3-Chloromethylpyridyl bipyridine fac-tricarbonyl rhenium: A thiol-reactive luminophore for fluorescence microscopy accumulates in mitochondria

Article abstract of DOI:10.1039/b802215a

The 3-chloromethylpyridyl bipyridine fac tricarbonyl rhenium cation is a thiol-reactive luminescent agent with a long luminescence lifetime and large Stokes shift that is demonstrated by co-localisation studies to accumulate in mitochondria. This represents the first application of a ³MLCT luminescent agent for the specific targeting of a biological entity in imaging. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/b802215a

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LETTER
www.rsc.org/njc | New Journal of Chemistry
3-Chloromethylpyridyl bipyridine fac-tricarbonyl rhenium:
a thiol-reactive luminophore for fluorescence microscopy accumulates in
mitochondriawz
Angelo J. Amoroso,a Richard J. Arthur,a Michael P. Coogan,*a
b
a
c
´
Jonathan B. Court, Vanesa Fernandez-Moreira, Anthony J. Hayes,
David Lloyd,b Coralie Milletb and Simon J. A. Popea
Received (in Montpellier, France) 8th February 2008, Accepted 18th April 2008
First published as an Advance Article on the web 23rd May 2008
DOI: 10.1039/b802215a
The 3-chloromethylpyridyl bipyridine fac tricarbonyl rhenium
cation is a thiol-reactive luminescent agent with a long lumines-
cence lifetime and large Stokes shift that is demonstrated by co-
localisation studies to accumulate in mitochondria. This repre-
sents the first application of a 3MLCT luminescent agent for the
specific targeting of a biological entity in imaging.
A rhenium complex functionalised with a chloromethyl unit
thus has the potential to provide a thiol-reactive, possibly
mitochondria-selective, probe with a long lifetime and a large
Stokes shift, with clear potential for biological imaging. There
have been previous reports5 of rhenium and ruthenium bisi-
mine complexes that are either thiol reactive or contain other
species, which could localise due to biological interactions. All
have long lifetimes and large Stokes shifts, making them
attractive candidates for biological imaging applications. A
rhenium quinoline complex5b has previously been bioconju-
gated to a peptide, and its localisation has been shown by
fluorescence microscopy to be identical to that of a fluorescein-
tagged peptide.
Following previous work,4a in which lipophilicising units
were incorporated into the axial pyridine of these rhenium
complexes, the fac-3-chloromethylpyridine rhenium tricarbo-
nyl bipyridyl complex (1) was chosen as a representative
chloromethyl rhenium fluorophore to study.
Traditional routes3 to rhenium tricarbonyl bisimines involve
the reaction of a bisimine (e.g. bipy, phen) with chlororhenium
pentacarbonyl, followed by activation of the resultant tricar-
bonyl chlorobisimine rhenium species, 2, by chloride abstrac-
tion and finally substitution with a ligand (typically phosphine
or pyridine) to give the cationic complexes that have the most
useful photophysics. Routes to chloromethylated species are
typically based around either the incorporation of a chloro-
methyl group into a prefabricated component of the molecule
or by the chlorination of an alcohol. As conditions for the
chlorination of alcohols are often harsh, incorporating the pre-
existing chloromethyl group of 3-chloromethylpyridine was
chosen. However, treatment of the activated intermediate fac-
[Re(CO)3(bpy)(MeCN)][BF4] (3) with 3-chloromethylpyridine
hydrochloride in the presence of base (triethylamine) gave not
the pyridine complex, but instead the precursor material fac-
[Re(CO)3(bpy)Cl][BF4] (2). Inclusion of silver tetrafluoro-
borate in the reaction mixture in an attempt to precipitate
the chloride, and thus prevent it from taking part in the
reaction, was unsuccessful, and again chloro complex 2 was
recovered. An attempt to provide a chloride-free chloromethyl
pyridine by treating the hydrochloride salt with silver tetra-
fluoroborate in a precipitative salt metathesis was again un-
successful, with a very slow reaction of the [Py-3-CH2Cl]ꢀ
HBF4/triethylamine system with 3 giving a very low yield of
Mitochondria, named from the Greek mitoB (thread) and
koudrion (grain), are believed to have their origin in a
symbiotic relationship between early eukaryotes and archaic
bacteria that were capable of performing roles unavailable to
the host cells.1a,b Mitochondria generate most of a cell’s energy
by ATP synthesis, and have many other important roles in
signalling, differentiation, cell division cycles and cell death.1c,d
As such, mitochondria are an important target for biological
investigation, and a wide variety of probes for their imaging
have been developed. One of the most important classes of
mitochondrial probe are the MitoTrackert probes,2 mem-
brane permeable thiol reactive dyes containing a chloromethyl
group that specifically stain mitochondria. Rhenium tricarbo-
nyl bisimine complexes are well know as triplet metal-to-
ligand charge transfer (3MLCT) luminescent materials with
useful photophysical properties, including large Stokes shifts,
long lifetimes and good quantum yields,3 that have long been
postulated but only recently demonstrated4 to have applica-
tions in biological imaging as fluorochromes in fluorescence
microscopy. These three advantages allow easy differentiation
of their emission from interfering autofluorescence.
