Glyco-functionalized dinuclear rhenium(i) complexes for cell imaging

Article abstract of DOI:10.1039/c6ob02559e

The design, synthesis and photophysical characterization of four new luminescent glycosylated luminophores based on dinuclear rhenium complexes, namely Glyco-Re, are described. The derivatives have the general formula [Re₂(μ-Cl)₂(CO)

Full text of DOI:10.1039/c6ob02559e

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Glyco-functionalized dinuclear rhenium(I)
complexes for cell imaging†
Cite this: DOI: 10.1039/c6ob02559e
Alessandro Palmioli,‡§a Alessandro Aliprandi,§b Dedy Septiadi,§b Matteo Mauro,*b
Anna Bernardi,a Luisa De Colab and Monica Panigati*a,c,d
The design, synthesis and photophysical characterization of four new luminescent glycosylated lumino-
phores based on dinuclear rhenium complexes, namely Glyco-Re, are described. The derivatives have the
general formula [Re2(μ-Cl)2(CO)6(μ-pydz-R)] (R-pydz = functionalized 1,2-pyridazine), where a sugar
residue (R) is covalently bound to the pyridazine ligand in the β position. Different synthetic pathways have
been investigated including the so-called neo-glycorandomization procedure, affording stereoselectively
glyco-conjugates containing glucose and maltose in a β anomeric configuration. A multivalent dinuclear
rhenium glycodendron bearing three glucose units is also synthesized. All the Glyco-Re conjugates are
comprehensively characterized and their photophysical properties and cellular internalization experiments
on human cervical adenocarcinoma (HeLa) cells are reported. The results show that such Glyco-Re
complexes display interesting bio-imaging properties, i.e. high cell permeability, organelle selectivity, low
cytotoxicity and fast internalization. These findings make the presented Glyco-Re derivatives efficient
phosphorescent probes suitable for cell imaging application.
Received 22nd November 2016,
Accepted 19th January 2017
DOI: 10.1039/c6ob02559e
rsc.li/obc
attention due to potential scientific fall-outs in biology, bio-
chemical and medical research.2 Moreover, metal complexes
Introduction
Carbohydrates play a crucial role in different important bio- could be endowed with high photoluminescence quantum
logical processes such as cell proliferation and differentiation yields even in air-equilibrated aqueous media and relatively
as well as cellular immune response. Therefore monitoring long lifetimes by appropriate molecular design. Furthermore,
their uptake process and distribution inside cellular compart- they may exhibit low toxicity and good cellular internalization
ments is of fundamental importance for the diagnosis of features.3
various diseases and for developing new therapeutic agents.1
In this respect, synergic combination of biomolecules, such
Luminescent organometallic complexes based on second as carbohydrates, with transition-metal complexes to generate
and third row transition metals possess unique spectroscopic luminescent bioconjugates represents a promising strategy to
and photophysical properties, such as high emission quantum obtain sensitive molecular tools to gain deeper insight into
yield, photostability, long-lived excited states and sensitivity to biological key-events such as cellular internalization processes,
the surrounding environment. Nowadays, all these features metabolism and biodistribution.
have been advantageously employed for bio-imaging purposes
Due to their high water solubility, carbohydrates can be
and this research topic has been attracting a great deal of favorably used to effectively solubilize apolar structures in
polar media. Furthermore, it is now well established that
carbohydrates are involved in a variety of molecular reco-
aDepartment of Chemistry, Università degli Studi di Milano, Via Golgi 19,
20133 Milano, Italy. E-mail: monica.panigati@unimi.it; Fax: +39 0250314405;
Tel: +30 0250314352
bISIS & icFRC, Université de Strasbourg & CNRS, 8 rue Gaspard Monge,
67000 Strasbourg, France. E-mail: mauro@unistra.fr
cMilan Unit of INSTM, Via Golgi 19, 20133 Milano, Italy
dIstituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche
(ISMAC-CNR), Via E. Bassini, 15, 20133 Milano, Italy
gnition phenomena of physiological and pathological rele-
vance in living organisms that could be imaged using glyco-
sylated luminescent probes.1b
Various luminescent transition-metal glycoconjugate com-
plexes based on zinc,4 ruthenium(II),5 iridium(III)6 and plati-
num(II)7 have been recently reported and amongst them phos-
phorescent rhenium(I) tricarbonyl polypyridine complexes are
the most extensively studied systems.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6ob02559e
Although the use of rhenium(I) glyco-conjugates as bio-
sensors in carbohydrate-related events goes back to 2006,8
quite surprisingly their application as luminescent probes for
‡Present address: Università degli Studi di Milano-Bicocca, Dipartimento di
Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy.
§These three authors contributed equally to the work.
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cell imaging has been little exploited to date. Indeed, only cent glyco-functionalized neutral dinuclear rhenium com-
three examples have been reported so far in the literature, to plexes are prepared. Hereafter, the effects of substituents
the best of our knowledge.9 Such examples deal with mono- bound to the diazine ligand, and the length of the spacer as
nuclear cationic complexes, containing either aromatic well as nature and number of the conjugated carbohydrates on
bipyridyl (bpy) or dipyridophenazine (dppz) ligands, which the photophysical properties are reported. Finally, the intra-
show cytotoxic activity with an IC50 value in the low micro- cellular distribution of the complexes by means of laser scan-
molar range.
ning confocal fluorescent microscopy is discussed.
In the last few years our groups have developed a novel
family of neutral dinuclear rhenium(I) derivatives (see Chart 1)
that exhibit intense emission in the visible region, originating
from a triplet-manifold excited state being a metal-to-ligand
charge transfer (3MLCT) in nature. These neutral derivatives,
which can be obtained with good reaction yields, are character-
ized by two Re(CO)3 units connected by a substituted diazine
ligand and two anionic ancillary ligands.10 Also, modulation
of the photophysical properties was promptly achieved by both
substituent effects on the diazinic moiety and ancillary brid-
ging ligands, reaching photoluminescence quantum yield
values, (Φ), as high as 0.53, when electron-rich diazine ligands
were employed and chlorine was chosen as the bridging ligand
(X = Cl).11
Owing to their highly interesting photophysical features
and photostability, including high Φ and the possibility to
stimulated them by two-photon excitation techniques, this
family of dinuclear complexes have been used as low-toxicity
luminescent tags for Peptido Nucleic Acid (PNA) labelling, and
successfully tested in cell imaging experiments,12 showing neg-
ligible cytotoxicity and efficient cell uptake in living cells with
different kinetics and localization depending on the nature of
the PNA chain and of the diazine substituent. The stiff “Re
(μ-Cl)2Re” skeleton, which leads to high photoluminescence
quantum yields in solution, allowed imaging at a low con-
centration (25–100 μM), and prevented the release of the chlo-
ride ligands which was often responsible for the toxicity as
reported for neutral mononuclear diimine rhenium chloride
complexes.13
Results and discussion
Design and synthesis
Two different dinuclear rhenium complexes (see Chart 2) were
selected as starting materials for the synthesis of Glyco-Re
derivatives 3–6 aiming to study their solubility, the photo-
physical behavior and the cellular uptake of these new glyco-
conjugates (see Chart 3). Indeed, the length, the degree of
branching and the flexibility of the spacer could be important
to control the affinity of the resulting conjugate with cell
surface receptors. Furthermore, the number and type of con-
nected sugars, as well as the chemical and biological stability
of the linker moiety are also crucial factors to consider in the
design of the bio-conjugated luminophores.
Complex 1 was already used as a luminescent probe for
PNA labelling12 while the new complex 2, which contains two
alkyl substituents in the β positions of the diazine ring, could
provide a hypsochromically shifted emission with a higher
quantum yield and longer lifetime compared to the analogous
mono-substituted complex 1.11 Both complexes were syn-
thesized using the previously reported two-step procedure
involving an inverse-type [4 + 2] Diels–Alder cycloaddition reac-
tion between the electron-poor 1,2,4,5-tetrazine and the appro-
priate functionalized alkynes with N2 loss.15 Following our pre-
viously reported procedure,10 the complexes 1 and 2 were pre-
pared by refluxing [Re(CO)5Cl] with 0.5 equivalents of the
corresponding 1,2-diazine in toluene solution.
