Rhenium(I) polypyridine dibenzocyclooctyne complexes as phosphorescent bioorthogonal probes: Synthesis, characterization, emissive behavior, and biolabeling properties

Article abstract of DOI:10.1016/j.jinorgbio.2015.02.018

We report the development of rhenium(I) polypyridine complexes appended with a dibenzocyclooctyne (DIBO) moiety as bioorthogonal probes for azide-modified biomolecules. Three phosphorescent rhenium(I) polypyridine DIBO complexes [Re(N^N)(CO)₃(py-C6-DIBO)][CF₃SO₃] (py-C6-DIBO = 3-(N-(6-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonylamino)hexyl)aminocarbonyl)pyridine; N^N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me₄-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph₂-phen) (3a)) and their DIBO-free counterparts [Re(N^N)(CO)₃(py-C6-BOC)][CF₃SO₃] (py-C6-BOC = 3-(N-(6-(tert-butoxycarbonylamino)hexyl)aminocarbonyl)pyridine; N^N = phen (1b), Me₄-phen (2b), Ph₂-phen (3b)) were synthesized and characterized. Upon photoexcitation, all the complexes displayed intense and long-lived yellow triplet metal-to-ligand charge-transfer (³MLCT) (dπ(Re) → π?(N^N)) emission. The DIBO complexes underwent facile reactions with benzyl azide in methanol at 298 K with second-order rate constants (k₂) in the range of 0.077 to 0.091 M^{- 1} s^{- 1}. As revealed from SDS-PAGE analysis, the DIBO complexes can selectively label azide-modified proteins and the resulting bioconjugates displayed strong phosphorescence upon photoexcitation. Results of inductively coupled plasma mass spectrometry (ICP-MS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays indicated that the DIBO complexes accumulated in Chinese Hamster Ovary (CHO) cells with considerable cytotoxic activity. Upon incubation of CHO cells with these complexes, relatively weak intracellular emission was observed. In contrast, upon pretreatment of the cells with 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-d-mannosamine (Ac₄ManNAz), intense emission was observed from the cell membrane and some internal compartments. The results suggest that the DIBO complexes are promising candidates for imaging azide-labeled biomolecules.