a Department of Chemistry, Cardiff University, Cardiff, Wales, UK
CF10 3AT. E-mail: cooganmp@cf.ac.uk;
Fax: +44 (0)2920 874030; Tel: +44 (0)2920 874066
b Cardiff School of Biosciences, Main Building, Museum Avenue,
Cardiff, Wales, UK CF10 3TL
c Confocal Microscopy Unit, Cardiff School of Biosciences, Life
Sciences Building, Cardiff, Wales, UK CF10 3US
w Electronic supplementary information (ESI) available: Co-localisa-
tion scatter plots. See DOI: 10.1039/b802215a
z Crystallographic data for 1: C19H14ClF6N3O3PRe, monoclinic, P21/
c, a = 13.7391(4), b = 11.5590(4), c = 14.6082(5) A, b = 100.743(2)1,
Z = 4, T = 150 K, m = 5.476 mmꢁ1, l = 0.71069 A (Mo-Ka), F(000)
= 1268, 2.96 o y o 27.45, 5171 reflections, 3737 unique [Rint
=
0.1529], R1 = 0.0503, wR2 = 0.1094 [I 4 2s(I)], R1 = 0.0798, wR2
= 0.1219 (all data). CCDC 685598. For crystallographic data in CIF
or other electronic format, see DOI: 10.1039/b802215a.
ꢂc
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Scheme 1 Attempted synthesis of 1.
2. The chloride ion source for the production of 2 must be by
displacement of chloride from the chloromethyl group, and it
is surprising that this is faster than substitution of the axial
acetonitrile of 3. Given the difficulty of converting 3 to 2, an
alternative approach was attempted via tosylation of the
analogous hydroxymethylpyridine complex [Re(CO)3(bpy)(3-
Py-CH2OH)][BF4] (4). However, a reaction with tosyl chloride
gave not a 3-tosyloxymethylpyridine complex but, again,
chloro complex 2 (Scheme 1). This entirely unexpected result
can only be rationalised in terms of an activation of the
pyridine ligand towards displacement during the reaction with
tosyl chloride, as the starting material is stable towards solu-
tions containing chloride (a concerted tosylation–substitution
is unlikely for a 3-hydroxymethylpyridine ligand). Finally,
following a recent report6 on the bromination of a similar
system with thionyl bromide, the hydroxy–chloride conversion
was successfully realised with thionyl chloride in DCM in 78%
yield, after aqueous work-up and precipitation of the product
as a hexafluorophosphate salt (Scheme 2).
Fig. 1 ORTEP representation of 1 (50% probabilities).