Complex 3, consisting of a β-O-glucoside derivative of the
dinuclear rhenium complex 2, was synthesized as shown in
Scheme 1. Commercial hex-3-ynol 7 was glycosylated with
penta-O-acetyl-D-glucose 8 and the resulting product 9 was de-
acetylated under Zemplen conditions, obtaining the β-hex-3-
ynyl-1-O-β-D-glucopyranoside-alkyne 10. Alkyne 10 was used for
the synthesis of the diazine ligand, following the well-estab-
lished literature procedure involving an inverse-type [4 + 2]
Diels–Alder cycloaddition reaction between the electron-poor
1,2,4,5-tetrazine and the alkyne dienophile, that occur with N2
These results prompted us to investigate the possibility to
develop new luminescent labels for novel glycosylated probes
based on these dinuclear rhenium complexes as simple,
chemically robust and easy-to-synthesize platforms that can
accommodate different carbohydrates.
Using the synthetic approach of the neo-glycorandomiz-
ation technique,14 which allows direct functionalization of
unprotected sugars in anomeric positions, a series of lumines-
Chart 1 General structure of dinuclear Re(I)-complexes bearing a brid-
ging 1,2-diazine ligand with different alkyl groups (R and R’) and two
ancillary anionic ligands (X = Cl, Br, I).
Chart 2 The dinuclear Re complexes 1 and 2 used as luminescent
labels.
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Chart 3 Structure of neutral dinuclear Re-glycoconjugates 3–6 synthesized in this study.
Scheme 1 Schematic synthetic pathway employed for preparing compound 3. Reagents and conditions: (a) BF3·Et2O, CH2Cl2 dry, 0 °C then room
temperature, 4 hours, yield 53%; (b) NaOMe 0.1 M, MeOH dry, room temperature, 1 hour, yield 81%; (c) 1,4-dioxane, 80 °C, 24 hours, yield 35%;
(d), toluene, reflux, 3 hours, yield 12%.
loss.15 Finally, the functionalized diazine ligand (11) was reaction of N-methylamino-oxy groups with an unprotected
treated with two equivalents of the organometallic precursor non-activated reducing sugar, affording a neo-glycoconjugate
Re(CO)5Cl obtaining the final β-O-D-glucoside-Re complex 3. under mild conditions.16 In addition, the formed neo-glycosi-
Both 3 and the corresponding diazine ligand 11 were isolated dic linkage was quite stable under physiological conditions
and purified by reverse phase chromatography. Complex 3 was and toward enzymatic hydrolysis. Overcoming protection/
isolated as a yellow solid which was found to be soluble at the activation/deprotection steps of traditional glycosylation reac-
mM level in organic polar solvents, such as MeOH and DMSO, tions, this method was used for the preparation of the glyco-
and in a 1 : 1 MeOH : H2O mixture. Although all synthetic conjugates 4 and 5 as reported in Scheme 2.
intermediates and final compounds have been successfully
The 2-(2-azidoethoxy) ethanol (13), obtained from commer-
isolated and fully characterized, this synthetic strategy has a cial 2-(2-chloroethoxy) ethanol (12) and NaN3 (Scheme 2), was
poor overall yield, principally because of the last two steps, first tosylated (14) and then treated with N-Boc-N-methyl-
which have reaction yields of 35% and 12%, respectively.
hydroxylamine and DBU to afford N-Boc-N-methyl-2-(2-azi-
For this reason the synthetic procedure shown in Scheme 1 doethoxy)ethoxy hydroxylamine (15). Then the azido group was
was not suitable for the synthesis of a large set of conjugates reduced under Staudinger conditions (PPh3 and water) and the
containing different natural monosaccharides and oligo- amine 16 was coupled with a dinuclear Re(I) complex 1
saccharides. Therefore a bifunctional linker (16, see Scheme 2), affording the intermediate 17a with a satisfactory overall yield.
that bears a primary amino group and an N-methylamino-oxy The N-Boc protecting group was removed in the presence of
group was developed. It could act as a robust and easily acces- CF3COOH in CH2Cl2 and the resulting N-methyl hydroxyl-
sible platform, able to accommodate both the photolumines- amine 17b was isolated as the trifluoroacetate salt in quantitat-
cent tag and the different carbohydrate moieties in a fast, con- ive yield. Finally, chemoselective glycosylation of 17b to afford
vergent and parallel manner. This linker allowed exploiting the 4 or 5 was performed in the presence of 3 eq. of unprotected
chemoselective glycosylation approach based on the one-step reducing sugar (D-glucose or D-maltose, respectively) and
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Scheme 2 Schematic synthetic pathways employed for the neoglyco-conjugates 4 and 5. Reagents and conditions: (a) NaN3, NaI, H2O, 60 °C,
4 days, quantitative yield; (b) TsCl, Et3N, CH2Cl2, 0 °C then room temperature, 3 hours, yield 80%; (c) 1,5-diazabiciclo(5.4.0)undec-5-ene (DBU), THF
then solvent-free, room temperature, 12 hours, yield 88%; (d) PPh3, Na2CO3, H2O, CH2Cl2, room temperature, 12 hours, yield 85%; (e) HATU, DIPEA,
CH2Cl2 (5% dry DMA), room temperature, 2 hours, yield 76%; (f) TFA, dry CH2Cl2, 1 hour, quantitative yield; (g) D-glucose or maltose, glacial AcOH,
dry MeOH, room temperature, 18 hours, yield 62% and 63% for 4 and 5, respectively.
25 eq. of AcOH in MeOH at room temperature overnight reported Scheme S1 of the ESI†) and linked with the dinuclear
(Scheme 2). The neo-glycoconjugate products were obtained Re(I) complex 1 using HATU/DIPEA as coupling reagents. The
stereoselectively in a β anomeric configuration with satisfac- Re(I)-glycodendrimer 6 was purely isolated after reverse-phase
tory yields (62% and 63% for
4 and 5, respectively). C18 chromatography purification. Complex 6 was soluble in
Compounds 4 and 5 are both soluble in water in a low mM water in the mM concentration.
range concentration.
Finally, for the synthesis of the multivalent glycoconjugate
6 (Scheme 3), the trivalent glucodendron 18 was prepared
from commercial pentaerythritol (the full synthetic scheme is
Photophysical characterization
The photophysical properties of the glyco-derivatives 3–5 and
of their parent complexes 1 and 2 were studied under dilute
condition (concentration of 1.0 × 10−5 M) in both air-equili-
brated and deareated 1,4-dioxane solution at room tempera-
ture. The multi-functionalized complex 6 was investigated in
aqueous solution due to its very low solubility into apolar sol-
vents. The corresponding photophysical data are listed in
Table 1.
At longer wavelengths, the electronic absorption spectra of
compounds 1–5 in dioxane display a broad absorption band
with the maxima in the range 347–359 nm with a moderate
intensity (ε ≈ 0.67–0.88 × 104 M−1 cm−1), whilst complex 6 in
distilled H2O displays an absorption maximum centred at
λabs = 323 (ε ≈ 0.55 × 104 M−1 cm−1). Such a transition can be
Scheme 3 Synthesis of the multivalent glycoconjugate 6. Reagents and
conditions: (a) 1, HATU, DIPEA, dry DMA, room temperature, 2 hours,
yield 68%.
Table 1 Photophysical properties obtained for compounds 1–6 in both air-equilibrated and deaerated dilute samples at room temperature
λabs (ε)
Compound
(nm, [×104 M−1 cm−1])
λem [nm]
τ [μs]
Φem (%)
kr [×105 s−1
]
knr [×105 s−1
]
1a
2a
3a
4a
5a
6b
Air
Deg
Air
Deg
Air
Deg
Air
Deg
Air
Deg
Air
359 (0.76)
—
348 (0.88)
—
347 (0.70)
—
352 (0.67)
—
355 (0.77)
—
323 (0.55)
—
585
585
562
562
570
570
591
588
589
589
594
594
0.378
1.27
0.458
3.26
0.451
2.73
0.419
3
8
3
20
3
19
3
12
4
11
<1
<1
0.63
0.61
0.68
0.66
0.78
0.66
7.24
2.45
2.98
4.81
6.36
66.0
1.18 (81%), 1.83 (19%)
0.436
1.36 (97%), 2.88 (3%)
0.011 (17%), 0.144 (74%), 0.504 (9%)
0.365 (24%), 0.138 (38%), 0.014 (38%)
Deg
a In 1,4-dioxane. b In distilled water.
