer-
gence of green luminescence, with the emission spectrum
of 1-Ubq nearly identical to that of 1-His and 1-Im (Table
localize position of
1 in the sequence 64-72 we
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1
15
Figure 5. Interaction of 1 with ubiquitin. A. Relative signal loss in the H- N HSQC NMR spectra of 1-Ubq compared to Ubq as a
function of amino acid sequence. Frontal view (B) and top view (C) of ribbon models of ubiquitin with the color presentation of
conformational perturbations. Colors for perturbation degrees: grey - none (<0.1); pink - low (0.1-0.35); deep pink - medium
(0.35-0.45); bright red - high (>0.45). Number of PDB entry for Ubq: 1UBQ. For clarity, the His-68 residue is shown by the red
pentagon.
synthesized Ac-ESTLHLVLR-NH peptide, mixed it with 1
further studies have shown that these adducts appear to
form aggregates in solution that eventually led to their
instability in aqueous media. As a result, the aggregated
complexes were not taken up by living cells and imaging
experiments could be performed on fixed cells only. The
aggregation may be provoked by the presence of rather
large polynuclear hydrophobic complexes in the adducts.
Taking into account substantially lower size of the mono-
nuclear Pt compounds we performed the synthesis and
investigation of covalent 1-HSA conjugate aiming at over-
coming the aggregation drawbacks.
2
and detected new ion that corresponds to the expected
conjugate ([peptide+1-Cl] = 1717.6 m/z). Finally, the set of
data obtained for the reaction of 1 with Ubq stronglyevi-
dences in favor of the complex binding to Ubq via His-68,
and makes possible application of 1 for regioselective la-
beling of histidine-containing peptides and proteins.
5
3
5
3
Preparation of waterꢀsoluble phosphorescent covalent conꢀ
jugates with functional proteins can be considered as another
prospective area of the complex 1 application, provided that
the conjugation does not have crucial effect onto the protein
functional properties. The assay of the proteinase K (proK)
and deoxyribonuclease I (DNase I) activity vs the correspondꢀ
ing conjugates with 1 display negligible effect of conjugation,
see SI (Table S6), but extrapolation of these observations onto
other proteins/enzymes evidently require additional tests.
Human serum albumin (HSA), the most abundant human
plasma protein, acts as a transporter for a variety of hy-
drophobic components in human organism. Additionally,
HSA, being endogenous protein, possesses low systemic
toxicity even if conjugated with exogenous molecules, and
thus, increases their tolerability.
Interaction between 1 and HSA proceeds similarly to
that between 1 and ubiquitin: addition of 1 to HSA leads,
on the one hand, to quenching the HSA fluorescence
(
Figure S17, A), and, on the other hand, to appearance of
intense green luminescence (Figure S17, A and B). It is
worth noting that aggregation properties of 1-HSA strong-
ly depend on stoichiometry of the reaction mixture: com-
plexes formed at the [1]:[HSA] molar ratios below 1:1 form
transparent solutions and do not precipitate under pre-
parative centrifugation at 14,000 rpm and retain their ab-
sorbance
and
luminescence
unchanged
(ratio
These considerations prompted us earlier to develop
water-soluble HSA-based phosphorescent adducts with
heterometallic copper subgroup supramolecular clusters
that appeared to be applicable in different bioimaging
Abs(after)/Abs(before) close to 1; Figure 6), while the re-
action mixtures with [1]:[HSA] above 1:1 form turbid solu-
tions and give a precipitate under preparative centrifuga-
tion (14,000 rpm, 10 min; Figure 6). This finding implies
that the conjugates containing one platinum ion per HSA
molecule are stable to aggregation while further binding
5
2
applications, including time-gating and 3D tissue recon-
5
3
struction by two-photon microscopy. Unfortunately,
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Bioconjugate Chemistry
major advantage of phosphors over conventional fluores-
of the platinum complex to available histidine residues
1
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6
leads to the increase in the overall hydrophobicity of the
conjugate and to the loss of solubility. Therefore we in-
vestigated the properties of 1-HSA conjugates formed at
stoichiometric composition ([1]:[HSA] = 1:1) of the reac-
tion mixture. The conjugates are soluble in water; gel
permeation chromatography (GPC) reveals both unimers
and dimers in the solution of 1-HSA, and their ratio is
shifted towards dimers compared to pure HSA (Figure
S18). This proves that aggregation of the conjugates ob-
tained is restricted to dimerization, without formation
larger aggregates that would cause precipitation. Alt-
hough we found that interaction of 1 with the imidazole
function is reversible, we observed neither trace amounts
of 1 in GPC experiments, nor any loss of absorbance after
extensive dialysis (Figure S19). Most probably, the equilib-
rium which was observed in organic solvents (DMSO) is
suppressed in water because of the complex insolubility in
aqueous solutions; therefore, the 1-HSA conjugates are in
kinetically ‘frozen’ state and thus protected from dissocia-
tion.
cent probes. Additionally because the lifetime of the most
organometallic phosphorescent complexes depends
1
1,55,56
strongly on the cell microenvironment
, PLIM is also
an appropriate technique to analyse the physiological
status of living cells and tissues. This is achieved through
the environment effects on the kinetics of the probe ex-
cited state deactivation that are difficult to measure via
steady-state methods. To demonstrate these benefits, we
performed recently developed simultaneous phosphores-
3
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0
cence/fluorescence lifetime imaging (PLIM/FLIM) . This
variant of lifetime-based imaging offers simultaneous
mapping of the cell metabolic components having auto-
fluorescence by FLIM and phosphorescent probe distribu-
tion in the cell by PLIM. The correlation between meta-
bolic activity, redox ratio and fluorescence lifetime during
stem cell differentiation, neurodegenerative diseases, and
5
7–60
carcinogenesis was earlier described
. In particular, it
has been shown that the fluorescence lifetime of the co-
enzyme NAD(P)H (nicotinamide adenine dinucleotide
(phosphate)) varies depending on the physiological state
61–67
of the sample
. Therefore, in this study the metabolic
information is derived from the FLIM data for NAD(P)H.
