A therapeutically viable photo-activated manganese-based CO-releasing molecule (photo-CO-RM)

Article abstract of DOI:10.1039/c2dt31588b

A new class of photochemically-activated CO-releasing molecule (photo-CO-RM), based on a Mn(CO)₄(C^N) system, is reported in this study. Three CO molecules are released per CO-RM molecule. Complex 3 is a fast releaser, thermally stable in the dark and a viable therapeutic agent.

Full text of DOI:10.1039/c2dt31588b

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COMMUNICATION
A therapeutically viable photo-activated manganese-based CO-releasing
molecule (photo-CO-RM)†
Jonathan S. Ward,a Jason M. Lynam,*a James W. B. Moir,b David E. Sanin,b Adrian P. Mountfordb and
Ian J. S. Fairlamb*a
Received 17th July 2012, Accepted 18th July 2012
DOI: 10.1039/c2dt31588b
therapeutic photo-CO-RMs. Crucially we have determined the
cell viability of the photo-CO-RMs prior to and following
photoirradiation.
Complex 2 was prepared as shown in Scheme 1 in 81% yield
(see ESI† for synthetic and characterisation details).7b Complex
2 crystallises from CH2Cl2/pentane (vapour diffusion), allowing
its structure to be determined by single crystal X-ray diffraction
(Fig. 1).9 Complex 2 was hydrolysed under mild conditions to
afford water compatible complex 3 in 84% yield.
A new class of photochemically-activated CO-releasing
molecule (photo-CO-RM), based on a Mn(CO)4(C^N) system,
is reported in this study. Three CO molecules are released
per CO-RM molecule. Complex 3 is a fast releaser, thermally
stable in the dark and a viable therapeutic agent.
Carbon monoxide (CO) is a well-established biological effector
molecule within the human body, e.g. CO binds and affects the
activity of proteins and receptors.1 Using it in a therapeutic sense
requires the safe and controlled delivery of CO via a suitable
molecular vehicle. Therapeutic metal-carbonyl complexes have
thus been designed as CO-releasing molecules (CO-RMs),
receiving significant attention since about 2003.2,3 There are
three classes of CO-RMs – those which: (i) thermally liberate
CO (thermo-CO-RMs); (ii) liberate CO by a trigger, that is by
hydrolytic (solvent) processes or other species, e.g. proteins
inter alia (triggered-CO-RMs);4 (iii) release CO from dark-and-
thermally-stable metal-carbonyl complex prodrugs under photo-
chemical conditions (photo-CO-RMs).5,6 Each class has their
advantages and disadvantages. Our research group has spent
considerable effort in developing several CO-RM classes that
release CO under physiological conditions, for which their cell
viability has been demonstrated.4c–e,4g,5i,10
CO-release from CO-RMs can be measured by a UV-vis
spectrometric deoxy-Mb → Mb-CO assay,10 with irradiation of
In 2007/8 we initiated studies on a Mn(CO)4(C^N) system
(C^N = ortho-metallated 2-phenylpyridine or benzoquinoline),7
which was found to be photochemically active in the presence of
a 2-electron donor ligand (e.g. DMSO and PPh3). Worthy of
note is Mann’s CO-RM, namely Mn(CO)4(S2CNMeCH2CO2H),8
which releases CO on exposure to myoglobin (Mb) under
thermal conditions. By contrast, the related Mn(CO)4(C^N)
systems are thermally stable both in the dark and presence of
myoglobin/other strong 2-electron donors. In this paper we
detail the synthesis of water compatible complexes 2 and
3 (Scheme 1), their CO-releasing ability and potential as
Fig. 1 X-ray structure of complex 2. Thermal ellipsoids set at 50% and
H-atoms omitted for clarity. Selected bond distances (Å): C1–Mn1
1.8148(14), C2–Mn1 1.8660(14), C3–Mn1 1.8263(14), C4–Mn1
1.8527(14), C5–Mn1 2.0528(13), C1–O1 1.1443(18), C2–O2 1.1330(17),
C3–O3 1.1500(17), C4–O4 1.1381(17), Mn1–N1 2.0672(11). Selected
bond angles (°): C1–Mn1–N1 172.00(5), C3–Mn1–N1 95.30(5),
C5–Mn1–N1 80.08(5). Selected torsion angles (°): Mn1–C5–C10–C11
3.62(14), C5–C10–C11–N1 −2.70(16), C10–C11–N1–Mn1 0.43(14).