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1
assigned to the spin allowed dπ(Re) → π*(diazine ring) MLCT observed in the luminescence properties are mainly to be
band, typical of this class of complexes.10,11 Also, the CT char- attributed to the efficiency of the nonradiative processes, knr.
acter of such transitions was supported by the modulation on
Even if the conjugation with sugar is not expected to
the absorption energy by both substituent effects on the directly influence the nature of the excited state, it is interesting
diazine ligand and the solvent polarity. Accordingly, complexes to note that deareated Φ and an excited-state lifetime for the
2 and 3, featuring a diazine containing two alkyl substituents glyco-substituted complex 3 were slightly lower than those
in the β positions, displayed a blue-shifted absorption band observed for the non-functionalized benchmark complex 2. The
with respect to the mono substituted complexes 1, 4 and 5. reason can be likely ascribed to the increased conformational
Indeed, the second electro-donating alkyl chain on the freedom due to the presence of the glucose moiety, which may
diazine ligand increases the energy level of the LUMO, favour nonradiative deactivation pathways as suggested by the
raising in this way the HOMO–LUMO gap and therefore the moderate increase of the knr for complex 3 (see Table 1).
energy of the 1MLCT excited state.11 A hypsochromically
In contrast, different findings were observed for complex
shifted absorption band was also observed for complex 6 but, 4–5 in comparison with the parental complex 1. The former
in this case, this feature is most likely due to the much higher showed a higher quantum yield and a slightly longer excited-
polarity of the solvent used (water vs. dioxane). It is interesting state lifetime with bi-exponential decay kinetics under de-
to note that the conjugation of the carbohydrate on the aerated conditions. This feature is most likely attributed to the
organometallic scaffold does not modify the absorption pro- formation of soft supramolecular aggregates by establishment
perties, as previously observed also for the analogues for PNA- of sugar–sugar interactions due to the amphiphilic nature
conjugates.12
of complexes 4 and 5 upon a freeze–pump procedure.
Upon excitation in the range 350–380 nm at room tempera- Indeed, the simultaneous presence of the apolar head core,
ture, all the samples showed broad and featureless emission in constituted by the dinuclear rhenium complex, and the hydro-
the yellow-orange region of the visible spectrum, which is philic sugar moiety, connected to each other by a flexible
3
assigned to the radiative decay of the MLCT excited state, as hydrophilic chain, is expected to promote self-organization of
comparison with closely related complexes (see Fig. 1).11 In the molecules in solution, as recently reported by some of
addition, the triplet nature of the radiative transition is further us.17 It is reasonable to think that such a self-assembling
supported by the oxygen quenching effect observed starting process is further driven by intermolecular hydrogen bonds
from degassed to air-equilibrated samples (Table 1). Similar to between the hydroxyl groups of the glucose and maltose moi-
what was observed in the absorption spectra, complex 1 and eties in an apolar media such as dioxane, affording large ves-
its related glyco-conjugates 4–5 display remarkably different icular-like structures with an inverse architecture in which the
emission features compared to derivatives 2–3. Indeed, a apolar “Re(CO)3” heads are oriented towards a dioxane solvent
hypsochromically shifted and more intense emission was media. Thus, the formation of such soft aggregates made by
recorded for complexes 2–3, which is accompanied by a higher amphiphilic molecules might be responsible for the observed
Φ and longer emission lifetime as a consequence of the higher photophysical features.
3
energy of the MLCT excited state (see Table 1). However, for
Additionally, the aggregation tendency of compound 4 and
1
all the complexes the values of Φ/τ, which represents the 5 was also confirmed by H NMR spectroscopy in a d4-metha-
product between the radiative rate constant (kr) and the nol solution at room temperature. In the H NMR spectra of a
1
efficiency of intersystem crossing (ηICS), were found in the dilute solution of either 4 or 5 (1 mM) no resonance signal of
range of 6.1–7.8 × 104
s
−1, indicating that the differences the N–H proton of the amide group of the linker was observed,
due to the H-D exchange with the solvent. However, upon
increasing the concentration of the complex up to 20 mM, a
rise of a triplet signal at about 8.00 ppm, attributed to the N–H
involved in stable intermolecular H-bond interactions, was
observed (see Fig. 2).
On the other hand, dilute samples of complex 6 in distilled
H2O display a very different behavior with a rather low photo-
luminescence with λem centred at 594 nm and a short-lived
excited state even after the degassing procedure.
In aqueous media, the amphiphilic compound 6 with a
cone-like shape is expected to aggregate into direct micellar
structures in which the apolar heads are oriented internally
into the supramolecular architecture. Such a configuration, in
which long-lived excited states located onto the organometallic
head are closely spaced, is expected to enhance triplet–triplet
annihilation processes yielding an increase of radiationless
Fig. 1 Normalized emission spectra of complexes 3 (blue trace), 4 (red
trace) and 5 (green trace) in deaerated 1,4-dioxane solution at a concen-
tration of 1.0 × 10−5 M at room temperature (λex 380–400 nm).
decay channels as demonstrated by the much higher knr con-
stant observed for 6.
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Fig. 2 1H NMR spectra recorded for compound 4 and 5 in methanol-d4 at a concentration range from 1 to 20 mM at room temperature. The red
symbol shows the variation of the resonance upon increasing the concentration.
Cellular uptake assays
the effect of the initial concentration of the complexes in the
staining media on the cellular behaviour at a specific incu-
bation time, we carried out concentration dependent uptake
experiments. Expectantly, increasing the concentration of the
emitting complex into the staining solution from 25 to 100 μM
results in an increase of the number of internalized molecules
as can be noticed through increment of emission intensity
signals. The results are displayed in Fig. 4 and S4 of the ESI†
for compound 4 and 5, respectively. The corresponding inten-
sity profile recorded for different concentrations of compound
4 in the incubating media is displayed in Fig. S5a of the ESI.†
Overall, we also found that there is no significant difference
in terms of intracellular distribution of the complexes since
for example migration of the luminescent probe towards the
nuclear region was not observed at the highest concentration
employed of 100 μM. Moreover, we were able to follow in real-
time the kinetics of internalization of our compounds by
means of a live cell imaging technique. The intensity plot over
different incubation times is displayed in Fig. S5b of the ESI.†
We found out that the uptake of compound 3, as an example,
occurred within a short span of time upon incubation. A low
intensity signal can be observed just a few seconds after the
incubation while intense staining patterns can be clearly seen
from the cytoplasmic region of the cells after 10 min of incu-
bation and they increased over time for 1 hour (Fig. 5, complex 3).
We also observed a similar trend for compound 4 and 5
(see Fig. S6 and S7† respectively). By comparing the intensity
In order to study the internalization and bio-imaging pro-
perties of the Glyco-Re(I) complexes as cellular probes, we per-
formed cellular uptake experiments of complexes 3–6 on the
human cervical carcinoma (HeLa) cell line. As depicted in
Fig. 3 and S3 of the ESI,† compound 3 was internalized by
living HeLa cells and accumulated mainly in the cytoplasmic
region and the corresponding intracellular emission was cen-
tered at 573 nm, as previously observed from photophysical
measurements in solution. Complex 4 and 5 display efficient
internalization into HeLa cells as well. In order to understand
Fig. 3 (a) Fluorescence confocal image of HeLa cells after the incu-
bation of compound 3 (50 μM in less than 1% DMSO containing PBS). (b)
The emission spectrum recorded from the cytoplasm of the cells. The
samples were excited at λexc = 405 nm.
Fig. 4 Confocal images of HeLa cells after the incubation of compound 4 at different concentrations (a) 25, (b) 50, and (c) 100 μM in less than 1%
v/v DMSO containing PBS. (d) The emission spectrum recorded from the cytoplasmic region of the cell. The samples were excited at λexc = 405 nm.