The simultaneous FLIM of NAD(P)H and PLIM of 1-HSA
makes it possible to evaluate the cell microenvironment
effect on the 1-HSA phosphorescence, as well as the 1-
3
4
HSA effect on the cells metabolism. The simultaneous
FLIM of NAD(P)H and PLIM of 1-HSA were used for the
analysis of live HeLa cells (Figure 7).
Figure 6. UV absorption and luminescence emission ratios
after and before centrifugation of 1-HSA solutions at 14000
rpm, and turbidity (τ = (100-T)/100, where T – solution
54
transmittance, %; ) of solution 1-HSA as a function of [1] :
[
HSA] molar ratio. Grayscale filling reflects solution turbidity
from transparent (white) to turbid (gray). Vertical dashed
line at [1] : [HSA] = 1.0 delineates area of stable solutions ([1]
:
[HSA] ≤ 1.0) from area of aggregation instability ([1] : [HSA]
>
1.0).
The 1-HSA conjugate demonstrates photophysical
Figure 7. Simultaneous FLIM of NAD(P)H and PLIM of 1-
HSA on live HeLa cells. (A) 1-HSA phosphorescence lifetime
imaging of experimental cells treated with 1-HSA; (B)
NAD(P)H fluorescence lifetime imaging of experimental cells
treated with 1-HSA; (C) NAD(P)H fluorescence lifetime im-
aging of control (nontreated) cells; (D) distribution of
NAD(P)H fluorescence lifetimes across the image in experi-
mental (treated with 1-HSA) and control (nontreated) cells;
(E) distribution of 1-HSA phosphorescence lifetime across
the image in experimental cells treated with 1-HSA.
characteristics similar to those of 1-Ubq and 1-His (Figure
S11) but the overall luminescence efficiency is superior
compared to those of the other substances: 1-HSA
demonstrates longer lifetimes (12 µs) and more than 10-
fold increase in quantum yield (Table 1). We believe that
both effects stem from more effective shielding of the
luminescent center from emission quenching by water
and lower vibrational deactivation of the triplet excited
state as a result of embedding of 1 into hydrophobic cavi-
ties present in the HSA globule.
The 1-HSA complex in a concentration of 0.3 mg/ml
was successfully internalized into live cells and distribut-
ed in cytoplasm (Figure 7A). The NAD(P)H lifetime dis-
tribution did not change when the cells were incubated
Long lifetimes of 1-HSA suggest its applicability in
5
2
time-resolved bioimaging (e.g. in time-gating or phos-
3
4
phorescent lifetime imaging, PLIM ), which benefits
from the ability to remove background fluorescence - the
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with 1-HSA as indicated by the comparison of NAD(P)H Cambridge Isotope Laboratories. All salts and acids used
1
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fluorescence lifetime distributions in experimental and
control cells (Figure 7D) while the average NAD(P)H life-
times in the experimental and control cells were 1.72 ±
for preparation of buffer solutions were produced by
“Vekton” (Russia) and had analytical-grade purity. The
platinum(II) precursor [Pt(2-phenylpyridine)dmsoCl] was
prepared according to the published procedure by the
reaction of potassium tetrachloroplatinate and 2-
phenylpyridine in water/2-ethoxyethanol solution fol-
lowed by heating at 80 °C in DMSO for 48 hours. Di-
chloromethane was purified and distilled using standard
procedure. Water was purified using Simplicity Water
Purification System Merck Millipore (type 1 water).
8
0
0
.09 and 1.68 ± 0.08 ns, respectively. The percentage of
the free and protein-bound NAD(P)H as well as its fluo-
rescence lifetime remained virtually unchanged in the
cells incubated with 1-HSA (Table S5) that indicates near-
ly no effect of the conjugate on the cell metabolism. The
average phosphorescence lifetime of 1-HSA in the live
cells was 7.24 μs and varies in the 6.8 to 7.8 μs range
across the image depending on the microenvironment
0
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1
1
Instrumentation. Solution H NMR, H- H COSY, and
P NMR spectra were recorded with a Bruker Avance III
31
(
Figure 7E). Luminescent platinum complexes can be suc-
cessfully used as oxygen sensors, in particular in the bio-
(
400 MHz) and a Bruker Avance III (500 MHz) spectrom-
1
1,55,56,68–73
1
logical and medical studies
due to their phospho-
eters (Bruker, Germany). Simulation of the H spectra was
performed using Dr. Kirk Marat SpinWorks (Canada)
and Bruker NMRSim programs. The ESI mass spectra
81
rescence quenching by oxygen. Recently, platinum phos-
phorescent probes have been adapted for the oxygen
+
1
1,56,70,71,74–77
measurements in cell cultures
, particularly in
microbioreactors , and in vivo tissue and organ
were obtained with a MaXis instrument (Bruker, Germa-
72
+
ny), in the ESI mode (solvent MeOH). Microanalyses
69,73,78
analysis
. The results clearly show that the 1-HSA
were carried out with a Euro EA-3028HT Elemental Ana-
lyzer (EuroVector, Italy). Gel permeation chromatography
probe retains its long lifetime in biological microenvi-
ronment and therefore is amenable for different time-
resolved imaging applications, in particular in the simul-
taneous PLIM/FLIM experiments due to a simple route to
preparation, effective internalization into live cells, the
absence of effect on cell metabolism and high sensitivity
to microenvironment.