Full text of DOI:10.1016/j.jinorgbio.2015.02.018

Rhenium(I) Polypyridine Dibenzocyclooctyne Complexes as Phosphorescent
Bioorthogonal Probes: Synthesis, Characterization, Emissive Behavior, and
Biolabeling Properties
Alex Wing-Tat Choi, Hua-Wei Liu, Kenneth Kam-Wing Lo
PII:
DOI:
Reference:
S0162-0134(15)00061-6
doi: 10.1016/j.jinorgbio.2015.02.018
JIB 9679
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
27 October 2014
24 February 2015
24 February 2015
Please cite this article as: Alex Wing-Tat Choi, Hua-Wei Liu, Kenneth Kam-Wing Lo,
Rhenium(I) Polypyridine Dibenzocyclooctyne Complexes as Phosphorescent Bioorthogo-
nal Probes: Synthesis, Characterization, Emissive Behavior, and Biolabeling Properties,
Journal of Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.02.018
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ACCEPTED MANUSCRIPT
Rhenium(I) Polypyridine Dibenzocyclooctyne Complexes as Phosphorescent
Bioorthogonal Probes: Synthesis, Characterization, Emissive Behavior, and
Biolabeling Properties
Alex Wing-Tat Choi, Hua-Wei Liu, and Kenneth Kam-Wing Lo*
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee
Avenue, Kowloon, Hong Kong (People’s Republic of China)
* Corresponding author: E-mail: bhkenlo@cityu.edu.hk; Fax: +852 3442 0522
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Abstract: We report the development of rhenium(I) polypyridine complexes
appended with a dibenzocyclooctyne (DIBO) moiety as bioorthogonal probes for
azide-modified biomolecules. Three phosphorescent rhenium(I) polypyridine DIBO
complexes [Re(N^N)(CO)3(py-C6-DIBO)][CF3SO3] (py-C6-DIBO
=
3-(N-(6-
(3,4:7,8-dibenzocyclooctyne-5-oxycarbonylamino)hexyl)aminocarbonyl)pyridine;
N^N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline
(Me4-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3a)) and their DIBO-
free counterparts [Re(N^N)(CO)3(py-C6-BOC)][CF3SO3] (py-C6-BOC = 3-(N-(6-
(tert-butoxycarbonylamino)hexyl)aminocarbonyl)pyridine; N^N = phen (1b), Me4-
phen (2b), Ph2-phen (3b)) were synthesized and characterized. Upon photoexcitation,
all the complexes displayed intense and long-lived yellow triplet metal-to-ligand
charge-transfer (3MLCT) (d(Re)  *(N^N)) emission. The DIBO complexes
underwent facile reactions with benzyl azide in methanol at 298 K with a second-
order rate constants (k2) in the range of 0.077 to 0.091 M1 s1. As revealed from
SDS-PAGE analysis, the DIBO complexes can selectively label azide-modified
proteins and the resulting bioconjugates displayed strong phosphorescence upon
photoexcitation. Results of inductively coupled plasma mass spectrometry (ICP-MS)
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays
indicated that the DIBO complexes accumulated in Chinese Hamster Ovary (CHO)
cells with considerable cytotoxic activity. Upon incubation of CHO cells with these
complexes, relatively weak intracellular emission was observed. In contrast, upon
pretreatment of the cells with 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine
(Ac4ManNAz), intense emission was observed from the cell membrane and some
internal compartments. The results suggest that the DIBO complexes are promising
candidates for imaging azide-labeled biomolecules.
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1. Introduction
The discovery of green fluorescent protein enables the visualization of protein
dynamics in living systems through a genetic tagging method [1]. However, this
strategy is not applicable to the study of non-protein biomolecules such as nucleic
acids, glycans, lipids, and small-molecule metabolites. To overcome this limitation, a
bioorthogonal chemistry approach has been designed as a versatile method to study
these biomolecules in their native environments. A number of chemical reporters and
their specific probes for bioorthogonal reactions have been identified and successfully
applied in biological studies [2,3]. Among different bioorthogonal reactions, the
strain-promoted alkyne-azide cycloaddition (SPAAC) between azide and strained
alkyne is one of the most extensively used reactions in bioorthogonal chemistry [4,5].
Azide is a particularly useful handle because of its small size, non-native nature, and
inertness toward biomolecules; for example, 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-
mannosamine (Ac4ManNAz) is a very commonly used unnatural sugar derivative
[6,7]. Upon cellular uptake, the acetyl groups are removed by carboxyesterases and
the mannose derivative is metabolized by glycosyltransferases, and eventually
expressed as an end-group of a glycan chain, leaving the azide group unmodified.
Thus, the azide group is able to undergo specific reactions with bioorthogonal probes
such as a strained alkyne. To date, the design of bioorthogonal probes to trace azide-
labeled biomolecules has been focused on affinity tags [8–11], fluorescent organic
dyes [12–15], and luminescent quantum dots [16]. Recently, we have prepared
iridium(III) dibenzocyclooctyne (DIBO) complexes as the first class of transition
metal-based bioorthogonal probes [17]. In view of the interesting photophysical and
biological properties of rhenium(I) polypyridine complexes [18], we anticipate that
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the modification of these complexes with a DIBO moiety will generate a new class of
phosphorescent bioorthogonal reagents for various biological applications. Herein,
we report the synthesis and characterization of three phosphorescent rhenium(I)
polypyridine DIBO complexes [Re(N^N)(CO)3(py-C6-DIBO)][CF3SO3] (py-C6-
DIBO
=
3-(N-(6-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonylamino)hexyl)-
aminocarbonyl)pyridine; N^N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-
1,10-phenanthroline (Me4-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen)
(3a)) and their DIBO-free counterparts [Re(N^N)(CO)3(py-C6-BOC)][CF3SO3] (py-
C6-BOC = 3-(N-(6-(tert-butoxycarbonylamino)hexyl)aminocarbonyl)pyridine; N^N =
phen (1b), Me4-phen (2b), Ph2-phen (3b)) (Scheme 1). The spectroscopic and