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Fig. 5 Confocal images of HeLa cell line (a) before and (b–f) after addition of compound 3 at a concentration of 100 μM in less than 1% v/v DMSO
containing PBS. Kinetics experiment shows fast internalization of the compound at different time periods (b) seconds, (c) 10 minutes, (d) 20 minutes,
(e) 40 minutes, and (f) 1 hour after incubation. The samples were excited at λexc = 405 nm.
profile over time for all of the complexes, we found out that towards the diffusion mechanism through the cell membrane.
the emission signal for cells stained with complex 5 shows the Further investigations in this direction are currently ongoing.
steepest increase over time (Fig. S5b of the ESI†). Such a
To further shed light on the ability of the presented com-
finding may underline a faster internalization process occur- plexes to act as bio-imaging probes in living cells, co-localiz-
ring for compound 5 compared to compound 3 and 4, keeping ation experiments were performed by means of co-staining
in mind that the experimental conditions were maintained the experiments with DAPI for the nucleus, ER-Tracker™ Red for
same and the photophysical properties of the three complexes, the endoplasmic reticulum and Phalloidin Alexa Fluor® 647
especially in terms of the luminescence quantum yield, are dye as the f-actin staining. As displayed in Fig. 6a–d, the
comparable (see Fig. S5† and photophysical data listed in results obtained with DAPI co-staining can exclude the pres-
Table 1). The higher cellular uptake of the more hydrophilic ence of all complexes inside the nuclear region whereas a
complex 5 could be associated with an active transport mech- perfect overlapping with the signals derived from ER-Tracker™
anism. Nonetheless, the fast internalization time points Red (overlap coefficient 0.87) was observed, indicating its local-
Fig. 6 Fluorescence confocal micrographs showing the distribution of compound 4 inside HeLa cells at a concentration of 100 μM in less than 1%
DMSO containing PBS as the incubation media. (a) and (e) 4, (b) DAPI staining of nucleus, (c) overlay (a) and (b), (d) orthogonal view of the image
showing 4 signals (yellow) coming from the inside of the cytoplasmic region of the cells which is stained with Phalloidin Alexa Fluor® 647 (red), (f)
ER-Tracker™ Red stains the endoplasmic reticulum, (g) overlay (e) and (f). The excitation wavelength for DAPI and compound 4 was 405 nm, while
ER-Tracker™ Red and Phalloidin Alexa Fluor® 647 were excited at 594 and 633 nm, respectively.
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appear to perturb the nature of the emitting excited state on
the complex moiety. More importantly, it affords an amphi-
philic character to the resulting glyco-conjugates, allowing the
formation of soft aggregates, which display a higher photo-
luminescent quantum yield and a longer excited state lifetime
than the corresponding parental luminescent rhenium
carbonyl complex.
Cellular internalization experiments on human cervical
adenocarcinoma cells (HeLa) have shown that these glyco-
conjugates have fascinating bio-imaging properties, i.e. high
cell permeability, organelle selectivity, low cytotoxicity, and
fast internalization demonstrating their valuable suitability as
efficient phosphorescent probes to be used in living cell
imaging application.
Fig. 7 Cellular viability studies after 1 hour incubation with different
complexes at a concentration of 50 μM in <1% DMSO containing PBS.
The compound name is indicated on the x axis.
Experimental
ization in the endoplasmic reticulum (Fig. 6e–g).18 Similar
results were also obtained for compound 3 and 5, as shown in
Fig. S8 and S9 in the ESI.†
Supporting figures and synthetic procedures including NMR
spectra can be found in the ESI.†
In sharp contrast, incubation with compound 6 of HeLa
cells under identical conditions resulted in no emission being
detected from the cells except for a typical autofluorescence
signal derived from NADH and FAD molecules19 (see Fig. S10,
ESI†). Even though, it is still not clear if the compound was
taken up but the emission was quenched in the cellular com-
partments due to quenching processes and aggregation
phenomena or not taken up at all. However, its rather poor
luminescence features make this compound much less attrac-
tive for bio-imaging purposes at this stage and it will not be
further investigated.
Noteworthily, typical healthy cell shapes and no sign of
apoptosis were observed during the incubation experiments.
This observation demonstrates the very low degree of toxicity
of the compounds inside the cellular compartments and orga-
nelles; a valuable feature for luminescent bio-imaging probes.
To further quantify such a finding, the cellular viability assay
based on electric current exclusion and pulse area analysis was
performed by means of CASY® equipment and the results are
depicted in Fig. 7. Interestingly, the number of viable cells
after 1 hour incubation with complexes 3–6 is found to be
close to the control experiments, confirming the low cyto-
toxicity of the complexes and their suitability as efficient phos-
phorescent labelling probes that can be used in cellular
imaging applications.
Materials and methods
Chemicals were purchased from commercial sources and used
without further purification, unless otherwise indicated. When
anhydrous conditions were required, the reactions were per-
formed in oven-dried glassware under a nitrogen atmosphere.
Anhydrous solvents were purchased from Sigma-Aldrich® with
a content of water ≤0.005%. THF was dried over Na/benzo-
phenone and freshly distilled prior to use. Thin-layer chrom-
atography (TLC) was performed on Silica Gel 60 F254 plates or
RP-C18 Silica plates (Merck) with UV detection (254 nm and
365 nm) or using appropriate developing solutions. Flash
column chromatography was performed on silica gel
230–400 mesh (Merck), according to the procedure described
in the literature. Automated flash chromatography was per-
formed on a Biotage® Isolera™ Prime system. NMR experi-
ments were recorded on a Bruker AVANCE 400 MHz instrument
at 298 K. Chemical shifts (δ) are reported in ppm downfield
from TMS as the internal standard, whereas coupling con-
stants (J) are stated in Hz. The H and 13C-NMR resonances of
1
compounds were assigned by means of COSY and HSQC
experiments. Mass spectra were recorded on an Apex II ICR
FTMS (ESI ionization-HRMS), Waters Micromass Q-TOF (ESI
ionization-HRMS), ThermoFischer LCQ apparatus (ESI ioniza-
tion) or Bruker Daltonics Microflex LT (MALDI-TOF apparatus).
Specific optical rotation values were measured using a Perkin-
Elmer 241, at 589 nm in a 1 mL cell. In the optimized copper(I)-
catalyzed azide–alkyne cycloaddition (CuAAC) procedure,
the starting materials and reagents were added to the reaction
Conclusions
Different glyco-conjugates based on luminescent dinuclear mixture as a solid or as a solution in water or THF. The water
rhenium complexes have been synthesized using glucose or was degassed by bubbling with nitrogen and THF was freshly
maltose derivatives and bio-orthogonal glyco-conjugation pro- distilled. The reagents were added to the reaction vessel in the
cedures. These new conjugates have been evaluated in terms following order: multivalent scaffold (1 eq., solid or in THF),
of their optical properties and suitability as bio-imaging dyes TBTA (0.2 eq., in THF), CuSO4·5H2O (0.1 eq., in H2O), sodium
in living HeLa cells. The conjugation with sugars increases the ascorbate (0.4 eq., in H2O) and finally the azide monovalent
solubility of the complexes in aqueous media and does not ligand (1.1 eq. per each triple bond, solid or in H2O). The final
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concentration of the multivalent scaffold was around 20 mM (ESI) m/z calculated for [C20H28O10Na]+: 451.16, found: 451.4
in a mixture of THF : H2O (1 : 1) and the reaction was stirred [M + Na]+. [α]2D5 = −17.85 (C = 0.685, CH3Cl).
overnight at room temperature under a nitrogen atmosphere,
Compound 10. A solution of compound (9) (740 mg,
protected from light and monitored by mass spectrometry 1.73 mmol) in freshly distilled MeOH (31.5 mL) was treated
(MALDI-Tof) until completion. When RP-C18 chromatography with a solution of NaOMe (3.5 mL, 1 M in MeOH). The reac-
was performed, the sample was loaded on a C18 prepacked tion was stirred at room temperature under a nitrogen atmo-
sample and purified using a Biotage system (column SNAP sphere monitoring the progress by TLC (hexane : AcOEt 1 : 1).
KP-C18-HS, gradient elution H2O : MeOH). Finally the solvent After 1 h the reaction was complete. The mixture was diluted
was removed by lyophilization obtaining the final purified with MeOH and neutralized by addition of Amberlite
product.