(
GPC) was performed with high performance liquid
chromatograph Shimadzu LC-20 Prominence (Shimadzu,
Japan) equipped with RF-20A and SDD-M20A detectors
and PSS PROTEEMA 300 column. Microcrystalline sam-
ples were used for the photophysical experiments in the
solid state. Steady-state photoluminescence spectra were
recorded on a Fluorolog-3 (JY Horiba Inc., Japan) spectro-
fluorimeter. Lifetimes measurements in solid state were
carried out with the Time-Correlated Single Photon
Counting (TCSPC) method and the results have been
treated with the Jobin-Yvon software package and the
Origin 8.1 program. Direct quantum yield measurements
for the crystalline samples were performed at room tem-
perature with an integrating sphere from Quanta-phi (JY
Horiba Inc., Japan). UV/Vis spectra and turbidity meas-
urements were recorded with a Shimadzu UV-1800 spec-
trophotometer. Turbidity (τ) was recalculated from solu-
CONCLUSIONS
In conclusion, this study presents the first example of
luminescence ignition resulting from chloride ligand sub-
stitution with imidazole ring in the platinum cyclomet-
alated complex, Pt(phpy)(PPh )Cl (1). The observed effect
3
encompasses various imidazole-containing biomolecules
such as histidine and histidine-containing peptides and
proteins. The complex 1 displays an extremely high selec-
tivity towards coordination of imidazole ring, which is
dictated by both thermodynamics and kinetics of the sub-
stitution reaction. This unique feature allows for site-
specific labeling of peptides and proteins using 1 with the
route that is orthogonal to classic bioconjugation schemes
via lysine, aspartic/glutamic acids or cysteine and does
not require any preliminary modification of a biomole-
cule. This approach has been tested on the site-specific
modification of ubiquitin (a small protein that contains
only one His residue in its sequence), and preparation of
nonaggregated HSA-based phosphorescent probe appli-
cable for live cell lifetime imaging in the FLIM/PLIM ex-
periments.
5
4
tions transmittance (T, %): τ = (100-T)/100. Emission
spectra in solution were recorded on a FluoMax-4 (JY
Horiba Inc., Japan) spectrofluorimeter. The pulse laser
DTL-399QT (Laser-export Co. Ltd., Russia; 351 nm, 50
mW, pulse width 6 ns, repetition rate 1 kHz), mono-
chromator MUM (LOMO, Russia; bandwidth of slit 1 nm),
photon counting head H10682 (Hamamatsu Photonics,
Japan) and multiple-event time digitizer P7887 (FAST
ComTec GmbH, Germany) were used for lifetime meas-
urements in solution. The absolute emission quantum
yield in solution was determined by the comparative
EXPERIMENTAL SECTION
82
method using 9,10-diphenylanthracene in cyclohexane
Materials and Reagents. 2-Phenylpyridine, tri-
phenylphosphine, imidazole, L-histidine, were obtained
from Sigma-Aldrich (USA) and Alfa Aesar (Great Britain).
Human serum albumin (HSA) and Recombinant Human
Ubiquitin (Ubq) were purchased from Sigma-Aldrich
(USA) and Boston Biochem (USA), respectively. The ex-
(
Φ = 0.97) as the reference with the refraction indexes of
r
cyclohexane, DMSO and water equal to 1.426, 1.477 and
8
3
1
.333 respectively.
Synthesis of 1. [Pt(2-phenylpyridine)dmsoCl] (100 mg,
.216mmol) and triphenylphosphine (57 mg, 0.216mmol)
0
1
5
were dissolved in 5 ml dichloromethane, then 1 ml hexane
was added. Resulting product was obtained as pale yellow
crystals by slow evaporation of solvent at 4°C. Yield: 119
pression and purification procedure for human N uni-
formly labeled ubiquitin was adapted from the work by
79 15
Lazar et al. N-ammonium chloride was purchased from
1
mg (85%). H NMR (400 MHz, CDCl , 25 °C): δ = 9.92 –
3
ACS Paragon Plus Environment

Page 11 of 22
.88 (m with broad Pt satellites, J
Bioconjugate Chemistry
1
95
3
1
9
= 26.1 Hz, 1 H ),
ter, and complex 1 (1.45 mg, 2.241 μmol) was dissolved in
Pt,H
3
3
3,9,4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
7
7
.87 (td, J
= 7.8, 1.4 Hz, 1 H ), 7.84 – 7.77 (m, 7 H ),
DMSO (1.0998 g), the concentrations of resulted solutions
were 0.48 mM and 2.24 mM respectively. The complex
solution was gradually added portionwise to the protein
solution, ultimately 200 µl of complex solution were add-
ed. After each addition, the mixture was gently stirred.
The mixture was incubated for 1 h, and then 2 ml of water
were added. DMSO was removed by dialysis with 6-8 kDa
MWCO membrane (Orange Scientific, Belgium) for 24 h
at room temperature with 5 water changes. Resulted solu-
tion was centrifuged at 4000g for 10 minutes. The sample
was lyophilized with freeze dryer. 1-Ubq was prepared
using the same methodology except that 2 kDa MWCO
dialysis membranes (Sigma-Aldrich, USA) were used.
H,H
3
5
3
11
.52 (d, J = 7.5 Hz, 1 H ), 7.43 (td, J =7.3, 1.9 Hz, 3 H ),
H,H
3
H,H
10
3
.37 (td, J
= 7.8, 1.9 Hz, 6 H ), 7.29 (dd, JH,H= 7.1, 6.0
H,H
2
3
6
Hz, 1 H ), 6.96 (t, J = 7.4 Hz, 1 H ), 6.68 (dd with broad
H,H
1
95
3
3
8
Pt satellites, J = 7.8, 3.2, J
= 55.5 Hz, 1 H ), 6.52 (td,
Pt,H
H,H
3
7
JH,H = 7.5, 1.0 Hz,1 H ) ppm. Please refer the Supporting
1
Information for proton assignments. H NMR (400 MHz,
1
95
(
CD ) SO, 25 °C): δ = 9.75 – 9.60 (m with broad Pt satel-
3 2
3
lites, 1H), 8.21 – 8.11 (m, 2H), 7.75 (d, J
= 7.3 Hz, 1H),
H,H
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.73 – 7.63 (m, 5H), 7.57 (t, J = 6.2 Hz, 1H), 7.54 – 7.40
H,H
3
(
m, 9H), 6.95 (t, J
= 7.4 Hz, 1H), 6.54 (dd with broad
H,H
1
95
3
3
Pt satellites, J = 7.1, 3.2 Hz, 1H), 6.48 (t, JH,H= 7.4 Hz,
H,H
3
1
1
H). P NMR (400 MHz, (CD ) SO, 25 °C): δ = 23.41 (d,
3 2
1
+
+
JP,Pt= 4321.5 Hz, 1P) ppm. ESI -MS(m/z): [M-Cl] found
DFT calculations of 1 and 1-Im The 1, 1-Im and 1-His
complexes were performed using the Gaussian 09 pro-
gram package at the density functional theory level
+
611.1187, calculated 611.1219, [M+Na] found 669.0760, cal-
culated 669.0805, [2M-Cl] found 1257.2122, calculated
+
84
85–87
1
257.2116. C29H23ClNPPt (647.01): calcd. C 53.83, H 3.58,
(
DFT) with the hybrid density functional B3LYP.