Fig. 2 Close contacts in 1.
with the mixed N,S-nucleophile cysteine methyl ester, exclu-
sive S-alkylation was observed (Scheme 3), as demonstrated
by the typical S-methylene shift of the 3-picolyl group in the
13C NMR spectrum (d 35.2), to give 6 in good yield (65%). In
order to demonstrate reactivity with more complex biologi-
cally-relevant thiols, chloromethyl complex 1 was treated with
reduced glutathione (GSH) in a methanol–water mixture at
pH 9 (Scheme 4). The formation of a glutathione–rhenium
conjugate, 7, was indicated by the formation of a water soluble
fluorescent complex, which was separated from unreacted 1 by
evaporation to dryness, dissolution in water and filtration,
before final purification by ion exchange chromatography
In order to determine that the required complex had finally
been synthesised, crystals that were suitable for X-ray analysis
were grown by slow evaporation of a chloroform solution.z
The structure of this species showed a fac-geometry of the
three carbonyl ligands, bipyridine describing the equatorial
plane and an axial chloromethylpyridine ligand completing the
octahedral geometry (Fig. 1), with two pairs of mutual Cl–C
close contacts (3.333 A) between the chlorines of one complex
and the carbonyl carbons of an adjacent complex (Fig. 2).
Chloromethyl complex 1 showed the typical photophysical
properties of a cationic rhenium tricarbonyl bisimine;1 excita-
tion at lmax 364 nm, emission at lmax 551 nm and a fluores-
+
(Amberlite IR120 resin, NH4 form). Its structure was con-
firmed by HRMS. No reaction of 1 with water was observed,
and it appeared to be hydrolytically stable under mild condi-
tions for many hours.
cence lifetime of
t = 131 ns. Having synthesised a
The thiol adducts 6 and 7 showed the typical photophysical
properties of a cationic rhenium tricarbonyl bisimine;1 excita-
tion at lmax 6 360 nm and 7 361 nm, emission at lmax 6 550 nm,
7 551 nm, and lifetimes of t = 6 122 ns, 7 116 ns. The
photophysical properties of the glutathione adduct 7 were
particularly relevant as it is likely that this would be a major
species produced from 1 in a mitochondrion.
chloromethylpyridine-substituted rhenium complex with a
(reasonably) long fluorescence lifetime and a large Stokes
shift, a variety of reactions with biologically-relevant potential
nucleophiles were then undertaken to see whether the desired
thiol specificity had been replicated. Complex 1 showed no
reactivity with the amine groups of simple amino acids (or
their esters) under mild, physiologically-relevant conditions,
or towards the N-terminus of simple peptides. In a reaction
Scheme 2 Synthesis of 1.
Scheme 3 Reaction of 1 with cysteine.
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greater than that of the plasma membrane, electrophoretic
uptake through each set of membranes should lead to high
concentrations of cationic lipophilic species in the mitochon-
dria, and thiol reactivity should then lead to immobilisation
due to reaction with reduced thiols in the mitochondria.
While it seemed likely that 1 was accumulating in mitochon-
dria, in order to prove this, studies were undertaken to show
that localisation was in the same organelles as those of a
known mitochondrial fluorescent probe. Although 1 had been
designed to mimic MitoTrackert dyes, it would be tautologi-
cal to use a chloromethyl dye to demonstrate the localisation
of another chloromethyl species, so instead, another dye,
TMRE, lacking this functional group but known to accumu-
late in mitochondria,9 was chosen. The patterns of localisation
(Fig. 5(a)–(c)) seen with TMRE and 1 under the same incuba-
tion conditions were essentially identical, with Fig. 4(a) and
Fig. 5(a) illustrating the ‘grainy’ nature of the organelles, and
Fig. 4(b)/(c) and Fig. 5(b)/(c) the similar locality within the
membrane, but in the periphery of the cytosol. Finally, co-
localisation experiments were attempted, in which 1 and
TMRE were incubated with the same cells in order to demon-
strate that they were localising in the same organelles. As the
emission wavelengths of TMRE and 1 are very close (588 and
551 nm, respectively), it was not possible to distinguish the
patterns of localisation of each fluorophore from the emission
characteristics, and instead the different excitation maxima
(555 and 364 nm, respectively) were utilised to allow the
localisation patterns of each fluorophore to be determined.