IR 120-H+ resin, then the beads were filtered off and washed
with MeOH. Finally the solvent was removed under reduced
pressure and the crude was purified by flash chromatography
Synthesis
[ReCl(CO)5] and complex 1 were prepared according to a litera- (DCM : MeOH 9 : 1) affording a pure compound (10) (420 mg,
1
ture method.12,20 Synthesis of 4-ethanoyl-5-ethyl-pyridazine 93%). H NMR (400 MHz, MeOD) δ 4.29 (d, J1,2 = 7.8 Hz, 1H,
was carried out according to a literature procedure,15 involving H1), 3.98–3.81 (m, 2H, H6a, H7a), 3.72–3.56 (m, 2H, H6b,
the synthesis of 1,2,4,5-tetrazine (from hydrazine hydrate and H7b), 3.37–3.32 (m, 1H, H3), 3.28–3.24 (m, 2H, H4, H5), 3.17
formamidine acetate) as the first step followed by its reaction (dd, J2,3 = 8.9, J2,1 = 7.8 Hz, 1H, H2), 2.46 (tt, J8,7 = 7.4, J8,11
=
with the alkyne. 2.3 Hz, 2H, H8), 2.13 (qt, J11,12 = 7.5, J8,11 = 2.3 Hz, 2H, H11),
Complex 2. A sample of [ReCl(CO)5] (117.1 mg, 0.324 mmol) 1.09 (t, J11,12 = 7.5 Hz, 3H, H12). 13C NMR (100 MHz, MeOD)
was dissolved in a toluene solution (20 mL) and treated with δ 104.36 (C1), 83.52, 78.01 (C3), 77.98 (C4), 76.61, 75.01 (C2),
0.5 eq. of 4-ethanoyl-5-ethyl-pyridazine (24.3 mg, 0.162 mmol). 71.57 (C5), 69.59 (C7), 62.71 (C6), 20.86 (C8), 14.64 (C12), 13.03
The solution was heated at the reflux temperature for three (C11); MS (ESI) m/z calculated for [C12H20O6Na]+: 283.27,
hours and then evaporated to dryness, giving a brownish solid. found: 283.3 [M + Na]+. [α]2D5: −35.2 (C = 0.481, MeOH).
The desired product was isolated as a yellow solid from the
Compound 11. Synthesis of glycosylated pyridazine was
crude by silica gel column chromatography using carried out according to a literature procedure, involving the
CH2Cl2 : AcOEt 4 : 1 as the eluent. IR (CH2Cl2) ν (CO): 2049 (m), synthesis of 1,2,4,5-tetrazine (from hydrazine hydrate and
2033 (s), 1945 (s), 1914 (m) cm−1 1H NMR (CD2Cl2, 300 K, formamidine acetate) as the first step followed by its reaction
.
400 MHz) δH (ppm) 9.65 (s, 1H, H3-pydz), 9.49 (s, 1H, H6- with the functionalized alkyne 10 (Scheme 1) The crude was
pydz), 4.06 (m, 2H), 3.10 (t, 2H), 2.97 (q, 2H), 1.42 (t, 3H). purified by RP-18 chromatography (H2O : MeOH gradient
Elemental Anal. Calcd for C14H12Cl2N2O7Re2: C 22.02, H 1.58, elution) affording a pure compound (11) (mg, 35% yield).
N 3.67. Found: C 22.12, H 1.53, N 3.65.
Compound 9. To a solution of penta-O-acetyl-D-glucose (8) HAr), 4.31 (d, J1,2 = 7.8 Hz, 1H, H1), 4.18 (dt, J7a,7b = 10.1, J7a,8
1H NMR (400 MHz, MeOD) δ 9.07 (s, 1H, HAr), 8.96 (s, 1H,
=
(1 g, 2.67 mmol, 1 eq.) and 3-hexyn-1-ol (7) (365 µL, 3.2 mmol, 6.5 Hz, 1H, H7a), 3.94–3.81 (m, 2H, H7b, H6a), 3.69–3.60 (m,
1.2 eq.) in anhydrous DCM (22 mL), BF3–Et2O (507 µL, 1H, H6b), 3.37–3.33 (m, 1H, H5), 3.26 (m, 2H, H3, H4), 3.15
4.0 mmol, 1.5 eq.) was added at 0 °C under a nitrogen atmo- (dd, J2,3 = 9.1, J1,2 = 7.8 Hz, 1H, H2), 3.08 (t, J7,8 = 6.4 Hz, 2H,
sphere. Then the mixture was slowly allowed to warm and stir H8), 2.81 (q, J11,12 = 7.6 Hz, 2H, H11), 1.29 (t, J11,12 = 7.6, 3H,
for 4 h at room temperature until the reaction was complete H12); 13C NMR (100 MHz, MeOD) δ 153.58, 152.72 (CHAr),
(TLC hex : AcOEt 7 : 3 Rf prod. = 0.26). Finally the reaction was 144.85, 139.64 (CqAr), 104.42 (C1), 78.13, 78.05 (C4, C5), 75.00
quenched by addition of Et3N (670 µL, 4.8 mmol, 1.8 eq.), (C2), 71.60 (C3), 69.01 (C7), 62.76 (C6), 30.56 (C8), 23.58 (C11),
stirred for additional 15 min and concentrated under reduced 14.22 (C12). MS (ESI) m/z calculated for [C14H22N2O6Na]+:
pressure. The crude was purified by flash chromatography 337.32, found: 337.4 [M + Na]+; [α]2D5: −16.01 (C = 0.316, MeOH).
(hexane : AcOEt gradient elution) affording a pure compound
Complex 3. Complex 3 (see Chart 3 for its structures and
(9) (740 mg, 68%, β-anomer). 1H NMR (400 MHz, CDCl3) δ 5.20 abbreviations) was prepared from [ReCl(CO)5], using the pre-
(t, J3,2 = 9.5 Hz, 1H, H3), 5.08 (t, J4,5 = 9.7 Hz, 1H, H4), 4.98 (dd, viously reported method. The crude was purified by RP-18
J2,3 = 9.5 Hz, J1,2 = 8.0 Hz, 1H, H2), 4.56 (d, J1,2 = 8.0 Hz, 1H, chromatography (H2O : MeOH gradient elution) affording a
H1), 4.26 (dd, J6a,6b = 12.3 Hz, J6a,5 = 4.7 Hz, 1H, H6a), 4.13 pure compound (3) (mg, 12% yield). 1H NMR (400 MHz,
(dd, J6a,6b = 12.3, J6b,5 = 2.4 Hz, 1H, H6b), 3.89 (dt, J7a,7b = 9.8, MeOD) δ 9.84 (s, 1H, HAr), 9.74 (s, 1H, HAr), 4.32 (d, J1,2 = 7.6
J7a,8 = 7.0 Hz, 1H, H7a), 3.69 (ddd, J5,4 = 9.9 Hz, J5,6a = 4.7 Hz, Hz, 1H, H1), 4.24–4.14 (m, 1H, H7a), 4.08–3.99 (m, 1H, H7b),
J5,6b = 2.4 Hz, 1H, H5), 3.62 (dt, J7a,7b = 9.8, J7b,8 = 7.5 Hz, 1H, 3.86 (dd, J6a,6b = 11.7, J6a,5 = 2.3 Hz, 1H, H6a), 3.56 (dd, J6a,6b
H7b), 2.43 (tt, J8,7 = 7.1 Hz, J8,11 = 2.3 Hz, 2H, H8), 2.18–2.10 11.7, J6b,5 = 6.8 Hz, 1H, H6b), 3.36–3.15 (m, 8H, H2, H3, H4,
(m, 2H, H11), 2.09 (s, 3H, AcO), 2.05 (s, 3H, AcO), 2.02 (s, 3H, H5, H8), 3.00 (q, J11,12 = 7.6 Hz, 2H, H11), 1.37 (t, J11,12
=
=
AcO), 2.00 (s, 3H, AcO), 1.10 (t, J12,11 = 7.5 Hz, 3H, H12). 13C 7.6 Hz, 3H, H12); 13C NMR (100 MHz, MeOD) δ 195.79, 191.96
NMR (100 MHz, CDCl3) δ 170.85, 170.46, 169.54, 169.50 (CO), (CO), 164.48, 163.16 (CAr), 151.05, 146.43 (C9, C10), 105.11
100.95 (C1), 83.21, 75.35 (C9, C10), 72.94 (C3), 71.97 (C5), (C1), 78.37, 78.14 (C4, C5), 74.68, 71.74 (C2, C3), 69.05 (C7),
71.33 (C2), 68.81 (C7), 68.53 (C4), 62.07 (C6), 20.90, 20.81, 63.03 (C6), 31.32 (C8), 24.07 (C11), 13.88 (C12); MS (ESI) 949.2
20.78, 20.76 (AcO), 20.24 (C8), 14.29 (C12), 12.52 (C11). MS [M + Na]+, HRMS (ESI) m/z calculated for [C20H22Cl2N2O12Re2Na]+:
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948.95311, found: 948.95418 [M + Na]+ (error: 1.1 ppm);
[α]D25: + 6.21 (C = 0.43, MeOH).