Plat-
N 2.16; found C 54.06, H 3.68, N 1.87.
inum atoms were described by Stuttgart-Dresden pseu-
dopotential basis set (SDD) while all electron Pople’s
88
Synthesis of 1-Im. Complex 1 (10 mg, 0.015 mmol) was
dissolved in CH Cl (5 ml), then imidazole (3.3 mg, 0.045
basis set 6-311+G* was used for all other atoms. The ge-
ometries of lowest singlet and triplet states were opti-
mized at the above mentioned level of the theory. The
excited states of the complexes were studied by time de-
pendent density functional theory (TDDFT) calculations.
2
2
mmol) was added. The reaction mixture was stirred at
room temperature for 30 min. It was then concentrated to
ca. 2 ml under vacuum and layered with ca. 1 ml of hex-
ane. The product was obtained as yellow-green crystals by
slow diffusion of hexane into dichloromethane solution at
4
Monitoring of reactions of 1 with amino acids using
UV-Vis and emission spectroscopy. The following
stock solutions were prepared: 1 mM solution of complex
1 in DMSO; 3 mM solution of the corresponding amino
acid in water. Two milliliters of DMSO, 100 μl of 1 solu-
tion and 100 μl of amino acid solution were mixed in 1 cm
quartz cuvette. Absorption and emission spectra were
taken at a regular time interval starting from the moment
as close as possible to the mixing point. The measure-
ments were carried out for histidine, cysteine, arginine
and equimolar arginine/histidine mixture.
1
°C. Yield: 10.9 mg (99 %). H NMR (400 MHz, (CD ) SO,
3
2
3
4
3
25 °C): δ = 8.17 (d, J
= 8.2 Hz, 1H ), 8.10 (t, J
= 7.8
H,H
H,H
12
3
3
5
Hz, 1H ), 7.78 (s, 1H ), 7.74 (d, J
= 7.8 Hz, 1H ), 7.61
H,H
3
9
3
(
dd, J
= 11.9, 7.4 Hz, 6H ), 7.50 (dd, J = 7.8, 6.2 Hz,
H,H H,H
11
3
10
3
3
H ), 7.38 (td, J
= 7.7, 2.2 Hz, 6H ), 7.31 (t, J
= 6.6
H,H
H,H
2
1
13,6
Hz, 1H ), 7.23 – 7.15 (m, 1H ), 7.03-6.99 (m, 2H ), 6.92 (s,
14
7,8
1
H ), 6.56-6.50 (m, 2H ). Please refer the Supporting
31
Information for proton assignments. P NMR (400 MHz,
1
(
CD ) SO:D O 5:1,25 °C): δ = 22.34 (d, JP,Pt= 4211.1 Hz, 1P)
3 2 2
+
+
ppm. ESI -MS(m/z): [M-Cl] found 679.1624, calculated
6
79.1593.
15
NMR experiments with 1-Ubq. The N labeled Ubq
stock solution (0.5 mM) was prepared by dissolving of
solid protein in 50 mM PBS pH 6.5. The stock solutions of
Synthesis of 1-His. The complex 1 (2 mg, 0.003 mmol)
was dissolved in DMSO-d6 (0.6 ml), and L-histidine (3
mg, 0.019 mmol) was dissolved in 200 μl deuterated wa-
ter; the concentrations of resulted solutions were 3.33
mg/ml (5.2 mM) and 15 mg/ml (97 mM) respectively. Ali-
quots of the amino acid solution were added to the solu-
tion of 1 in a NMR-tube, and the mixtures were incubated
at room temperature for 2 h. The H and P NMR-spectra
were measured for solutions with different {histidine/1}
ratios (Figure S7). H NMR (400 MHz, (CD ) SO:D O =
1
(1.5 mM) were prepared by dissolving of the solid com-
-
6
plex (0.77 mg, 1.2×10 mol) in DMSO-d6 (877 mg). Two
probes were prepared — the mixture of the Ubq solution
and 1 solution (the Ubq:1 ratio was 1:1; the H O:DMSO-d6
ratio was 3:1), and the mixture of the Ubq solution and
DMSO-d6 (the H O/DMSO-d6 ratio was 3:1). After incu-
bating for 2 hours H- N HSQC NMR spectra spectra were
2
1
31
2
1
15
1
1
15
3
2
2
recorded. H- N HSQC spectra were measured on a
3
4
3
1
1
0:1, 25 °C): δ = 8.20 (d, J = 7.9 Hz, 1H ), 8.12 (t, J
=
H,H
H,H
Bruker AVANCE III spectrometer (500.03 MHz H, 50.67
MHz N) at 298 K using 5 mm BBO probehead; the spec-
tra were obtained using standard Bruker “hsqcetfpf3gp”
3
12
5
1
5
8
.5 Hz, 1H ), 7.95 – 7.73 (m, 1H ), 7.77 (d, J = 7.6 Hz, 1H ),
9
1,11
7.67 – 7.58 (m, 6H ), 7.57 – 7.49 (m, 4H ), 7.43 – 7.37 (m,
10
3
2
3
1
15
6
H ), 7.30 (dd, J = 13.5, 6.4 Hz, 1H ), 7.01 (td, J = 8.1,
H,H
H,H
7,8
pulse program. H and N hard pulse fields were 19.7 and
6.7 kHz, respectively. Spectral widths were 7.0 and 2.8
6
13
3
3
.1 Hz, 1H ), 6.96 – 6.59 (m, 1H ), 6.54 (m, 2H ), 3.42 –
.28 (m, 1H ), 3.29 – 2.45 (m, 2H ). Please refer the Sup-
1
16
14,15
1
15
kHz for the direct H and indirect N dimensions. The
data matrix consisted of 4096 (direct dimension) by 512
(indirect dimension) real data points. Number of acquisi-
tions was 16 (for three hour) or 80 (for overnight meas-
31
porting Information for proton assignments. P NMR
(
(
400 MHz, (CD ) SO:/D O = 10:1, 25 °C): δ = 22.34 – 22.07
3 2 2
m, J = 4200 Hz, 1P) ppm. ESI -MS(m/z): [M-Cl] found
1
+
+
P,Pt
8
9
766.1933, calculated 766.1914.
urement). The spectra were processed by NMRPipe and
analyzed by NMRDraw and Sparky.