Using 405 nm laser excitation, only emission from 1 was
observed, whereas using 543 nm excitation, only emission
from TMRE was observed, and thus the large difference in
Stokes shift between these fluorophores, which emit essentially
indistinguishable light, allowed the differentiation of their
localisation. While it is trivial to demonstrate in vitro that 1
(or its thiol derivatives) will not display significant emission
when excited at 543 nm, and that, conversely, TMRE will not
emit when excited at 405 nm, it would only be by the
observation of minor differences in the pattern of localisation
for each fluorophore that this differential excitation technique
could be validated. While it is impractical to demonstrate
exactly the nature of the species present in the mitochondria,
the observed luminescence is compatible with that of the
glutathione adduct 7 (although 1 and all its derivatives have
similar photophysical properties). Observation of the fluores-
cence patterns in the population treated with both 1 and
TMRE showed that the pattern of emission observed from
exciting at both wavelengths was essentially identical (Fig. 6
and Fig. 7), indicating either that co-localisation had occurred
Scheme 4 Reaction of 1 with glutathione.
With confirmation that 1 represents a potentially useful
thiol-reactive luminescent dye, imaging experiments in
Saccharomyces Cerevisiae IFO 0233 (yeast) cells were carried
out, as yeast is a well characterised organism and the sites of
localisation of the rhenium dyes might be identifiable with
reference to the literature. Yeast cells were incubated in
standard growth media after treatment with a DMSO solution
of 1, then fixed and examined by confocal microscopy. While
the dye was clearly accumulated in a proportion of the cells,
the general level of uptake was poor (Fig. 3(a)), but interest-
ingly it appeared that the cells which had incorporated the
fluorophore were in the process of budding (Fig. 3(b) and (c)).
While a dye which is only taken up by cells at a certain stage of
the life cycle is possibly of limited use it was interesting to note
that in cells which had taken up 1 at low levels, localisation
(Fig. 3(d)) had occurred in specific organelles, which are likely
to be mitochondria, given the precedent for the localisation of
cationic thiol reactive dyes.
Yeast cells are unlike mammalian cells in that they possess a
rigid cell wall7 that may slow or prevent the permeation of 1,
and thus further studies were undertaken using cultured
mammalian cells. Human mammary adenocarcinoma cells
(MCF-7)8 were incubated with 1, and the uptake and localisa-
tion were again studied by confocal fluorescence microscopy.
In this case, the entire population showed a good uptake of 1,
with no evidence of toxicity, as the cell population evidently
maintained a healthy membrane potential, concentrating
cationic complex 1 on the cytostolic side of the limiting plasma
membrane, visualised by superimposition of the fluorescence
image on a transmitted light image, obtained using Nomarski
differential interference contrast optics. Furthermore, localisa-
tion in specific organelles was evident (Fig. 4(a)–(c)). These are
believed to be mitochondria, since the cationic, lipophilic,
thiol-reactive nature of 1 would be expected to lead to high
levels being localised in mitochondria. Because the transmem-
brane potential of the inner mitochondrial membrane1e is
Fig. 3 Confocal fluorescence microscopy images of 1 incubated with yeast cells.
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Fig. 4 Confocal fluorescence microscopy images of 1 incubated with MCF-7 cells.
Fig. 5 Confocal fluorescence microscopy images of TMRE incubated with MCF-7 cells.
or that the technique was flawed and could not distinguish
between the two fluorophores.
We thank the EPSRC National Mass Spectrometry Service
Centre, Swansea for mass spectra of the labile rhenium
carbonyl complexes and the EPSRC LSI (EP/D080401/1) for
financial support.
Careful examination of the images, however, identified a few
cells in which preferential uptake of 1 had occurred. In these
cases, excitation at 405 nm led to fluorescence, while excitation
at 543 nm did not (Fig. 8). These figures are shown with no
transmitted light image for clarity, and with the observed
emission obtained at the different excitation wavelengths
illustrated in blue for 405 nm and red for 543 nm, combined
with an overlayed image. Thus, in the overlayed image, red or
blue indicate non-coincidence of localisation and pink or
purple indicate co-localisation. Except for a single cell in
Fig. 8, all the cells studied showed predominantly pink-to-
purple overlays, and the cross-sections of intensity (see, e.g.,
Fig. 7(c)) showed good agreement with this visual inspection.