Compound 16. To a solution of compound (15) (60 mg,
0.23 mmol, 1 eq.) in DCM (2 mL), PPh3 (113 mg, 0.43 mmol, 1.9
Compound 13. To a solution of 2-(2-chloroethoxy)ethanol eq.), Na2CO3 (46 mg, 0.23 mmol, 1 eq.) and H2O (400 µL) were
(12) (423 µL, 4 mmol, 1 eq.) in water (5 mL), NaN3 (782 mg, added, and then the reaction mixture was vigourously stirred at
12 mmol, 3 eq.) and NaI (60 mg, 0.4 mmol, 0.1 eq.) were r.t. overnight. The solvents were removed under reduced
added. The reaction mixture was stirred at 60 °C for 96 h. The pressure and the crude was purified by flash chromatography
mixture was saturated with NaCl and extracted with DCM (eluent DCM : MeOH 9 : 1 + 1% 7 N NH3 in MeOH) obtaining a
(3 × 10 mL), and the organic phase was dried over Na2SO4, fil- pure compound (16) (46 mg, 85% yield) as a yellowish oil.
tered, concentrated under nitrogen flow and used without 1H NMR (400 MHz, MeOD) δ 4.02–3.97 (m, 2H, H4), 3.69–3.63
further purification.
(m, 2H, H3), 3.53 (t, J = 5.3 Hz, 2H, H2), 3.11 (s, 3H, NMe), 2.79
Compound 14. To a solution of compound (13) (4 mmol, (t, J1,2 = 5.3 Hz, 2H, H1), 1.49 (s, 9H, tBu). 13C NMR (100 MHz,
1 eq.) in DCM (25 mL), TsCl (1.15 g, 6 mmol, 1.5 eq.) and then MeOD) δ 158.55 (CO), 82.73 (Cq), 74.63 (C4), 73.38 (C2),
Et3N (1.7 mL, 12 mmol, 3 eq.) were added under nitrogen at 69.57 (C3), 42.08 (C1), 37.04 (NMe), 28.52 (tBu); MS (ESI) m/z
0 °C, and then the mixture was stirred at r.t. for 3 h. The crude calculated for [C10H23N2O4]+: 235.30, found: 235.1 [M + H]+.
solution was washed with saturated NaHCO3 (3 × 20 mL) and
Compound 17a. To a solution of compound (1) (48 mg,
brine (20 mL), dried on anhydrous Na2SO4, filtered and evapo- 62 µmol, 1.1 eq.) in dry DCM (2.5% DMA dry) (2 mL + 70 µL),
rated under reduced pressure. Flash chromatography purifi- HATU (32 mg, 85 µmol, 1.5 eq.) and then freshly distilled
cation (hex : AcOEt gradient elution) gave a pure compound DIPEA (29 µL, 17 µmol, 3 eq.) were added under a nitrogen
1
(14) (1.09 g, 80% yield). H NMR (400 MHz, CDCl3) δ 7.80 (d, atmosphere. After 15 min a solution of compound (16) (13 mg,
JAB = 8.2 Hz, 2H, AA′BB′, Ar–HAA′), 7.35 (d, JAB = 8.2 Hz, 2H, 56 µmol, 1 eq.) in dry DCM (600 µL) was added and the reac-
AA′BB′, Ar–HBB′), 4.25–4.08 (m, 2H, H4), 3.76–3.64 (m, 2H, tion was stirred at r.t. under nitrogen for 1 h. The crude was
H3), 3.64–3.57 (m, 2H, H2), 3.32 (t, J1,2 = 5.0 Hz, 2H, H1), 2.45 coevaporated with toluene and CHCl3, and purified by flash
(s, 3H, Me); 13C NMR (100 MHz, CDCl3) δ 145.05, 133.03 (Cq), chromatography (hexane : AcOEt gradient elution) obtaining a
1
129.99, 128.12 (CHAr), 70.31 (C2), 69.25 (C4), 68.83 (C3), pure compound (17a) (47 mg, 72% yield). H NMR (400 MHz,
50.76 (C1), 21.78 (Me); MS (ESI) m/z calculated for CDCl3) δ 9.67–9.55 (m, 2H, H-10, H11), 8.00 (dd, J9,10 = 5.9,
[C11H15N3O4SNa]+: 308.06, found: 308.1 [M + Na]+.
J9,11 = 2.3 Hz, 1H, H9), 7.10 (bt, 1H, NH), 4.01–3.94 (m, 2H,
of H1), 3.70–3.62 (m, 2H, H2), 3.62–3.56 (m, 2H, H3), 3.42 (m,
N-Boc-N-methylhydroxylamine.
To
solution
N-methylhydroxylamine hydrochloride (630 mg, 7.54 mmol, 2H, H4), 3.13 (s, 3H, NMe), 2.98 (t, J7,6 = 7.5 Hz, 2H, H7),
1 eq.) in dry DCM (36 mL), di-tert-butyl dicarbonate (1.3 g, 2.44–2.37 (m, 2H, H5), 2.20–2.10 (m, 2H, H6), 1.47 (s, 9H,
6.03 mmol, 0.8 eq.) was added, and then the mixture was OtBu); 13C NMR (100 MHz, CDCl3) δ 193.62, 193.46, 190.66
treated with Et3N (1 mL, 7.54 mmol, 1 eq.) and stirred at r.t. (CORe), 171.63 (CONH), 163.02, 161.22 (C10, C11), 157.04
under a nitrogen atmosphere for 4 hours. Finally, the crude (OCON), 149.01 (C8), 132.00 (C9), 81.90 (Cq), 73.24 (C1), 69.54
solution was washed with 0.1 M NaHSO4 (3 × 25 mL) and brine (C3), 67.99 (C2), 39.12 (C4), 36.87 (NMe), 34.16 (C5), 31.94
(3 × 25 mL). The organic phase was dried on anhydrous (C7), 28.41 (tBu), 25.13 (C6); MS (ESI) m/z calculated for
Na2SO4, filtered and evaporated under reduced pressure giving [C24H30Cl2N4O11Re2Na]+: 1017.02, found: 1017.2 [M + Na]+ and
pure N-Boc-N-methylhydroxylamine (583 mg, 52% yield). 993.5 [M − H]−.
1H NMR (400 MHz, CDCl3) δ 6.68 (bs, 1H, OH), 3.15 (s, 3H,
NMe), 1.49 (s, 9H, tBu).
Compound 17b. A solution of compound (17a) (47 mg,
47 µmol, 1 eq.) in dry DCM (2.4 mL) was treated with TFA
Compound 15. To
a
solution of N-Boc-N-methyl- (181 µL, 2.36 mmol, 50 eq.) under a nitrogen atmosphere at
hydroxylamine (360 mg, 2.45 mmol, 1.4 eq.) in dry THF 0 °C, and then the mixture was stirred at room temperature for
(2 mL), DBU (393 µL, 2.63 mmol, 1.5 eq.) and a solution of 1 hour until the reaction was complete (TLC, AcOEt : MeOH
compound 14 (500 mg, 1.75 mmol, 1 eq.) in dry THF (1.5 mL) 9 : 1, Rf prod. = 0.28). The crude was diluted with CHCl3 and co-
were added. Then the mixture was stirred and left under the evaporated with toluene (3×) and CHCl3 (3×) affording com-
flow of nitrogen until the solvent was removed, and then the pound 17b in quantitative yield that was used without further
neat reaction was stirred overnight at room temperature. The purification. 1H NMR (400 MHz, MeOD) δ 9.96 (dd, J9,11
crude was suspended in AcOEt (30 mL) and the organic phase 2.3 Hz, J10,11 = 0.9 Hz, 1H, H11), 9.88 (dd, J9,10 = 5.9 Hz, J10,11
=
=
was washed with 0.1 M NaHSO4 (3 × 10 mL), 0.1 M NaOH 0.9 Hz, 1H, H10), 8.11 (dd, J9,10 = 5.9 Hz, J9,11 = 2.3 Hz, 1H,
(3 × 10 mL) and brine (10 mL), and then dried over anhydrous H9), 4.24–4.14 (m, 2H, H1), 3.80–3.71 (m, 2H, H2), 3.56 (t,
Na2SO4 and filtered. The evaporation of the solvent under J3,4 = 5.5 Hz, 2H, H3), 3.37 (t, J3,4 = 5.5 Hz, 2H, H4), 3.00–2.94
reduced pressure gave a pure compound (15) (400 mg, 88% (m, 5H, NMe, H5), 2.35 (t, J6,7 = 7.1 Hz, 2H, H7), 2.15–2.03 (m,
yield). 1H NMR (400 MHz, CDCl3) δ 4.05–3.97 (m, 2H, H4), 2H, H6). 13C NMR (100 MHz, MeOD) δ 195.63, 191.77 (CO),
3.74–3.61 (m, 4H, H2, H3), 3.43–3.34 (m, 2H, H1), 3.10 (s, 3H, 175.33 (CONH), 165.01 (C11), 163.12 (C10), 151.16 (C8), 133.64
Me), 1.47 (s, 9H, tBu). 13C NMR (100 MHz, CDCl3) δ 157.11 (C9), 74.27 (C1), 71.38 (C3), 70.52 (C2), 40.25 (C4), 36.70
(CO), 81.44 (Cq), 73.68 (C1), 70.12, 68.92 (C2, C3), 50.83 (C1), (NMe), 35.71 (C7), 32.73 (C5), 26.24 (C6).