8
9
90
Preparation of the 1-HSA and 1-Ubq conjugates.
HSA (32.57 mg, 0.489 μmol) was dissolved in 1 ml of wa-
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Bioconjugate Chemistry
Page 12 of 22
MALDI mass-spectroscopy of 1-Ubq conjugate. Mass Chromatography was carried out using 10 mM PBS (pH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
spectra were recorded by AB SCIEX TOF/TOF 5800
MALDI spectrometer in linear mode. The instrument was
calibrated using a Mass Standards Kit for Calibration of
AB Sciex TOF/TOF Instruments (AB Sciex). The stock
solutions of the samples for MALDI experiments (100 µM)
were prepared by dissolving pure Ubiquitin or lyophi-
lizate of 1-Ubq conjugate in ultrapure water and dilution
with 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich, USA)
up to concentration 10 µM. The samples (0.5 µl) were
spotted onto a steel plate and air-dried at room ambience.
Then 0.5 µl of sinapinic acid matrix solution (Sigma-
Aldrich, 10 mg/ml in 70% acetonitrile and 0.1% TFA) was
spotted on samples and air-dried at room ambience. The
accuracy of the mass peak measurements in linear mode
was about 5 Da.
6.6) as an eluent. The flow rate was 0.5 ml/min.
Dialysis of 1-HSA. Solid 1-HSA (10 mg) was dissolved
in 10 ml of water (1 mg/ml, 150 μM), and an aliquot was
taken. The remainder of the solution was placed in dialy-
sis tube (6-8 kDa MWCO membrane). The dialysis was
carried out for 6 days and 21 outer solution changes were
performed. Aliquots of the 1-HSA solution were taken
periodically and corresponding UV/Vis absorption and
luminescence emission spectra were recorded.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cell cultures preparation. La cells were cultivated in
F-12 culture medium (PAN-Biotech, Germany) with 10%
fetal bovine serum (PAN-Biotech, Germany). Cells were
cultivated on µ-dishes having a glass bottom (Ibidi, Ger-
many) and on open 8 wells µ-slides with a glass bottom
(
Ibidi, Germany) in CO2-incubator with 5% CO2 at 37°C.
Trypsinolysis of 1-Ubq conjugate and its fingerprint
mass-spectroscopy. The stock solutions of Ubiquitin
and 1-Ubq conjugate were diluted up to concentration 50
μM in the 100 mM ammonium bicarbonate buffer
After 24-hour pre-incubation 1-HSA was added to exper-
imental cell samples for final concentration of 0.3 mg/ml.
1
-HSA was dissolved in F-12 medium. Culture medium
without 1-HSA was added to control cell samples. After
this all samples were cultivated during 24 h under the
same conditions. The culture medium was then removed
and the cells were rinsed three times with DPBS.
(
NH HCO , Sigma-Aldrich, USA) followed by addition of
4 3
solution of proteomic grade trypsin (0.2 μg/ μl in 50mM
NH HCO , Sigma-Aldrich, USA), 0.2 μg of trypsin per 2 μg
4
3
of the sample. Solutions were incubated for 4 hours at
7°C, hydrolysis was stopped by adding to sample an
Simultaneous FLIM of NAD(P)H and PLIM of 1-
HSA. We used Nikon TE 2000 inverted fluorescence mi-
croscope (Nikon, Japan) equipped with a confocal scanner
DCS-120 and a Simple-Tau 150 time correlated single pho-
ton counting (TCSPC) system (Becker and Hickl GmbH,
Berlin, Germany) to simultaneously record FLIM of
NAD(P)H and PLIM of 1-HSA. The detailed approach to
3
equal volume of 0.1% TFA. The samples obtained (0.4 µl)
were spotted on a steel plate with 0.4 µl of a 2,5-
dihydroxybenzoic acid matrix (Sigma-Aldrich, USA) and
air-dried at room ambience. High-resolution mass spectra
were recorded using a Fourier Transform Ion Cyclotron
Resonance Mass Spectrometer (Varian 902-MS) equipped
by 9.4 T magnet (FTMS) in positive MALDI mode. The
instrument was calibrated using ProteoMass Peptide
MALDI-MS Calibration Kit (Sigma-Aldrich, USA). The
accuracy of the mass peak measurements after recalibra-
tion with trypsin autolysis peak (842, 5094 Dа) was better
than 2 ppm. Analysis of the MS data was carried out with
FTDocViewer software (Varian, USA) and identification
of peptides was performed using Mascot software
perform combined PLIM/FLIM has been published earli-
34,35
er.
405nm ps laser (BDL-405 SMN, Becker&Hickl
GmbH, Berlin, Germany) was used for simultaneous
NAD(P)H and 1-HSA excitation. The fluorescence and the
phosphorescence signals were recorded with HPM-100-40
detector (Becker&Hickl GmbH, Berlin, Germany). All im-
ages were collected with oil immersion Nikon objective
(
Nikon S Fluor, 40x, 1.3, Japan). The fluorescence and the
phosphorescence signals were separated by a 510 LP di-
chroic mirror (Chroma, USA). TCSPC data were analyzed
by SPCImage 5.4 software (Becker and Hickl GmbH, Ber-
lin, Germany).
(
www.matrixscience.com).