While no statistical pixel analysis was undertaken (scatter
plots are included in the ESIw), it is beyond reasonable doubt
that the images obtained are characteristic of the co-localisa-
tion of 1 and TMRE. They therefore demonstrate that 1 is a
mitochondrion-selective 3MLCT rhenium dye, a 3MLCT com-
plex that is designed for a specific biological target, and one
that may have a promising future for 3MLCT biological
imaging agents.
Experimental
General
All starting materials, reagents and solvents were purchased
from commercial suppliers and used as supplied, unless other-
wise stated. NMR spectra were recorded on a Bruker DPX 400
at 400 MHz for proton and 60 MHz for carbon, unless
otherwise reported. IR spectra were recorded on a Perkin-
Elmer 1600 FT IR as thin films or Nujol mulls, and are
reported in wavenumbers. Mass spectra were recorded on a
VG Fisons Platform II instrument or at the EPSRC National
Mass Spectrometry Service in Swansea (HRMS). Elemental
analyses were performed by Warwick Analytical Services. All
photophysical data were obtained on a HORIBA Jobin Yvon
Fluorolog spectrometer fitted with a JY TBX picosecond
photodetection module and a Hamamatsu R5509-73 detector
(cooled to ꢁ80 1C using a C9940 housing). The pulsed laser
Fig. 6 Co-localisation of 1 and TMRE in MCF-7: (a) exciting at 405, (b) exciting at 543, (c) superimposed.
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Fig. 7 Colocalisation of 1 and TMRE in MCF-7 close up and cross section.
Fig. 8 Colocalisation of 1 and TMRE in MCF7 showing single uptake in one (bottom) cell.
source was a Continuum Minilite Nd:YAG configured for 355
nm output. Lifetimes were obtained using a HORIBA Jobin
Yvon FluoroHub single photon counting module. Solvents
were purified according to ref. 10. 4 was prepared according to
ref. 4a.
concentration 20 nM) for 2 h at room temperature, before
being washed three times in PBS, mounted and viewed as
above by confocal microscopy.
Co-localisation experiments. MCF-7 adenocarcinoma cells
were harvested as indicated above, resuspended in EMEM
culture medium and incubated with both TMRE (final con-
centration 100 nM) and 1 (final concentration 20 mg mlꢁ1) for
1.5 h at room temperature, before being washed three times in
PBS, mounted and viewed as above by confocal microscopy.
Images were recorded in each case with excitation at either 405
or 543 nm and detection between 500–600 nm.
Cell culture and imaging
Yeast cell incubation with 1. Saccharomyces cerevisiae IFO
0233 cells were harvested by centrifugation (1000 g, 3 min),
washed twice in phosphate buffer saline (PBS, pH 7.4), before
being resuspended in PBS, incubated with 1 (final concentra-
tion 100 mg mlꢁ1) for 30 min in 0.3% yeast extract, 1%
peptone and 1% glucose, rinsed in PBS (X3) and mounted
on a slide. Preparations were viewed using a Leica TCS SP2
AOBS confocal scanning laser microscope using a ꢃ63 or
ꢃ100 objective, with excitation at 405 nm and detection at
520–570 nm.