36.91 (NMe), 28.38 (tBu). MS (ESI) m/z calculated for
Complex 4. To a solution of compound 17b (23 mg,
22.8 µmol, 1 eq.) and D-glucose (12 mg, 69 µmol, 3 eq.) in dry
[C10H20N4O4Na]+: 283.13, found: 283.1 [M + Na]+.
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MeOH (230 µL), glacial AcOH (33 µL, 0.57 mmol, 25 eq.) was 1H NMR (400 MHz, MeOD) δ 10.00 (dd, J = 2.2, 0.9 Hz, 1H,
added and then the mixture was stirred at r.t. overnight. The H27), 9.89 (dd, J = 5.9, 0.9 Hz, 1H, H26), 8.14 (dd, J = 5.9,
crude mixture was directly loaded on a C-18 column and puri- 2.3 Hz, 1H, H25), 8.03 (s, J = 6.9 Hz, 3H, H11), 4.60–4.55 (m,
fied by RP-18 chromatography (H2O : MeOH gradient elution) 6H, H10), 4.53 (s, 6H, H13), 4.29 (d, J = 7.8 Hz, 3H, H1),
affording a pure compound (4) (15 mg, 62% yield). 1H NMR 4.01–3.94 (m, 3H, H7a), 3.93–3.89 (m, 6H, H9), 3.89–3.85 (m,
(400 MHz, MeOD) δ 9.99 (dd, J15,17 = 2.3 Hz, J16,17 = 0.9 Hz, 1H, 3H, H6a), 3.74–3.63 (m, 14H, H6b, H7b, H8, H19), 3.56–3.48
H17), 9.88 (dd, J15,16 = 5.9 Hz, J16,17 = 0.9 Hz, 1H, H16), 8.14 (m, 4H, H17, H18), 3.46 (s, 6H, H14), 3.42 (s, 2H, H16),
(dd, J15,16 = 5.9 Hz, J15,17 = 2.3 Hz, 1H, H15), 8.02 (bt, JNH,10
=
3.39–3.36 (m, 3H, H3), 3.35–3.32 (m, 2H, H20), 3.30–3.25 (m,
5.7 Hz, 1H, NH), 3.99 (d, J1,2 = 8.9 Hz, 1H, H1), 3.92 (t, J7,8 = 4.5 6H, H4, H5), 3.20 (dd, J = 9.1, 7.8 Hz, 3H, H2), 3.02–2.91 (m,
Hz, 2H, H7), 3.86 (dd, J6a,6b = 12.0 Hz, J5,6a = 2.2 Hz, 1H, H6a), 2H, H21), 2.35 (t, J = 7.1 Hz, 2H, H23), 2.12–2.02 (m, 2H, H22);
3.70 (dd, J6a,6b = 12.0, J5,6b = 5.1 Hz, 1H, H6b), 3.67–3.60 (m, 2H, 13C NMR (100 MHz, MeOD) δ 195.69, 195.61, 191.79, 191.71
H8), 3.55 (t, J9,10 = 5.2 Hz, 2H, H9), 3.49 (t, J1,2 = 8.9 Hz, 1H, H2), (ReCO), 174.92 (CONH), 165.09, 163.20 (C26, C27), 151.28
3.42–3.32 (m, 4H, H3, H4, H10), 3.23 (ddd, J4,5 = 9.5 Hz, J5,6b
=
(C24), 146.02 (C12), 133.75 (C25), 125.99 (C11), 104.48 (C1),
5.1 Hz, J5,6a = 2.2 Hz, 1H, H5), 3.02–2.94 (m, 2H, H11), 2.74 (s, 78.05, 78.01 (C3, C5), 75.11 (C2), 72.18 (C17), 71.64 (C4), 71.38
3H, NMe), 2.42–2.32 (m, 2H, H13), 2.15–2.02 (m, 2H, H12). 13C (C8), 71.25 (C18), 70.81 (C16), 70.51 (C19), 70.34 (C9), 70.03
NMR (100 MHz, MeOD) δ 195.68, 191.78 (CO), 174.98 (CONH), (C14), 69.81 (C7), 65.32 (C13), 62.77 (C6), 51.35 (C10), 46.58
165.04, 163.15 (C16, C17), 151.28 (Cq), 133.69 (C15), 95.45 (C1), (C15), 40.49 (C20), 35.75 (C22), 32.69 (C21), 26.31 (C22); HRMS
79.61 (C4), 79.25 (C5), 73.04 (C7), 71.61 (C2), 71.27 (C3), 70.39 (ESI-Qtof) m/z calculated for [C62H92Cl2N12O33Re2Na2]2+:
(C9), 70.16 (C8), 62.79 (C6), 40.44 (C10), 39.23 (NMe), 35.75 1011.2061, found: 1011.1946 [M + 2Na]2+ (error: 11 ppm); [α]2D5:
(C13), 32.69 (C11), 26.35 (C12). HRMS (ESI) m/z calculated for −12.43 (C = 0.725, MeOH).
[C25H32Cl2N4O14Re2Na]+: 1079.02751, found: 1079.02718
Photophysical characterization
[M + Na]+ (error: −0.3 ppm); [α]2D5: −3.03 (C = 0.515, MeOH).
Complex 5. To a solution of compound 17b (23 mg,
Steady-state measurements. Absorption spectra were
22.8 µmol, 1 eq.) and D-maltose (25 mg, 69 µmol, 3 eq.) in dry measured on a double-beam Shimadzu UV-3600 UV-vis-NIR
MeOH (230 µL), glacial AcOH (33 µL, 0.57 mmol, 25 eq.) was spectrophotometer and baseline corrected. Steady-state emis-
added and then the mixture was stirred overnight at room sion spectra were recorded on a HORIBA Jobin-Yvon IBH
temperature. The crude mixture was directly loaded on a C-18 FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon
column and purified by RP-18 chromatography (H2O : MeOH arc lamp and a TBX-4-X single-photon-counting as an exci-
gradient elution) affording a pure compound (5) (15 mg, 62% tation source and a detector, respectively. Emission spectra
yield). 1H NMR (400 MHz, MeOD) δ 9.99 (dd, J15,17 = 2.3 Hz, were corrected for source intensity (lamp and grating) and
J16,17 = 0.9 Hz, 1H, H17), 9.88 (dd, J15,16 = 5.9 Hz, J16,17
=
emission spectral response (detector and grating) by standard
0.9 Hz, 1H, H16), 8.15 (dd, J15,16 = 5.9, J15,17 = 2.3 Hz, 1H, correction curves. The quantum yield measurements were per-
H15), 8.04 (bt, JNH,10 = 5.4 Hz, 1H, NH), 5.16 (d, J1′,2′ = 3.8 Hz, formed by using a Hamamatsu Photonics absolute photo-
1H, H1′), 4.01 (d, J1,2 = 8.9 Hz, 1H, H1), 3.92 (t, J7,8 = 4.4 Hz, luminescence quantum yield system Quantaurus C11347-11
2H, H7), 3.89–3.79 (m, 2H, H6a, H6′ab), 3.72–3.52 (m, 10H, equipped with a continuous wave xenon light source (150 W),
H8, H6b, H2, H3, H3′, H4, H4′, H9), 3.45 (dd, J2′,3′ = 9.7, a motorized monochromator, a CCD photonic multichannel
J1′,2′ 3.8 Hz, 1H, H2′), 3.41–3.33 (m, 3H, H10, H5), 3.29–3.23 analyzer and employing a commercially available dedicated
(m, 2H, H5′), 3.01–2.94 (m, 2H, H11), 2.75 (s, 3H, NMe), 2.38 measurement software (Hamamatsu Photonics, Japan). The
(t, J12,13 = 7.1 Hz, 2H, H13), 2.14–2.04 (m, 2H, H12). 13C NMR photoluminescence quantum yields were measured exciting
(100 MHz, MeOD) δ 195.67, 195.60, 191.78, 191.71 (CO), 174.98 the samples between 380–420 nm.