Fluorescent titration of HSA with complex 1. The
HSA stock solution (30 μM) was prepared by dissolving of
solid protein (6.2 mg) in ultrapure water (3.1125 g) and
stored at room temperature for 24h. The stock solutions
of 1 in DMSO (1 mM; 700 μM; 300 μM; 100 μM; 30 μM)
were prepared. The stock solutions of protein and 1 were
mixed in appropriate amounts to give a series of 27 mix-
tures with the HSA:1 ratio from 1:0 to 1:15. The
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge at http://pubs.acs.org.
AUTHOR INFORMATION
H O:DMSO(18:1) ratio was kept constant for all solutions.
2
Corresponding Author
Concentration of protein was 1.5 μM; concentration of
platinum complex varied from 0 μM to 22.5 μM. The solu-
tions were incubated for 24h. Then each solution was di-
vided into two parts. One part was centrifuged (10 min,
0000 g) and the other one was investigated without cen-
trifugation. Absorption, emission and excitation spectra
were obtained for each solution in 10 mm quartz cuvette.
*E-mail: sergey.tunik@spbu.ru; stunik@inbox.ru; Fax: +7
(
812) 3241258
ACKNOWLEDGMENT
1
All the research presented in this paper, excluding bioimag-
ing experiments, were supported by Russian Science Founda-
tion, grant No 16-43-03003. The bioimaging study
(FLIM/PLIM experiments) has been done with the support
from Saint-Petersburg State University research grants No
1.37.153.2014 and No 1.50.1043.2014 using TC SPS system
Analytical chromatography of 1-HSA. The 1-HSA so-
lutions prepared for titration experiment were investigat-
ed by analytical gel-permeation chromatography (GPC).
ACS Paragon Plus Environment

Page 13 of 22
Bioconjugate Chemistry
(
Becker and Hickl GmbH, Berlin Germany) in the Max Dle-
Fagagnini, A.; Gotte, G.; Merlino, A. J. Inorg. Biochem. 2015,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
brück Center for Molecular Medicine (Berlin, Germany). The
NMR, photophysical, analytical, and crystallographic meas-
urements were performed using the following core facilities
at St. Petersburg State University Research Park: Centre for
Magnetic Resonance, Centre for Optical and Laser Materials
Research, Centre for Chemical Analysis and Materials Re-
search, Computing Centre, and X-ray Diffraction Centre and
Chromas Core Facility. Lifetime measurements were carried
out using scientific equipment of the analytical center of
nano- and biotechnologies of SPbSPU.
146, 37–43.
(
(
(
27)
28)
29)
Ferraro, G.; Massai, L.; Messori, L.; Merlino, A. Chem.
Commun. 2015, 51 (46), 9436–9439.
Ferraro, G.; Massai, L.; Messori, L.; Merlino, A. Chem.
Commun. (Camb). 2015, 51 (46), 9436–9439.
Sullivan, D. J. J.; Gluzman, L. Y.; Goldberg, D. E. Science
1996, 271 (5246), 219–222.
Jones, A. L.; Hulett, M. D.; Parish, C. R. Immunol. Cell Biol.
2005, 83 (2), 106–118.
Engesser, L.; Kluft, C.; Briët, E.; Brommer, E. J. P. Br. J.
Haematol. 2008, 67 (3), 355–358.
Kuhli, C.; Scharrer, I.; Koch, F.; Hattenbach, L.-O. Am. J.
Ophthalmol. 2003, 135 (2), 232–234.
Bartneck, M.; Fech, V.; Ehling, J.; Govaere, O.; Warzecha, K.
T.; Hittatiya, K.; Vucur, M.; Gautheron, J.; Luedde, T.;
Trautwein, C. et al. Hepatology 2016, 63 (4), 1310–1324.
Shcheslavskiy, V. I.; Neubauer, A.; Bukowiecki, R.; Dinter,
F.; Becker, W. Appl. Phys. Lett. 2016, 108 (9), 91111.
Kalinina, S.; Breymayer, J.; Schäfer, P.; Calzia, E.;
Shcheslavskiy, V.; Becker, W.; Rück, A. J. Biophotonics 2016,
(30)
(
(
(
31)
32)
33)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
REFERENCES
(
(
(
(
(
1)
Fernández-Moreira, V.; Thorp-Greenwood, F. L.; Coogan,
M. P. Chem. Commun. 2010, 46 (2), 186–202.
Zhao, Q.; Huang, C.; Li, F. Chem. Soc. Rev. 2011, 40 (5),
2508–2524.
2)
3)
4)
5)
(
34)
Thorp-Greenwood, F. L. Organometallics 2012, 31 (16),
(35)
5
686–5692.
Coogan, M. P.; Fernández-Moreira, V. Chem. Commun.
014, 50 (4), 384–399.
9
(8), 800–811.
2
(
(
(
36)
37)
38)
Edwards, G. L.; Black, D. S. .; Deacon, G. B.; Wakelin, L. P.
Can. J. Chem. 2005, 83 (6–7), 980–989.
Appletorq, T. G.; Pescb, J. F. J.; Wienken, M.; Menzer, S.;
Lippert, B. Inorg. Chem. 1992, 31 (21), 4410–4419.
Streltsov, V. A.; Epa, V. C.; James, S. A.; Caine, J. M.;
Kenche, V. B.; Barnham, K. J. Chem. Commun. 2013, 49,
Demas, J. N.; DeGraff, B. A. Coord. Chem. Rev. 2001, 211 (1),
317–351.
(6)
7)
Lo, K. K.-W.; Choi, A. W.-T.; Law, W. H.-T. Dalt. Trans.
2012, 41 (20), 6021.
(
Ma, D.-L.; Ma, V. P.-Y.; Chan, D. S.-H.; Leung, K.-H.; He,
H.-Z.; Leung, C.-H. Coord. Chem. Rev. 2012, 256 (23), 3087–
1
1364–11366.
3
113.
(
(
39)
40)
Hureau, C.; Faller, P. Dalt. Trans. 2014, 43, 4233–4237.
Eryazici, I.; Moorefield, C. N.; Newkome, G. R. Chem. Rev.