fac-[Re(bpy)(CO)3PyCH2Cl](PF6) (1)
A solution of fac-[Re(bpy)(CO)3PyCH2OH](BF4)4a (150 mg,
0.2 mmol) in 3 ml of thionyl chloride was stirred under a
nitrogen atmosphere overnight. The mixture was cooled in an
ice bath and a saturated aqueous NH4PF6 solution was added
very slowly, dropwise, with extremely rapid stirring until a
yellow solid precipitate formed. The precipitate was separated
from the mixture of solvents by filtration and washed several
times with water to give 1 as a yellow solid (132 mg, 78.3%
yield), m.p. 180 1C. dH (CD3CN) 4.58 (2H, s, CH2), 7.33 (1H,
m, PyH5), 7.82 (2H, m, bpyH5,50), 7.91 (1H, d, J = 4.5 Hz,
PyH4), 8.20 (1H, d, J = 5.5 Hz, PyH6), 8.32 (3H, m, PyH2
and bpyH4,40), 8.42 (2H, d, J = 6.4 Hz, bpyH3,30) and 9.18
(2H, d, J = 5.4 Hz, bpyH6,60). dC (CD3CN) 41.1 (CH2), 124.2
(2C(5,50), bipy), 126.1 (1C(5), py), 128.3 (2C(3,30), bipy), 136.6
(1C(3), py), 139.0 (1C(4), py), 140.7 (2C(4,40), bipy), 151.0,
151.1 (1C(2), py, 1C(6), py), 153.4 (2C(6,60), bipy), 155.2
Human cell incubation with 1. Human mammary adenocar-
cinoma cells (MCF-7),8 obtained from the European Collec-
tion of Cell Cultures, Porton Down, Wiltshire, UK, were
maintained as adherent cultures in Eagle’s Minimum Essential
Medium (EMEM) supplemented with 10% foetal bovine
serum, penicillin and streptomycin. Cells were detached from
the plastic flask using trypsin and EDTA, and were suspended
in an excess volume of growth medium. While suspended in
this medium, cells were incubated with 1 (final concentration
100 mg mlꢁ1) over ice for 30 min. Cells were then washed three
times in PBS, harvested by centrifugation (3 min, 800 g),
mounted on a slide and viewed as above by confocal micro-
scopy.
(2C(2,20), bipy), 191.2 (1C, COapical
) and 195.0 (2C,
COequatorial). nmax (Nujol): 2027s, 1922sb and 1895sb (CO).
m/z (ESI) 553.9 [MH]+ and 427.0 [M ꢁ PF6 ꢁ PyCH2Cl]+.
Theoretical isotope pattern 552.0 (52%), 553.0 (11%), 554.0
(100%), 550.0 (22%), 556.0 (31%) and 557.0 (8%); observed
Human cell incubation with TMRE. MCF-7 adenocarcino-
ma cells were harvested as indicated above, resuspended in
EMEM culture medium and incubated with TMRE (final
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isotope pattern 551.9 (50%), 552.9 (11%), 553.9 (100%), 554.9
(22%), 556.0 (30%) and 557.0 (5%). HRMS (ESI) calculated
[M ꢁ PF6]+ = 552.0248; measured [M ꢁ PF6]+ = 552.0252.
ReC19H14O3N3ClPF6 requires C, 32.65; H, 2.02; N, 6.01;
found C, 32.46; H, 1.92; N, 5.93%.
(2C(3,30), bipy), 137.6 (1C(3), py), 140.3 (1C(4), py), 141.2
(2C(4,40), bipy), 150.9 (1C(6), py), 151.7 (1C(2), py)), 153.9
(2C(6, 60), bipy), 157.0 (2C(2,20), bipy), 171.6, 174.2, 174.9,
176.4 (4C(2COOH, 2CON), 192.0 (1C, COapical) and 196.0
(2C, COequatorial). nmax (CH3CN): 2022s, 1915sb and 1897sb
(CO). m/z (ESI) 825.1 [MH]+ and 427.0 [M ꢁ PF6
ꢁ
fac-[Re(bpy)(CO)3PyCH2SCH2CH(NH2)COOMe](PF6) (6)
PyCH2SCH2CH[NHCO(CH2)2CH(NH2)COOH]CONHCH2-
COOH]+. Theoretical isotope pattern 823.1 (54%), 824.1
(21%), 825.1 (100%), 826.1 (36%) and 827.1 (11%). Actual
isotope pattern 823.1 (58%), 824.1 (20%), 825.1 (100%), 826.1
(34%) and 827.1 (12%). HRMS (ESI) calculated [M ꢁ PF6]+
= 823.1319; measured [M ꢁ PF6]+ = 823.1333.