aDepartment of Chemistry, University of York, Heslington, York, YO10
5DD, UK. E-mail: jason.lynam@york.ac.uk, ian.fairlamb@york.ac.uk;
Fax: +44 (0)1904 322516; Tel: +44 (0)1904 324091
bCentre for Immunology and Infection, Department of Biology,
University of York, Heslington, York, YO10 5DD, UK
†Electronic supplementary information (ESI) available: Details of all
experimental procedures, characterisation data, kinetic experiments and
biological assays. CCDC reference number 887264. For ESI and crys-
tallographic data in CIF or other electronic format see DOI:
10.1039/c2dt31588b
Scheme 1 Photo-CO-RMs developed in this study; (i) Mn(CO)5(Bn),
toluene, 75 °C, (ii) Et3N (3 eqv.), LiBr (10 eqv.), CH3CN/H2O (98 : 2,
v/v), 2 h, 25 °C.
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Table 1 CO-release ability of complexes
2
and
3
under
photoirradiation
Irradiation source
hv (365 nm), t1/2 (s);
hv (400 nm), t1/2 (s);
conc. (μM)b
Complex
conc. (μM)a
1740; 40
960; 40
2
3
—
—
300; 10
360; 40
a UV Lamp (6 W; the effective power is significantly less). b LED
(2.4 W).
Fig. 3 Left: Photochemical-induced release of CO from complex 3 (c
= 4.81 × 10−5 mol dm−3) in CH3CN using an LED (at 400 nm, 2.4 W)
system monitored by UV-vis spectroscopy over time (total irradiation
time in brackets). Right: Deoxy-Mb → Mb-CO conversion monitored
(Q-bands) by UV-vis spectroscopy over time, with stepwise induced
photochemical irradiation of complex 3 (c = 10 μM) using a LED
(400 nm, 2.4 W).
Fig. 2 Deoxy-Mb → Mb-CO conversion monitored (Q-bands) by UV-
vis spectroscopy over time – photochemical induced release of CO using
an LED (at 400 nm, 2.4 W) from complex 3 (c = 10 and 40 μM). Photo-
chemical activation was at t = 45 min, the LED cycle was on for 2 min
and off for 3 min.
the photo-CO-RMs using a UV-lamp (365 nm). The t1/2 values
for the release of CO from 2 and 3 are listed in Table 1. An
alternative experimental setup was also developed in this study
allowing UV cuvettes to be directly irradiated with an LED
system (at 400 nm; see ESI† for details). The advantage of this
setup is that it is more controlled and convenient to use than a
UV-lamp, typically used in TLC analyses (e.g. power and time
settings can be controlled, and the system is compatible with
96 well plates and Falcon tubes).
Fig. 4 Decomposition of 3 in H2O/CH3CN, followed by ESI-MS
analysis (see ESI† for mass spectra over time).
The Deoxy-Mb → Mb-CO conversion cleanly occurs on
irradiation of 3 (Fig. 2).11
and is most likely diacid 5, formed by a CO migration/insertion
process from 3 via proposed intermediate III. Hydrolysis of III
would be expected to afford II and 5. Interestingly, the CO inser-
tion to give III would explain in part the measurement of three
liberated molecules of CO. It is interesting to note that II is
isostructural with the known Mn(CO)3(scorpionate) photo-
CO-RMs.5b Recent studies by Continuous Wave EPR spectro-
scopy have demonstrated that such complexes decompose to
give paramagnetic MnII species.14 The appearance of a yellow
colour, which occurs on exposure of 3 to light indicates that
paramagnetic species are formed (cf. signal broadening is
observed by 1H NMR spectroscopic analysis).
Having comprehensively established the CO-release behaviour
of 3, its cell viability against RAW 264.7 murine macrophages
(Alamar Blue assay) and its effect on cell lysis (lactate dehydro-
genase (LDH) activity) was determined (Fig. 5). The first assay
measures the ability of living cells to reduce Resazurin, whereas
the latter provides quantification of cell lysis based on the
The CO-release profiles at 3 concentrations of 10 and 40 μM
indicate that approximately three CO ligands are released from 3.