photophysical properties of the complexes were studied. The DIBO complexes
underwent facile reactions with benzyl azide and the reaction kinetics were
investigated. The bioorthogonal labeling properties of these complexes toward azide-
modified bovine serum albumin (BSA), human serum albumin (HSA), and apo-
transferrin (aTf) were also examined. Additionally, the cellular uptake properties and
cytotoxicity of these complexes were studied by inductively coupled plasma mass
spectrometry (ICP-MS) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (MTT) assays, respectively. Furthermore, the bioorthogonal labeling
properties of one of the DIBO complexes were examined using Chinese Hamster
Ovary (CHO) cells pretreated with Ac4ManNAz.
2. Results and Discussion
2.1. Design and Synthesis of Complexes
Regarding the design of the DIBO complexes 1a  3a, the DIBO moiety was attached
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to the pyridine ligand through a hexyl spacer-arm to reduce the steric bulkiness of the
rhenium(I) polypyridine unit. The pyridine ligand py-C6-DIBO was synthesized from
the reaction of 3,4:7,8-dibenzocyclooctyn-5-yl 4-nitrophenyl carbonate with 3-(6-
aminohexyl)aminocarbonylpyridine (py-C6-NH2). The DIBO complexes 1a  3a and
the DIBO-free complexes 1b  3b were obtained from the reaction of
[Re(N^N)(CO)3(CH3CN)][CF3SO3] with the pyridine ligands py-C6-DIBO and py-
C6-BOC, respectively, in refluxing THF. The samples were purified by column
chromatography and recrystallization from CH2Cl2/diethyl ether.
They were
1
characterized by H NMR, positive-ion ESI-MS, IR spectroscopy, and microanalysis.
All the complexes were stable in air and soluble in most organic solvents such as
CH2Cl2 and alcohols. One of the basic requirements for luminescent probes is their
stability in the medium and environment where they are used [19]. Based on the ESI-
MS data, all the complexes in this work remained stable in aqueous solutions
(containing 1% DMSO for solubility reasons).
2.2. Photophysical Properties
The electronic absorption spectral data of the complexes are summarized in Table 1.
All the complexes displayed intense spin-allowed intraligand (1IL) (  *) (N^N
and pyridine ligands) absorption bands at ca. 250  338 nm and metal-to-ligand
charge-transfer (1MLCT) (d(Re)  *(N^N)) absorption shoulders at ca. 366  394
nm [2049]. It is noteworthy that the DIBO complexes revealed an additional
absorption shoulder at ca. 305 nm, which was assigned to a (  *) transition of the
DIBO moiety.
Upon photoexcitation, all the complexes showed intense and long-lived green
to yellow emission. The photophysical data are summarized in Table 2 and the
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emission spectra of the DIBO complexes 1a  3a in CH3CN at 298 K are shown in
Fig. 1. In fluid solutions at 298 K, most of the complexes exhibited reduced emission
energy, quantum yields, and lifetimes on changing the solvent from the more polar
CH3CN to the less polar CH2Cl2. These findings, together with the dependence of the
emission energy on the * orbital energy level of the N^N ligands, point to an
emissive state of triplet metal-to-ligand charge-transfer (3MLCT) (d(Re)  *(N^N))
character. However, the structural features and long emission lifetimes of the Me4-
phen complexes in fluid solutions under ambient conditions (Table 2) suggest the
involvement of triplet intraligand (3IL) (  *) (Me4-phen) character in their
emissive states.
2.3. Reaction with Benzyl Azide
The azide-targeting properties of the DIBO complexes were examined using benzyl
azide as a model substrate (Scheme 2). The SPAAC reaction kinetics of the DIBO
complexes with benzyl azide in methanol at 298 K were studied by monitoring the
exponential decay of the absorption feature of DIBO at ca. 305 nm and the results are
summarized in Table 3. The formation of the triazole complexes was confirmed by
electrospray ionization mass spectrometry (ESI-MS) (Table 4) and the plots of the
pseudo first-order rate constants (kobs) of the DIBO complexes versus the
concentrations of benzyl azide are shown in Fig. 2. The second-order rate constants
(k2) were determined to range from 0.077 to 0.091 M1 s1 (Table 3), which are
comparable to those of other DIBO-azide systems such as dibenzocyclooctynol (k2 =
0.057 M1 s1) [10] and [Ir(ppy-COOH)2(bpy-TEG-DIBO)][PF6] (k2 = 0.069 M1 s1)
[17]. This result reflects that the bulky rhenium(I) polypyridine unit did not adversely
affect the reactivity of the DIBO unit due to the long spacer-arm.
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2.4. Protein-labeling Studies
The biomolecular targeting properties of the DIBO complexes were studied using
complex 1a as an example. This complex was used to label BSA, HSA, and aTf that
had been modified with azidoacetic acid (Scheme 3). The resultant bioconjugates 1-
BSA, 1-HSA, and 1-aTf were purified by size exclusion chromatography and
ultrafiltration. As revealed from the SDS-PAGE analysis (Fig. 3), the protein-azide
conjugates were successfully labeled by the DIBO complexes, giving rise to
phosphorescent bands in the gels. The absence of similar bands for the unmodified
protein samples confirmed that the labeling originated from the specific reaction
between the DIBO moiety of the complexes and the azide units of the conjugates. The
photophysical properties of the bioconjugates 1-BSA, 1-HSA, and 1-aTf were studied
and the results are summarized in Table 5. All the bioconjugates showed a blue-
shifted emission band compared with free complex 1a in CH3CN. This observation
was ascribed to the increased hydrophobicity of the surroundings of the complex after
binding to the proteins. Notably, the emission of all the bioconjugates showed
biexponential decay, which is common for biomolecules labeled with phosphorescent
transition metal complexes due to their heterogeneous microenvironments on the
protein molecule [35,50,51].
2.5. Cellular Uptake Properties and Live-cell Confocal Imaging
The cellular uptake properties of the DIBO complexes were studied using ICP-MS.