L-Cystine methyl ester (59 mg, 0.4 mmol) was added to a de-
gassed solution of 1 (50 mg, 0.07 mmol) and triethylamine
(0.04 ml, 0.3 mmol) in 10 ml of acetonitrile. After stirring for 1
h under a flow of nitrogen, the solvent was removed and the
dark solid remaining was washed with water (2 ꢃ 2 ml) to
afford 6 as a dark yellow solid (44 mg, 65.4% yield), m.p. 185
1C. dH (CD3CN) 2.22–2.39 (2H, m, CH2(Cys)), 3.39 (1H, m,
CH), 3.50 (2H, s, CH2), 7.18 (1H, m, PyH5), 7.70–7.75 (3H,
m, PyH5 and bpyH5,50), 8.03 (1H, d, J = 5.5 Hz, PyH6), 8.09
(1H, s, PyH2), 8.19 (2H, m, bpyH4,40), 8.28 (2H, d, J = 8.1
Hz, bpyH3,30) and 9.16 (2H, d, J = 5.3 Hz, bpyH6,60). dC
(CD3CN) 32.0 (CH2(Cys)), 35.2 (CH2), 51.5 (CH3), 54.2 (CH),
124.5 (2C(5,50), bipy), 126.2 (1C(5), py), 128.7 (2C(3,30), bipy),
138.0 (1C(3), py), 140.1 (1C(4), py), 141.0 (2C(4,40), bipy),
150.3 (1C(6), py), 151.6 (1C(2), py), 153.8 (2C(6,60), bipy),
155.6 (2C(2,20), bipy), 192.3 (1C, COapical) and 195.3 (2C,
COequatorial). nmax (CH3CN): 2036s and 1931sb (CO). m/z
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(ESI) 653.1 [MH]+ and 427.0 [M
ꢁ
PF6
ꢁ
PyCH2SCH2CH(NH2)COOMe]+. Theoretical isotope pattern
651.1 (55%), 652.1 (18%), 653.1 (100%), 654.1 (29%) and
655.1 (10%), observed isotope pattern 651.1 (58%), 652.1
(16%), 653.1 (100%), 654.1 (27%) and 655.1 (9%). HRMS
(ESI) calculated [M
ꢁ
PF6]+
= 653.0863; measured
[M ꢁ PF6]+ = 653.0868.
fac-[Re(bpy)(CO)3Py-3-CH2SCH2CH
[NHCO(CH2)2CH(NH2)COOH]CONHCH2COOꢁ] (7)
L-Glutathione (44 mg, 0.14 mmol) was added to a yellow
suspension of 1 (50 mg, 0.07 mmol), and triethylamine (0.04
ml, 0.28 mmol) introduced in 15 ml of de-gassed methanol.
Next, 3 ml of de-gassed water was added to the suspension,
affording a dark solution, from which a dark precipitate
appeared after few minutes of stirring at 40 1C. The mixture
of solvents was removed under vacuum and the dark solid
remaining was dissolved in water, from which the impurities
were filtered off. It was then dissolved in the smallest possible
volume of water and passed through an ion exchange column
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+
(Amberlite IR120 H resin, NH4 form) that had previously
been washed with aqueous ammonium chloride, eluting with
water. The yellow coloured fraction that eluted immediately
was collected, evaporated and dried under vacuum to give 7 as
a yellow solid (26 mg, 43.7% yield), m.p. 168 1C. dH (D2O)
1.98 (2H, m, CH2(11)), 2.23–2.32 (4H, m, CH2(6), CH2(10)),
3.62 (5H, m, PyCH2,CH2(2), CH(12)), 4.10 (1H, m, CH(3)),
7.08 (1H, t, J = 7.7 Hz, PyH5), 7.62–7.69 (3H, m, PyH4,
bpyH5,50), 8.08–8.10 (4H, m, bpyH4,40, PyH(6,2)), 8.22 (2H,
d, J = 8.2 Hz, bpyH3,30) and 9.17 (2H, d, J = 5.5 Hz,
bpyH6,60). dC (D2O) 26.4 (1C(10)), 31.6 (1C (11)), 32.0
(1C(3)), 38.9 (CH2(glu)), 43.5 (1C(2)), 52.6 (1C(12)), 54.3
(1C(6)), 124.6 (2C(5,50), bipy), 126.5 (1C(5), py), 128.7
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1102 | New J. Chem., 2008, 32, 1097–1102