This demonstrates that the degraded species could contain one
remaining CO ligand vide infra. Irradiation of 3 in a stepwise
manner revealed that CO release could be switched on and off.
Again, three CO ligands are released per molecule of 3. In the
absence of protein, irradiation of 3 in CH3CN led to decompo-
sition, showing that CO release is not protein assisted (Fig. 3).
A similar trend is seen in H2O/DMSO (see ESI†).
Investigation of the species produced by photoirradiation of 3
in H2O/CH3CN12 by ESI-MS13 showed that the starting complex
was rapidly converted to I and one equivalent of CO (not quan-
tified) within the first few seconds of irradiation (Fig. 4). The
disappearance of I is noted after 2 min total irradiation time.
The formation of II (and 4) is noted as an intermediate,
which accumulates and then degrades to give uncharacterised
Mn species and CO. A peak at m/z 274 accumulates over time,
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scorpionate-derived,§ and related photo-CO-RMs.5b,f,6 The cell
viability and LDH activity measurements on photo-CO-RM 3
were excellent in the assays used. The finding that the photo-
irradiated species derived from 6 were less toxic than non-
activated 6 was quite remarkable, and moreover, surprising.
Further studies employing the LED photoirradiation equipment
are on-going, as are time-resolved infrared spectroscopic experi-
ments on the manganese(I) carbonyl complexes reported in this
paper.
Fig. 5 Cell viability of RAW 264.7 murine macrophages, as measured
by Alamar blue assay, in the presence of photo-CO-RMs 2, 3 and 6,
prior to and following photoirradiation at 400 nm (LED) (8 min, 2.4 W
in DMSO before addition to cells). Experiments were run in triplicate
against a positive control (100% cell viability) and 1% Triton control
(0% cell viability). Error bars are included (average standard deviation
values).
Acknowledgements
We acknowledge funding from BBSRC for a PhD studentship
(J. S. W.), and thank Dr B. E. Moulton7a (supported by EPSRC),
Ms. O. Rogers and Dr A. K. Duhme-Klair for preliminary
experiments and discussion. We thank Dr A. C. Whitwood for
X-ray diffraction support.
Notes and references
‡Findings detailed in this paper were presented by I.J.S.F. at the 6th
International Symposium on Bioorganometallic Chemistry (ISBOMC),
held in Toronto, Canada, July 8th–12th 2012.
§Whilst it is unlikely that the chelating-scorpionate ligand is displaced
by other 2-electron donor ligands, to generate similar MnI species, we
cannot rule it out.
Fig. 6 Complex 6, a potential degradation product of 2–b.
1 (a) R. Alberto and R. Motterlini, Dalton Trans., 2007, 1651;
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measurement of LDH activity released from the cytosol of dead
cells. Complex 2 and related hydroxy-derivative 6 are included
for comparison (see ESI† for the synthesis and characterisation
of 6). Complex 6 was considered a potential biodegradation
product from either 2 or 3, which could form on cleavage of the
ether bond in biological systems (Fig. 6).
In keeping with previous studies, 2, 3 and 6 were tested over
three concentrations (10, 50 and 100 μM).4 We deemed it appro-
priate to test cell viability prior to and following photoirradia-
tion, which allows the toxicity of the degradation species to also
be determined.
Complex 3 exhibits excellent viability at all the concentrations
tested, no difference was noted with photoirradiation, showing
that the degradation species (formed within 8 min irradiation)
resulting from 3 are viable in this assay. Complex 2 is viable at
10 μM; however following photoirradiation at higher concen-
trations we note that cell viability is reduced (to ca. 80%).
Finally, complex 6 is more toxic at 50 μM < 10% of the cells are
viable. Interestingly, at both 10 and 50 μM the cells are more
viable following photoirradiation of 6.
The results from the LDH assay show that 2 and 3 do not
induce release of LDH at 10, 50 and 100 μM concentrations.
Complex 6 was found to induce the release of LDH at 50 μM
(ca. 63% compared to the Triton control). The data is consistent
with the cell viability assay results. There is no evidence for for-
mation of 6 from either 2 or 3, in the assays tested or ESI-MS
study.