After incubation with the complexes at 37 C for 1 h, an average CHO cell contained
0.32 to 2.07 fmol of rhenium (Table 6), which is comparable to related rhenium(I)
polypyridine complexes such as [Re(phen)(CO)3(py-TU-DPAT)][CF3SO3] (py-TU-
DPAT
=
3-(2-(4-hydroxy-3-(2,2’-
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dipicolylaminomethyl)phenyl)ethylthioureidyl)pyridine) (2.80 fmol of rhenium) [33]
and
[Re(phen)(CO)3(py-DA)][PF6]
(py-DA=3-(N-(2-amino-5-
methoxyphenyl)aminomethyl)pyridine) (0.35 fmol of rhenium) [36]. This result
suggests that the modification of rhenium(I) polypyridine complexes with a DIBO
moiety did not have substantial effects on their cellular uptake efficiency. The cellular
uptake efficiency of the complexes was found to be dependent on the diimine ligands
and follow the order: Ph2-phen > Me4-phen > phen (Table 6), which is in accordance
with the decreasing hydrophobic character of the ligands. The cytotoxicity of the
DIBO complexes toward CHO cells over an incubation period of 48 h was
investigated using the MTT assay and the IC50 values ranged from 2.94 to 9.55 M
(Table 6). The Ph2-phen and Me4-phen complexes exhibited higher cytotoxicity,
which is in accordance with their higher uptake efficiency. With reference to the
cytotoxic activity of related organometallic rhenium(I) complexes [52], complexes
1a – 3a are relatively cytotoxic. Since these complexes are both luminescent and
cytotoxic, and exhibit bioorthogonal specificity due to the appended DIBO unit, they
can serve as an interesting class of theranostic reagents for a wide range of biological
studies.
Laser-scanning confocal microscopy images of live CHO cells treated with
the DIBO complex 1a (5 M, 1 h) showed that the complex was effectively
internalized and localized in the cytoplasmic region (Fig. 4). The cellular uptake
pathways of the complexes were studied using the cytoskeletal inhibitor colchicine
and ATPase inhibitor potassium nitrate [53]. As revealed from the confocal
microscopy images (Fig. 4), the intracellular emission intensity of the CHO cells did
not display any significant difference after pretreatment with the two reagents.
Additionally, a similar result was found when the cells were incubated at 4 C (Fig. 4).
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These findings indicate that the DIBO complexes were internalized into CHO cells
through an energy-independent passive diffusion pathway.
Since Ac4ManNAz is metabolically converted by CHO cells to N-azidoacetyl
sialic acids, which are located on the cell surface [9,13,14], pretreatment of the cells
with this reagent is expected to lead to the localization of the complex in the plasma
membrane. Confocal microscopy images of Ac4ManNAz-treated and -untreated CHO
cells were very similar upon incubation with complex 1a for a relatively short
incubation time (1 h) (Fig. 5a). This has been attributed to the less efficient labeling
of azido sialic acid residues within the cell-surface glycans. This was supported by
the ICP-MS measurements which showed that the Ac4ManNAz pretreatment only
slightly increased the rhenium uptake from 0.26  0.02 to 0.35  0.05 fmol per
average CHO cell. In other words, the complex did not have sufficient time to react
with the surface glycans. Interestingly, upon increasing the incubation time to 24 h,
while the CHO cells without pretreatment of Ac4ManNAz did not show any
significant changes in emission intensity, those that were pretreated with Ac4ManNAz
displayed more intense intracellular emission (Fig. 5b), implying increased uptake.
This was also confirmed by ICP-MS measurement, which showed that pretreatment
with Ac4ManNAz increased the uptake of rhenium from 0.23  0.02 to 0.57  0.06
fmol per average CHO cell; the dependence of uptake on the incubation time is
illustrated in Fig. 6. It is noteworthy that Ac4ManNAz-treated cells with prolonged
incubation time (24 h) exhibited intense emission from the cell membrane and internal
compartments which is likely to be Golgi apparatus (Fig. 5b). While the observed
membrane labeling was ascribed to the specific reaction of the DIBO unit of complex
1a with the membrane-bound azido-sialic acid, the enhanced emission of the internal
compartments is most likely due to internalization and intracellular trafficking of the
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glycans labeled by the luminescent complex. Similar observations of surface-glycan
labeling followed by subsequent cellular internalization have been reported for other
fluorescent bioorthogonal probes [13] and a related iridium(III) DIBO complex we
reported previously. [17]
3. Conclusions
In this work, rhenium(I) polypyridine complexes containing a DIBO moiety were
developed as new bioorthogonal probes for azides. Upon photoexcitation, all the
complexes displayed intense and long-lived green to yellow emission. The k2 values
for the reactions of the DIBO complexes with benzyl azide were determined to range
from 0.077 to 0.091 M1 s1. Also, these complexes were used to label azide-modified
proteins including BSA, HSA, and aTf. The DIBO complexes were found to
accumulate in CHO cells with moderate cytotoxicity. Upon incubation of CHO cells
with these complexes, relatively weak intracellular emission was observed. In
contrast, upon pretreatment of the cells with Ac4ManNAz, the cell membrane and
internal compartments showed intense emission. The observation of membrane
labeling is due to the specific reaction of the DIBO unit of complex 1a with the azido-
sialic acid located on the surface of the cells. The enhanced emission of the internal
compartments is probably related to the intracellular trafficking of the labeled glycans.
Our results indicated that the DIBO complexes are promising reagents for imaging