(CONH), 165.03, 163.17 (C16, C17), 151.27 (C14), 133.75 (C15),
Lifetime measurements. Time-resolved measurements were
102.93 (C1), 95.38 (C1′), 81.06, 78.96, 78.21, 75.09, 74.80, performed using either time-correlated single-photon counting
74.19, 73.06 (C7), 71.47, 71.19, 70.41 (C8), 70.16 (C9), 62.72 (TCSPC) electronics PicoHarp300 or Multi Channel Scaling
(C6), 62.28 (C6′), 40.43 (C10), 39.38 (NMe), 35.77 (C13), (MCS) electronics NanoHarp 250 of PicoQuant FluoTime 300
32.70 (C11), 26.38 (C12). HRMS (ESI) m/z calculated for (PicoQuant GmbH, Germany), equipped with a PDL 820 laser
[C31H48Cl2N4O19Re2Na]+: 1241.08057, found 1241.08278 pulse driver. A pulsed laser diode LDH-P-C-405 (λex = 405 nm,
[M + Na]+ (error: 1.8 ppm). [α]2D5: +15.54 (C = 0.605, MeOH).
pulse FWHM <70 ps, repetition rate 200 kHz–40 MHz) was
Compound 6. To a solution of compound 1 (18 mg, used to excite the sample and was mounted directly on the
23 µmol, 1.2 eq.) and HATU (11 mg, 29 µmol, 1.5 eq.) in DMA sample chamber at 90°. The photons were collected by using a
dry (600 µL), freshly distilled DIPEA (10 µL, 57 µmol, 3 eq.) was PMA-C-192 photomultiplier (PMT) single-photon-counting
added under a nitrogen atmosphere. After 15 min a solution of detector. The data were acquired by using the commercially
compound (21) (24 mg, 19 µmol, 1 eq.) in dry DMA (400 µL) available software EasyTau (PicoQuant GmbH, Germany),
was added and the reaction was stirred at r.t. under nitrogen while data analysis was performed using the commercially
for 1 h. The crude was directly loaded on a C-18 column and available software FluoFit (PicoQuant GmbH, Germany). The
purified by RP-18 chromatography (H2O : MeOH gradient quality of the fit was assessed by minimizing the reduced
elution) obtaining a pure compound (6) (26 mg, 68% yield). χ2 function (χ2 < 1.2 and <1.5 for mono- and bi-exponential
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model, respectively) and by visual inspection of the random (50 μM in less than 1% DMSO containing PBS) were gently
distribution of the weighed residuals. For multi-exponential added onto cells. After incubation at 37 °C for 1 hour, the
decays, the intensity, namely I(t), has been assumed to decay incubating media was removed and the cell layer on glass
as the sum of individual single exponential decays (eqn (1)):
cover slips was gently washed (three times) with PBS and fixed
with 4% paraformaldehyde (PFA) in PBS solution for 10 min.
ꢀ
ꢁ
n
X
t
τi
IðtÞ ¼
αi exp ꢀ
ð1Þ
Kinetic of internalization
i¼1
The culture media of live cells grown onto glass bottom dishes
was removed and 2 mL of the complex staining solution
(100 μM in less than 1% DMSO containing PBS) was added.
The cells were subsequently imaged by using a fluorescence
confocal microscopy setup for a ten-minute acquisition time
for a total duration of 60 minutes.
where τi are the decay times and αi are the amplitude of the
components at t = 0. In the tables, the percentages of the pre-
exponential factors, αi, are listed upon normalization.
Fluorescence confocal microscopy
The experiments were carried out by incubating approximately
50 000 HeLa cells with various concentrations of compound
solutions (25–100 μM in less than 1% v/v of DMSO/Phosphate
Buffer Saline, PBS) for 1 h under normal biological conditions
(37 °C and 5% CO2). After the incubation, the stained cells were
imaged directly using a fluorescence confocal microscopy setup.
All of the fluorescence images were obtained by using a Zeiss
LSM 710 confocal microscope set up with a 63× magnification,
and a numerical aperture, NA, 1.3 of Zeiss LCI Plan-NEOFLUAR
water immersion objective lens (Zeiss GmbH) equipped with a
spectrometer. Image acquisition was directly performed by excit-
ing the samples using a continuous wave (cw) laser at 405 nm.
The emission of the complexes was collected in the range from
540 to 720 nm and also scanned in lambda-mode (for acquiring
the emission spectra). For co-localization experiments, the
samples previously co-stained with different dyes, DAPI (exci-
tation/emission wavelength: 358 nm/461 nm), ER Tracker Red
(excitation/emission wavelength: 587 nm/615 nm), and Alexa
Fluor® 647 Phalloidin (excitation/emission wavelength: 650 nm/
668 nm) were excited independently at 405, 594 and 633 nm,
and the emission signals were collected according to their
corresponding emission profiles. Image processing was per-
formed by using Zen 2011 software (Zeiss GmbH). False colour
images were adjusted to better distinguish different complexes
and complexes from cellular organelles.
Organelle staining
The cell layer was washed twice with PBS and kept in 0.1%
Triton X-100 (Sigma Aldrich) in PBS for 5 minutes and after-
wards in 1% bovine serum albumin, BSA (Sigma Aldrich), in
PBS for 20 min. The cell layer on the glass cover slip was
stained with Phalloidin Alexa Fluor® 647 (Invitrogen), for
f-actin/membrane staining, for 20 min, in the dark at room
temperature, and washed two times with PBS. For visualizing
the nuclear region, the cell nucleus was stained with 4′,6-di-
amidino-2-phenylindole carboxamidine (DAPI) and washed
twice with PBS. In the case of 4, ER Tracker Red dye
(Invitrogen) was added to visualize the endoplasmic reticulum.
The cover slips were mounted onto glass slides for microscopy
measurements.
Cell viability studies
Cellular viability was measured by using an automatic cell
counter CASY (Roche Innovatis AG, Bielefeld, Germany).
Approximately 50 000 cells were grown in 2 ml of culture
media in 6 well plates at 37 °C, in a 5% CO2 environment for
48 hours. The culture media was removed and replaced with
1 ml staining solution of complex 2, 3, 4, and 5 (50 μM in less
than 1% DMSO in PBS). Subsequently after 1 hour of incu-
bation, the staining solution was transferred to Eppendorf
tubes and 0.5 ml of trypsin were added. To detach the cell
from the surface of the plate, the cells were incubated for
5 minutes under the same conditions mentioned before.
Subsequently, 0.5 ml of PBS was added to neutralize trypsin.
The cell suspensions together with the first solutions were col-
lected and removed into an Eppendorf tube. 100 µl of the cell
suspension was dissolved in 10 ml of CASY ton solution and
measurement was performed. The positive control of the cells
grown without the complexes was also performed. All experi-
ments were repeated 3 times.
Cell culture
All materials for cell culture were purchased from Gibco.
Human cervical carcinoma, HeLa, cells were cultured in media
containing 88% Dulbecco’s Modified Eagle Medium (DMEM),
10% Fetal Bovine Serum (FBS), 1% penicillin–streptomycin
and 1% L-glutamine (200 mM) at 37 °C and under 5% CO2 con-
ditions for 48 hours until reaching 70 to 80% cell confluency.
Subsequently, the cells were washed twice with Phosphate
Buffer Solution (PBS, Gibco), trypsinated and approximately
50 000 cells were reseeded on a monolayer glass cover slip in a
six-well plate culture dish and glass bottom dishes (MatTek).
Fresh culture media (2 mL) was added gently and the cells
were grown overnight.
Author contribution
A. P., A. A., and D. S. contributed equally to the work as
follows: A. P. planned and performed the synthesis of all the
Rhenium complex incubation
The culture media was removed and 2 mL of a new staining compounds and of the complexes, supervised by A. B.;
solution containing the corresponding rhenium complexes A. A. provided the spectroscopic and photophysical characteriz-
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Paper
ation of the conjugates, supervised by M. M.; D. S. performed
cellular uptake experiments and acquired the confocal
microscopy images, all supervised by L. D. C. M. P., M. M.,
A. P., D. S. and A. B. planned the experiments, analysed and
discussed the data and wrote the paper.
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The University of Strasbourg is kindly acknowledged for finan-
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