4 (a) K. S. Davidge, G. Sanguinetti, C. H. Yee, A. G. Cox, C. E. McLeod,
C. E. Monk, B. E. Mann, R. Motterlini and R. K. Poole, J. Biol. Chem.,
2008, 284, 4516, and references cited therein (b) D. Scapens, H. Adams,
T. R. Johnson, B. E. Mann, P. Sawle, R. Aqil, T. Perrior and
R. Motterlini, Dalton Trans., 2007, 4962; (c) I. J. S. Fairlamb,
A. K. Duhme-Klair, J. M. Lynam, B. E. Moulton, C. T. O’Brien,
P. Sawle, J. Hammad and R. Motterlini, Bioorg. Med. Chem. Lett., 2006,
16, 995; (d) I. J. S. Fairlamb, J. M. Lynam, B. E. Moulton, I. E. Taylor,
A. K. Duhme-Klair, P. Sawle and R. Motterlini, Dalton Trans., 2007,
3603; (e) A. J. Atkin, S. Williams, P. Sawle, R. Motterlini, J. M. Lynam
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I. J. S. Fairlamb and J. M. Lynam, Dalton Trans., 2009, 4351.
5 (a) H. W. Peindy N’Dongo, I. Ott, R. Gust and U. Schatzschneider,
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U. Schatzschneider, R. Gust, I. Ott, L. A. Onambele, A. Prokop and
I. Neundorf, Bioconjugate Chem., 2010, 21, 1288; (d) K. Splith,
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J. M. Lynam, Organometallics, 2011, 30, 4643. See also ref. 6 for earlier
references.
In conclusion‡ we have identified two biologically compatible
photo-CO-RMs 2 and 3. Their CO-release profiles compare well
against other reported photo-CO-RMs. Studies by ESI-MS indi-
6 Key review: U. Schatzschneider, Inorg. Chim. Acta, 2011, 374, 19.
7 (a) B. E. Moulton, PhD thesis, University of York, 2008; (b) M. I. Bruce,
B. L. Goodall and I. Matsuda, Aust. J. Chem., 1975, 28, 1259.
8 S. H. Crook, B. E. Mann, A. J. Meijer, H. Adams, P. Sawle, D. Scapens
and R. Motterlini, Dalton Trans., 2011, 40, 4230.
+
cate that an intermediate, Mn(CO)3(NCCH3)3 , is formed on
photoirradiation of 3, which is an isostructural variant of
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9 Crystal Data for 2. C18H12NO7Mn, M = 409.23, triclinic, a = 7.0000(4)
Å, b = 11.2458(6) Å, c = 12.1356(8) Å, α = 67.181(6)°, β = 83.042(5)°,
γ = 86.381(4)°, V = 873.93(9) Å , T = 110.00(10), space group P1
(no. 2), Z = 2, μ(Mo Kα) = 0.797, 9348 reflections measured, 5500
unique (Rint = 0.0247) which were used in all calculations. The final wR2
was 0.0831 (all data) and R1 was 0.0322 (>2sigma(I)).
10 A. J. Atkin, J. M. Lynam, B. E. Moulton, P. Sawle, R. Motterlini,
N. M. Boyle, M. T. Pryce and I. J. S. Fairlamb, Dalton Trans., 2011, 40,
5755.
11 The photo-CO-RMs do not release CO in the presence of myoglobin and
sodium dithionite until photoirradiation is initiated. A recent paper indi-
cates that sodium dithionite can initiate CO-release from some CO-RMs,
see: S. McLean, B. E. Mann and R. K. Poole, Anal. Biochem., 2012, 427,
36.
3
ˉ
12 It was necessary to substitute DMSO with CH3CN (i.e. a similar 2-elec-
tron donor ligand), as the former was found to be incompatible with
ESI-MS analysis with (DMSO)2H+ saturating the detector).
13 It was necessary to reduce the skimmer voltage which diminishes M-CO
fragmentation (see ESI† for mass spectra and optimisation). In all the MS
experiments conducted the ion peak at m/z 230 appears, which could be
formed by an ionisation process, in addition to degradation induced by
photoirradiation. The ion peaks at m/z 274 and m/z 409 are real intermedi-
ate species (in solution).
14 H.-M. Berends and P. Kurz, Inorg. Chim. Acta, 2012, 380, 141.
This journal is © The Royal Society of Chemistry 2012
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