azide-labeled biomolecules. We believe that the bioorthogonal labeling properties of
these complexes can be further enhanced by increasing the reactivity of the DIBO
moiety and/or decreasing the cellular uptake rate of the complexes. The design of
related phosphorescent transition-metal DIBO complexes as bioorthogonal probes for
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azide-modified molecules is underway.
4. Experimental Section
4.1. Materials and Synthesis
All solvents were of analytical reagent grade and purified according to standard
procedures [54]. Diimine ligands including phen, Me4-phen, and Ph2-phen,
trifluoroacetic acid, potassium nitrate, colchicines, iodoacetic acid, and N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) were purchased
from Acros. Nicotinoyl chloride hydrochloride, sodium azide, tert-butyl-N-(6-
aminohexyl)carbamate hydrochloride, N-hydroxybenzotriazole (HOBt), and MTT
were obtained from Sigma. Benzyl azide was purchased from Alfa Aesar. BSA
(Fraction V, RIA and ELISA Grade), HSA (Fraction V, High Purity), and aTf (Human
Plasma) were obtained from Calbiochem. All these chemicals were used without
further purification.
[Re(N^N)(CO)3(CH3CN)][CF3SO3] [55] and 3,4:7,8-
dibenzocyclooctyn-5-yl 4-nitrophenyl carbonate [9] were prepared according to
reported procedures. All buffer components were of biological grade and used as
received. Autoclaved Milli-Q water was used for the preparation of aqueous solutions.
CHO cells were obtained from American Type Culture Collection. F12-nutrition
mixture, fetal bovine serum (FBS), phosphate buffered saline (PBS) , trypsin-EDTA,
Novex® sharp pre-stained protein standard, Ac4ManNAz, and penicillin/streptomycin
were purchased from Invitrogen. The growth medium for cell culture contained F12-
nutrition mixture with 10% FBS and 1% penicillin/streptomycin.
Py-C6-BOC: A mixture of nicotinoyl chloride hydrochloride (1.01 g, 5.62 mmol) and
tert-butyl-N-(6-aminohexyl)carbamate hydrochloride (1.42 g, 5.62 mmol) in pyridine
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(10 mL) was stirred under an inert atmosphere of nitrogen at room temperature for 24
h. Water (100 mL) was added to quench the reaction, and the product was extracted
with CH2Cl2 (150 mL  3). The combined organic layer was dried over MgSO4 and
evaporated to dryness yielding a yellow solid, which was purified by column
chromatography on silica gel. The desired product was eluted with hexane/ethyl
acetate (1:4, v/v). The solvent was removed under vacuum to afford the product as a
1
white solid. Yield: 766 mg (42%). H NMR (300 MHz, CD3OD, 298 K):  8.96 (s,
1H, H2 of pyridine), 8.67 (d, 1H, J = 3.3 Hz, H6 of pyridine), 8.23 (d, 1H, J = 8.1 Hz,
H4 of pyridine), 7.56  7.52 (m, 1H, H5 of pyridine), 3.41  3.30 (t, 2H, J = 6.9 Hz,
py-3-CONHCH2), 3.05  3.00 (t, 2H, J = 6.9 Hz, (CH3)3OCONHCH2), 1.66  1.39 (m,
17H, CH2(CH2)4CH2 and CH3 of Boc). Positive-ion ESI-MS ion cluster at m/z 322
{M + H+}+.
Py-C6-NH2: A mixture of py-C6-BOC (478 mg, 1.46 mmol) and trifluoroacetic acid
(1 mL) in CH2Cl2 (6 mL) was stirred under an inert atmosphere of nitrogen at room
temperature for 30 min. The solution was evaporated to dryness yielding a pale
yellow solid, which was purified by column chromatography on silica gel. The
desired product was eluted with CH2Cl2/MeOH (5:1, v/v). The solvent was removed
1
under vacuum to afford the product as a white solid. Yield: 289 mg (90%). H NMR
(300 MHz, CD3OD, 298 K):  8.98 (s, 1H, H2 of pyridine), 8.67 (d, 1H, J = 2.4 Hz,
H6 of pyridine), 8.25 (d, 1H, J = 7.8 Hz, H4 of pyridine), 7.54 (dd, 1H, J = 7.8 and 2.4
Hz, H5 of pyridine), 3.43  3.30 (m, 2H, py-3-CONHCH2), 2.94 (t, 2H, J = 7.5 Hz,
NH2CH2), 1.72  1.42 (m, 8H, CH2(CH2)4CH2). Positive-ion ESI-MS ion cluster at
m/z 222 {M + H+}+.
Py-C6-DIBO:
A mixture of py-C6-NH2 (150 mg, 0.69 mmol), 3,4:7,8-
dibenzocyclooctyn-5-yl 4-nitrophenyl carbonate (261 mg, 0.69 mmol), and
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triethylamine (2 mL) in CH2Cl2 (15 mL) was stirred under an inert atmosphere of
nitrogen at room temperature for 4 h. The solution was evaporated to dryness yielding
a pale grey solid, which was purified by column chromatography on silica gel. The
desired product was eluted with CH2Cl2/MeOH (5:1, v/v). The solvent was removed
1
under vacuum to afford the product as a white solid. Yield: 231 mg (73%). H NMR
(300 MHz, CDCl3, 298 K):  8.96 (s, 1H, H2 of pyridine), 8.61 (d, 1H, J = 5.1 Hz, H6
of pyridine), 8.00 (d, 1H, J = 7.8 Hz, H4 of pyridine), 7.44 (d, 1H, J = 7.5 Hz, H5 of
pyridine), 7.34  7.20 (m, 8H, C6H4 of DIBO), 7.05 (br, 1H, py-3-CONH), 5.44  5.42
(m, 2H, OCONH and OCH of DIBO), 3.34  3.23 (m, 2H, py-3-CONHCH2), 3.18 
3.10 (m, 3H, OCONHCH2 of DIBO and CH2 of DIBO), 2.83 (dd, 1H, J = 15 and 3.9
Hz, CH2 of DIBO), 1.50  1.22 (m, 8H, CH2(CH2)4CH2). Positive-ion ESI-MS ion
cluster at m/z 469 {M + H+}+.
[Re(N^N)(CO)3(py-C6-DIBO)][CF3SO3] (N^N = phen (1a), Me4-phen (2a), Ph2-
phen (3a)): A mixture of [Re(N^N)(CO)3(CH3CN)][CF3SO3] (0.13 mmol) and py-
C6-DIBO (63 mg, 0.13 mmol) in THF (30 mL) was refluxed under an inert
atmosphere of nitrogen for 12 h. Subsequent recrystallization of the complex from
CH2Cl2/diethyl ether resulted in the formation of the complex as yellow crystals.
1
Complex 1a. Yield: 100 mg (70%). H NMR (300 MHz, CD3OD, 298 K):  9.75 (d,
2H, J = 5.1 Hz, H2 and H9 of phen), 8.91 (d, 2H, J = 8.4 Hz, H4 and H7 of phen),
8.62  8.59 (m, 2H, H2 and H6 of pyridine), 8.21  8.17 (m, 4H, H3, H5, H6, and H8
of phen), 8.08  8.07 (m, 1H, H4 of pyridine), 7.49 (d, 1H, J = 7.8 Hz, H5 of pyridine),
7.37  7.23 (m, 8H, C6H4 of DIBO), 5.27  5.26 (m, 1H, OCH of DIBO), 3.32  3.06
(m, 5H, CH2 and OCONHCH2 of DIBO and py-3-CONHCH2), 2.74 (dd, 1H, J = 15
and 3.9 Hz, CH2 of DIBO), 1.51  1.28 (m, 8H, CH2(CH2)4CH2). IR (KBr) /cm–1:

3447 (m, NH), 2031 (s, CO), 1919 (s, CO), 1161 (m, CF3SO3 ), 1030 (m,
13

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

CF3SO3 ). Positive-ion ESI-MS ion cluster at m/z 918 {M  CF3SO3 }+. Elemental
analysis calcd (%) for C45H37N5O9SF3Re1.5H2O0.5(CH3CH2)2O: C 49.91; H 4.01; N
1
6.19. Found: C 49.85; H 4.03; N 6.43. Complex 2a. Yield: 80 mg (53%). H NMR
(300 MHz, CD3OD, 298 K):  9.48 (s, 2H, H2 and H9 of Me4-phen), 8.66  8.60 (m,
2H, H2 and H6 of pyridine), 8.37 (s, 2H, H5 and H6 of Me4-phen), 8.10 (d, J = 6.0 Hz,
H4 of pyridine), 7.52  7.04 (m, 9H, C6H4 of DIBO and H5 of pyridine), 5.27 (s, 1H,
OCH of DIBO), 3.21  3.06 (m, 5H, CH2 and OCONHCH2 of DIBO and py-3-
CONHCH2), 2.86 (s, 6H, CH3 at C4 and C7 of Me4-phen), 2.74  2.70 (m, 7H, CH2 of
DIBO and CH3 at C3 and C8 of Me4-phen), 1.53  1.35 (m, 8H, CH2(CH2)4CH2). IR

(KBr) /cm–1: 3447 (m, NH), 2031 (s, CO), 1918 (s, CO), 1161 (m, CF3SO3 ),


1030 (m, CF3SO3 ). Positive-ion ESI-MS ion cluster at m/z 974 {M  CF3SO3 }+.
Elemental analysis calcd (%) for C49H45N5O9SF3Re1.5H2O: C 51.17; H 4.21; N 6.09.
1
Found: C 50.79; H 4.21; N 6.40. Complex 3a. Yield: 105 mg (64%). H NMR (300
MHz, CD3OD, 298 K):  9.83  9.81 (m, 2H, H2 and H9 of Ph2-phen), 8.75  8.74 (m,
2H, H2 and H6 of pyridine), 8.20  8.15 (m, 5H, H4 of pyridine and H3, H5, H6, and
H8 of Ph2-phen), 7.73  7.63 (m, 10H, C6H5 of Ph2-phen), 7.50  7.25 (m, 9H, C6H4
of DIBO and H5 of pyridine), 5.24 (s, 1H, OCH of DIBO), 3.24  3.07 (m, 5H, CH2
and OCONHCH2 of DIBO and py-3-CONHCH2), 2.74 (d, 1H, J = 11 Hz, CH2 of
DIBO), 1.54  1.32 (m, 8H, CH2(CH2)4CH2). IR (KBr) /cm–1: 3448 (m, NH), 2032


(s, CO), 1920 (s, CO), 1158 (m, CF3SO3 ), 1030 (m, CF3SO3 ). Positive-ion ESI-

MS ion cluster at m/z 1071 {M  CF3SO3 }+. Elemental analysis calcd (%) for
C57H45N5O9SF3Re2H2O: C 54.54; H 3.93; N 5.58. Found: C 54.70; H 4.22; N 5.82.
[Re(N^N)(CO)3(py-C6-BOC)][CF3SO3] (N^N = phen (1b), Me4-phen (2b), Ph2-
phen (3b)): A mixture of [Re(N^N)(CO)3(CH3CN)][CF3SO3] (0.13 mmol) and py-
14

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C6-BOC (42 mg, 0.13 mmol) in THF (30 mL) was refluxed under an inert atmosphere
of nitrogen for 12 h. Subsequent recrystallization of the complex from CH2Cl2/diethyl
ether resulted in the formation of the complex as yellow crystals. Complex 1b. Yield:
1
85 mg (71%). H NMR (300 MHz, CD3OD, 298 K):  9.76 (d, 2H, J = 5.1 Hz, H2
and H9 of phen), 8.95 (d, 2H, J = 8.4 Hz, H4 and H7 of phen), 8.65  8.60 (m, 2H, H2
and H6 of pyridine), 8.24  8.15 (m, 5H, H3, H5, H6, and H8 of phen and H4 of
pyridine), 7.39  7.35 (m, 1H, H5 of pyridine), 3.25 (t, 2H, J = 7.2 Hz, py-3-
CONHCH2), 3.00 (t, 2H, J = 6.6 Hz, NHCH2), 1.51  1.30 (m, 17H, CH2(CH2)4CH2
and CH3 of Boc). IR (KBr) /cm–1: 3443 (m, NH), 2031 (s, CO), 1923 (s, CO),


1165 (m, CF3SO3 ), 1031 (m, CF3SO3 ). Positive-ion ESI-MS ion cluster at m/z 772

{M  CF3SO3 }+. Elemental analysis calcd (%) for C33H35N5O9SF3ReH2O: C 42.21;
H 3.97; N 7.46. Found: C 42.46; H 4.24; N 7.34. Complex 2b. Yield: 71 mg (56%).
1H NMR (300 MHz, CD3OD, 298 K):  9.46 (s, 2H, H2 and H9 of Me4-phen), 8.67 
8.61 (m, 2H, H2 and H6 of pyridine), 8.41 (s, 2H, H5 and H6 of Me4-phen), 8.18 (d, J
= 5.7 Hz, H4 of pyridine), 7.38 (t, J = 4.5 Hz, H5 of pyridine), 3.30  3.26 (m, 2H, py-
3-CONHCH2), 3.10  2.98 (m, 2H, NHCH2), 2.91 (s, 6H, CH3 at C4 and C7 of Me4-
phen), 2.77 (s, CH3 at C3 and C8 of Me4-phen), 1.54  1.34 (m, 17H, CH2(CH2)4CH2
and CH3 of Boc). IR (KBr) /cm–1: 3463 (m, NH), 2032 (s, CO), 1919 (s, CO),