(8)
Yu, M.; Zhao, Q.; Shi, L.; Li, F.; Zhou, Z.; Yang, H.; Yi, T.;
Huang, C. Chem. Commun. 2008, 102 (18), 2115.
2008, 108 (6), 1834–1895.
(
(
9)
Zimmermann, T.; Rietdorf, J.; Pepperkok, R. FEBS Lett.
(41)
Sasaki, I.; Bijani, C.; Ladeira, S.; Bourdon, V.; Faller, P.;
Hureau, C. Dalt. Trans. 2012, 41, 6404–6407.
Williams, J. A. G. In Photochemistry and Photophysics of
Coordination Compounds II; Springer Berlin Heidelberg:
Berlin, Heidelberg, 2007; pp 205–268.
Gauthier, M. A.; Klok, H.-A. Chem. Commun. 2008, 32 (23),
2591–2611.
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem. Int. Ed. 2002, 41 (14), 2596–2599.
Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int.
Ed. 2001, 40 (11), 2004–2021.
Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem. Int. Ed.
2008, 47 (52), 10030–10074.
Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P.
Chem. – A Eur. J. 2014, 20 (1), 34–41.
Kratz, F. J. Control. Release 2008, 132 (3), 171–183.
Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki,
K.; Kanai, M. J. Am. Chem. Soc. 2016, 138 (34), 10798–10801.
Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67
2003, 546 (1), 87–92.
10)
Zimmermann, T. In Microscopy Techniques; Springer Berlin
Heidelberg, 2005; pp 245–265.
(
42)
(11)
Baggaley, E.; Botchway, S. W.; Haycock, J. W.; Morris, H.;
Sazanovich, I. V.; Williams, J. A. G.; Weinstein, J. A. Chem.
Sci. 2014, 5 (3), 879–886.
(
(
(
(
(
43)
44)
45)
46)
47)
(
(
12)
13)
Kit-Man Siu, P.; Ma, D.-L.; Che, C.-M. Chem. Commun.
2005, 18 (8), 1025–1027.
Wu, P.; Wong, E. L.-M.; Ma, D.-L.; Tong, G. S.-M.; Ng, K.-
M.; Che, C.-M. Chem. Eur. J. 2009, 15 (15), 3652–3656.
Che, C.-M.; Zhang, J.-L.; Lin, L.-R. Chem. Commun. 2002, 97
(14)
(
21), 2556–2557.
Vaidyanathan, V. G.; Nair, B. U. Eur. J. Inorg. Chem. 2005,
005 (18), 3756–3759.
Ma, D.-L.; Che, C.-M.; Yan, S.-C. J. Am. Chem. Soc. 2009, 131
5), 1835–1846.
(
(
15)
16)
2
(
(
48)
49)
(
(17)
Man, B. Y.-W.; Chan, D. S.-H.; Yang, H.; Ang, S.-W.; Yang,
F.; Yan, S.-C.; Ho, C.-M.; Wu, P.; Che, C.-M.; Leung, C.-H.;
Ma, D.-L. Chem. Commun. 2010, 46 (45), 8534.
(50)
(
1), 425–479.
(
18)
Leung, K.-H.; He, H.-Z.; Chan, D. S.-H.; Fu, W.-C.; Leung,
C.-H.; Ma, D.-L. Sens. Actuators B-Chem. 2013, 177, 487–
(
(
51)
52)
Pickart, C. M. Annu. Rev. Biochem. 2001, 70 (1), 503–533.
Chelushkin, P. S.; Krupenya, D. V.; Tseng, Y.-J.; Kuo, T.-Y.;
Chou, P.-T.; Koshevoy, I. O.; Burov, S. V.; Tunik, S. P. Chem.
Commun. 2014, 50 (7), 849–851.
Chelushkin, P. S.; Nukolova, N. V.; Melnikov, A. S.;
Serdobintsev, P. Y.; Melnikov, P. A.; Krupenya, D. V.;
Koshevoy, I. O.; Burov, S. V.; Tunik, S. P. J. Inorg. Biochem.
4
92.
(19)
Wong, C.-Y.; Chung, L.-H.; Lin, S.; Chan, D. S.-H.; Leung,
C.-H.; Ma, D.-L. Sci. Rep. 2014, 4, 7136.
(53)
(
20)
Ma, D.-L.; Wong, W.-L.; Chung, W.-H.; Chan, F.-Y.; So, P.-
K.; Lai, T.-S.; Zhou, Z.-Y.; Leung, Y.-C.; Wong, K.-Y. Angew.
Chem. Int. Ed. 2008, 47 (20), 3735–3739.
2015, 149, 108–111.
(
(
(
(
21)
Li, C.; Yu, M.; Sun, Y.; Wu, Y.; Huang, C.; Li, F. J. Am. Chem.
Soc. 2011, 133 (29), 11231–11239.
Iakovidis, A.; Hadjiliadis, N. Coord. Chem. Rev. 1994, 135, 17–
(
54)
Chelushkin, P. S.; Lysenko, E. A.; Bronich, T. K.; Eisenberg,
A.; Kabanov, V. A.; Kabanov, A. V. J. Phys. Chem. B 2007, 111
(29), 8419–8425.
Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd
edition; Springer US: Boston, MA, 2006.
22)
23)
24)
6
3.
Espósito, B. P.; Najjar, R. Coord. Chem. Rev. 2002, 232 (1),
37–149.
(
(
55)
56)
1
Baggaley, E.; Sazanovich, I. V.; Williams, J. A. G.; Haycock,
J. W.; Botchway, S. W.; Weinstein, J. A. RSC Adv. 2014, 4
Valensin, D.; Gabbiani, C.; Messori, L. Coord. Chem. Rev.
2012, 256 (19), 2357–2366.
(
66), 35003–35008.
Rück, A.; Hauser, C.; Mosch, S.; Kalinina, S. J. Biomed. Opt.
014, 19 (9), 96005.
König, K.; Uchugonova, A.; Gorjup, E. Microsc. Res. Tech.
(25)
26)
Messori, L.; Marzo, T.; Merlino, A. J. Inorg. Biochem. 2015,
(
(
57)
58)
1
53, 136–142.