1162 (m, CF3SO3 ), 1031 (m, CF3SO3 ). Positive-ion ESI-MS ion cluster at m/z 828

{M

CF3SO3 }+.
Elemental
analysis
calcd
(%)
for
C37H43N5O9SF3ReH2O0.5CH3OH: C 44.55; H 4.69; N 6.93. Found: C 44.69; H 4.97;
1
N 7.29. Complex 3b. Yield: 60 mg (43%). H NMR (300 MHz, CD3OD, 298 K): 
9.81  9.80 (m, 2H, H2 and H9 of Ph2-phen), 8.77  8.73 (m, 2H, H2 and H6 of
pyridine), 8.22  8.18 (m, 5H, H4 of pyridine and H3, H5, H6, and H8 of Ph2-phen),
15

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7.68  7.47 (m, 11H, C6H5 of Ph2-phen and H5 of pyridine), 3.34  3.30 (m, 2H, py-3-
CONHCH2), 3.05  2.98 (m, 2H, NHCH2), 1.53  1.32 (m, 17H, CH2(CH2)4CH2 and
CH3 of Boc). IR (KBr) /cm–1: 3448 (m, NH), 2033 (s, CO), 1920 (s, CO), 1162


(m, CF3SO3 ), 1030 (m, CF3SO3 ). Positive-ion ESI-MS ion cluster at m/z 924 {M 

CF3SO3 }+. Elemental analysis calcd (%) for C45H43N5O9SF3Re3H2O: C 47.95; H
4.38; N 6.21. Found: C 48.17; H 4.33; N 6.56.
4.2. Instrumentation and Methods
1H NMR spectra were recorded on a Varian Mercury 300 MHz NMR spectrometer at
298 K. Positive-ion ESI mass spectra were recorded on a Perkin-Elmer Sciex API 365
mass spectrometer. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR
spectrophotometer. Elemental analyses were carried out on a Vario EL III CHN
elemental analyzer. Electronic absorption and steady-state emission spectra were
recorded on a Hewlett-Packard 8453 diode array spectrophotometer and a SPEX
FluoroLog 3-TCSPC spectrophotometer equipped with a Hamamatsu R928 PMT
detector, respectively. Emission lifetimes were measured in the Fast MCS lifetime
mode with a NanoLED N-375 as the excitation source. All the solutions for
photophysical studies were degassed with at least four successive freeze-pump-thaw
cycles and stored in a 10-cm3 round-bottomed flask equipped with a sidearm 1-cm
fluorescence cuvette and sealed from the atmosphere by a Rotaflo HP6/6 quick-
release Teflon stopper. Luminescence quantum yields were measured by the optically
dilute
method
[56]
using
a
degassed
CH3CN
solution
of
[Re(phen)(CO)3(pyridine)][CF3SO3] (em = 0.18, ex = 355 nm) as the standard
solution [57]. Details on MTT assays and ICP-MS have been reported previously
[33,58].
16

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4.3. Kinetics Studies
Kinetic experiments for the reactions of the DIBO complexes with benzyl azide were
performed under pseudo first-order conditions. The DIBO complexes (20 M) were
mixed in a 1:100, 1:150, 1:200, or 1:250 molar ratio with benzyl azide in MeOH at
298 K and the reactions were monitored by UV-Visible (UV-Vis) spectroscopy at ca.
305 nm. The kobs values were determined by fitting the experimental data to the first-
order rate equation, [A] = [A]oe−kt. The k2 values were determined by plotting kobs
versus [benzyl azide] and the rate constant corresponds to the determined slope.
4.4. Preparation of Azidoacetic Acid
A mixture of iodoacetic acid (1.86 g, 10 mmol) and sodium azide (1.43 g, 22 mmol) in
H2O (25 mL) was stirred slowly in dark at room temperature for 48 h. The mixture
was acidified with 1 M HCl and the solution was extracted with diethyl ether (50 mL
 3). The organic phase was washed with saturated NaHSO3 and then dried over
MgSO4. The solvent was evaporated to dryness yielding azidoacetic acid as a yellow
oil. Yield: 483 mg (48%). 1H NMR (300 MHz, CDCl3, 298 K):  3.96 (s, 2H, CH2).
4.5. Modification of BSA, HSA, and aTf with Azidoacetic Acid
A mixture of the protein (BSA, HSA, or aTf) (0.10 mol), azidoacetic acid (450.0 g,
4.5 mol), EDC (1.4 mg, 9.0 mol), and HOBt (1.2 mg, 9.0 mol) in 50 mM
potassium phosphate buffer at pH 7.4 (500 L) was stirred in the dark at room
temperature for 24 h. The solution was loaded onto a PD-10 size exclusion column.
Volume fractions between 2.5 and 5.0 mL were collected. Finally, the bioconjugates
were washed successively with potassium phosphate buffer using an YM-50 centricon,
concentrated to 1.0 mL, and stored at 4 °C.
17

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4.6. Labeling of Azide-modified Proteins
In a typical reaction, complex 1a (0.21 mg, 0.20 mol) in anhydrous DMSO (20 L)
was added to the azide-modified BSA, HSA, or aTf (0.02 mol) dissolved in 50 mM
potassium phosphate buffer at pH 7.4 (980 L). The solution was stirred in the dark at
room temperature for 24 h. Any precipitate was removed by centrifugation and the
supernatant was loaded onto a PD-10 size exclusion column. The first colored band
was collected and washed successively with potassium phosphate buffer using an YM-
50 centricon, concentrated to 1.0 mL, and stored at 4 °C.
4.7. Live-cell Confocal Microscopy and Bioorthogonal Imaging in CHO Cells
CHO cells in growth medium were seeded on a sterilized coverslip in a 60-mm tissue
culture dish and grown at 37 C under a 5% CO2 atmosphere for 48 h. The culture
medium was then removed and replaced with medium/DMSO (99:1, v/v) containing
complex 1a (5 M). After incubation for 1 h, the medium was removed and the cell
layer was washed gently with PBS (1 mL  3). The coverslip was mounted onto a
sterilized glass slide and then imaged using a Leica TCS SPE confocal microscope. In
the bioorthogonal imaging experiments, the cells were pretreated with Ac4ManNAz
(50 M) in growth medium for 72 h, and finally washed with PBS (1 mL  3).
5. Abbreviations
Ac4ManNAz
aTf
1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine
apo-transferrin
BSA
bovine serum albumin
18

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CHO
Chinese Hamster Ovary
DIBO
dibenzocyclooctyne
HSA
human serum albumin
IL
intraligand
Me4-phen
MLCT
Ph2-phen
phen
3,4,7,8-tetramethyl-1,10-phenanthroline
metal-to-ligand charge-transfer
4,7-diphenyl-1,10-phenanthroline
1,10-phenanthroline
py-C6-BOC
py-C6-DIBO
3-(N-(6-(tert-butoxycarbonylamino)hexyl)aminocarbonyl)pyridine
3-(N-(6-(3,4:7,8-dibenzocyclooctyne-5-
oxycarbonylamino)hexyl)aminocarbonyl)pyridine
strain-promoted alkyne-azide cycloaddition
SPAAC
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1016/j.jinorgbio.2014.xx.xxx
Acknowledgements
We thank the Hong Kong Research Grants Councils (Project No. CityU 102212) and
City University of Hong Kong (Project No. 9667090) for financial support. We thank
Mr. Tat-Ming Wong for his involvement in the early stage of this project. A.W.-T.C.
acknowledges the receipt of a Postgraduate Studentship, a Research Tuition
Scholarship, and a Student Research Excellence Award administered by City
University of Hong Kong.
19

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Table 1
Electronic absorption spectral data of the rhenium(I) polypyridine complexes at 298 K.
abs [nm] ( [M–1 cm–1])
complex solvent

CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
257 sh (30 365), 277 (38 725), 288 sh (34 460), 306 (25 385),
334 sh (5940), 375 sh (4005)
1a
1b
2a
259 sh (31 165), 274 (41 920), 285 sh (38 860), 304 (29 245),
330 sh (6655), 367 sh (3970)
257 sh (27 585), 277 (29 790), 295 sh (16 155), 334 sh (6140),
380 sh (4085)
254 sh (22 710), 274 (26 840), 290 sh (15 775), 325 sh (6225),
368 sh (3560)
254 (33 715), 282 (46 375), 306 (32 055), 328 sh (10 840), 369
sh (4245)
250 (34 945), 286 (46 340), 304 (33 300), 320 sh (12 260), 366
sh (3710)
CH2Cl2
CH3CN
CH2Cl2
251 (30 520), 282 (33 815), 325 sh (12 610), 373 sh (4305)
247 (33 530), 280 (35 020), 313 sh (14 755), 366 sh (3880)
276 sh (44 430), 289 (57 335), 304 sh (46 980), 338 sh (16
265), 384 sh (8970)
2b
3a
CH3CN
264 sh (37 375), 288 (58 395), 303 sh (45 995), 334 sh (16
780), 382 sh (7995)
CH2Cl2
CH3CN
271 sh (34 445), 294 (49 710), 337 sh (20 650), 394 sh (9245)
258 sh (35 105), 291 (51 485), 334 sh (20 495), 386 sh (9295)
3b
25

ACCEPTED MANUSCRIPT
Table 2
Photophysical data of the rhenium(I) polypyridine complexes at 298 K.
complex
solvent

em [nm]
 [s]
1.86
1.24
2.55
1.68
3.03
4.57
9.27
8.27
7.24
4.20
7.19
4.43
em
0.45
0.11
0.63
0.15
0.07
0.03
0.28
0.06
0.30
0.09
0.24
0.09
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
528
1a
545
528
1b
545
492,a 510
485,a 512
490,a 511
486,a 512
542
2a
2b
3a
556
542
3b
556
a Shoulders.
26

ACCEPTED MANUSCRIPT
Table 3
The k2 values for the DIBO complexes upon reaction with benzyl azide in MeOH at
298 K.
k2 [M1 s1]
complex
1a
2a
3a
0.077
0.085
0.091
27

ACCEPTED MANUSCRIPT
Table 4
ESI-mass spectrometry data for the DIBO complexes after the reactions with benzyl
azide in MeOH at 298 K.
complex
formula for
[M  CF3SO3 ]
mass calculated for
mass found for
 +
 +
 +
[M  CF3SO3 ]
[M  CF3SO3 ]
+
ReC51H44N8O6
ReC55H52N8O6
ReC63H52N8O6
1051.3
1107.4
1203.4
1051.5
1a
2a
3a
+
+
1107.6
1203.9
28

ACCEPTED MANUSCRIPT
Table 5
Photophysical data of conjugates 1-BSA, 1-HSA, and 1-aTf in degassed 50 mM
potassium phosphate buffer pH 7.4 at 298 K.
conjugate
em/nm
535
o/s
em
0.96 (82%), 2.75 (18%)
0.91 (77%), 2.11 (23%)
1.04 (77%), 2.83 (23%)
0.089
0.15
1-BSA
1-HSA
1-aTf
534
534
0.087
29