2
(
Picone, D.; Donnarumma, F.; Ferraro, G.; Russo Krauss, I.;
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 22
2011, 74 (1), 9–17.
759.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(59)
Stringari, C.; Cinquin, A.; Cinquin, O.; Digman, M. A.;
Donovan, P. J.; Gratton, E. Proc. Natl. Acad. Sci. U. S. A.
(74)
(75)
Dmitriev, R. I.; O’Donnell, N.; Papkovsky, D. B. Bioconjug.
Chem. 2016, 27 (2), 439–445.
2011, 108 (33), 13582–13587.
Botchway, S. W.; Charnley, M.; Haycock, J. W.; Parker, A.
W.; Rochester, D. L.; Weinstein, J. A.; Williams, J. A. G.
Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (42), 16071–16076.
Saito, T.; Asakura, N.; Kamachi, T.; Okura, I. J. Porphyr.
Phthalocyanines 2007, 11 (3), 160–164.
Schafer, R.; Gmitro, A. F. Biomed. Opt. Express 2015, 6 (2),
639.
Zhao, Q.; Zhou, X.; Cao, T.; Zhang, K. Y.; Yang, L.; Liu, S.;
Liang, H.; Yang, H.; Li, F.; Huang, W. Chem. Sci. 2015, 6 (3),
1825–1831.
Lazar, G. a; Desjarlais, J. R.; Handel, T. M. Protein Sci. 1997,
6 (6), 1167–1178.
Godbert, N.; Pugliese, T.; Aiello, I.; Bellusci, A.; Crispini, A.;
Ghedini, M. Eur. J. Inorg. Chem. 2007, No. 32, 5105–5111.
Kirk, M. SpinWorks 3.1.3; 2010.
Brouwer, A. M. Pure Appl. Chem. 2011, 83 (12), 2213–2228.
LeBel, R. G.; Goring, D. A. I. J. Chem. Eng. Data 1962, 7 (1),
100–101.
Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G. A. et al. Gaussian 09, Revision
D.01, Gaussian, Inc.; 2013.
Becke, A. D. J. Chem. Phys. 1993, 98 (7), 5648.
Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37 (2), 785–
789.
Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J.
J. Phys. Chem. 1994, 98 (45), 11623–11627.
Andrae, D.; Haussermann, U.; Dolg, M.; Stoll, H.; Preuss, H.
Theor. Chim. Acta 1990, 77 (2), 123–141.
Delaglio, F.; Grzesiek, S.; Vuister, G.; Zhu, G.; Pfeifer, J.; Bax,
A. J. Biomol. NMR 1995, 6 (3), 277–293.
(
60)
Yaseen, M. A.; Sakadžić, S.; Wu, W.; Becker, W.; Kasischke,
K. A.; Boas, D. A. Biomed. Opt. Express 2013, 4 (2), 307.
Blacker, T. S.; Mann, Z. F.; Gale, J. E.; Ziegler, M.; Bain, A. J.;
Szabadkai, G.; Duchen, M. R. Nat. Commun. 2014, 5, 801–
(61)
(76)
(77)
(78)
8
06.
(
62)
Evans, N. D.; Gnudi, L.; Rolinski, O. J.; Birch, D. J. S.;
Pickup, J. C. J. Photochem. Photobiol. B Biol. 2005, 80 (2),
1
22–129.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
(
(
63)
64)
65)
66)
Butte, P. V.; Fang, Q.; Jo, J. A.; Yong, W. H.; Pikul, B. K.;
Black, K. L.; Marcu, L. J. Biomed. Opt. 2010, 15 (2), 27008.
Sanchez, W. Y.; Prow, T. W.; Sanchez, W. H.; Grice, J. E.;
Roberts, M. S. J. Biomed. Opt. 2010, 15 (4), 46008.
Yu, J.-S.; Guo, H.-W.; Wang, C.-H.; Wei, Y.-H.; Wang, H.-
W. J. Biomed. Opt. 2011, 16 (3), 36008.
Gehlsen, U.; Oetke, A.; Szaszák, M.; Koop, N.; Paulsen, F.;
Gebert, A.; Huettmann, G.; Steven, P. Graefe’s Arch. Clin.
Exp. Ophthalmol. 2012, 250 (9), 1293–1302.
Stringari, C.; Edwards, R. A.; Pate, K. T.; Waterman, M. L.;
Donovan, P. J.; Gratton, E. Sci. Rep. 2012, 2, 309–314.
Papkovsky, D. B. Sens. Actuators B-Chem. 1995, 29 (1), 213–
(79)
(80)
(81)
(82)
(83)
(
(
67)
68)
(84)
218.
(69)
Lebedev, A. Y.; Cheprakov, A. V.; Sakadžić, S.; Boas, D. A.;
Wilson, D. F.; Vinogradov, S. A. ACS Appl. Mater. Interfaces
(85)
(86)
2009, 1 (6), 1292–1304.
(
(
70)
71)
Fercher, A.; Borisov, S. M.; Zhdanov, A. V.; Klimant, I.;
Papkovsky, D. B. ACS Nano 2011, 5 (7), 5499–5508.
Kurokawa, H.; Ito, H.; Inoue, M.; Tabata, K.; Sato, Y.;
Yamagata, K.; Kizaka-Kondoh, S.; Kadonosono, T.; Yano, S.;
Inoue, M. et al. Sci. Rep. 2015, 5, 10657.
(87)
(88)
(89)
(90)
(
(
72)
73)
Jin, P.; Chu, J.; Miao, Y.; Tan, J.; Zhang, S.; Zhu, W. AIChE J.
2013, 59 (8), 2743–2752.
Goddard, T. D.; Kneller, D. G. SPARKY 3, University of
California, San Francisco.
Sakadžić, S.; Roussakis, E.; Yaseen, M. A.; Mandeville, E. T.;
Srinivasan, V. J.; Arai, K.; Ruvinskaya, S.; Devor, A.; Lo, E.
H.; Vinogradov, S. A. et al. Nat. Methods 2010, 7 (9), 755–
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