Osmium(II) Tethered Half-Sandwich Complexes: pH-dependent Aqueous Speciation and Transfer Hydrogenation in Cells

Abstract

Aquation is often acknowledged as a necessary step for metallodrug activity inside the cell. Hemilabile ligands can be used for reversible metallodrug activation. We report a new family of osmium(ii) arene complexes of formula [Os(η6-C6H5(CH2)3OH)(XY)Cl]+/0 (1-13) bearing the hemilabile η6-bound arene 3-phenylpropanol, where XY is a neutral N,N or an anionic N,O- bidentate chelating ligand. Os-Cl bond cleavage in water leads to the formation of the hydroxido/aqua adduct, Os-OH(H). In spite of being considered inert, the hydroxido adduct unexpectedly triggers rapid tether ring formation by attachment of the pendant alcohol-oxygen to the osmium centre, resulting in the alkoxy tethered complex [Os(η6-arene-O-κ1)(XY)] n+. Complexes 1C-13C of formula [Os(η6:κ1-C6H5(CH2)3OH/O)(XY)]+ are fully characterised, including the X-ray structure of cation 3C. Tether-ring formation is reversible and pH dependent. Osmium complexes bearing picolinate N,O-chelates (9-12) catalyse the hydrogenation of pyruvate to lactate. Intracellular lactate production upon co-incubation of complex 11 (XY = 4-Me-picolinate) with formate has been quantified inside MDA-MB-231 and MCF7 breast cancer cells. The tether Os-arene complexes presented here can be exploited for the intracellular conversion of metabolites that are essential in the intricate metabolism of the cancer cell.

Full text

Chemical
Science
rsc.li/chemical-science
Volume 12
Number 27
21 July 2021
Pages 9249–9562
ISSN 2041-6539
EDGE ARTICLE
Ana M. Pizarro et al.
Osmium(II) tethered half-sandwich complexes:
pH-dependent aqueous speciation and transfer
hydrogenation in cells

Osmium(II) tethered half-sandwich complexes: pH-
dependent aqueous speciation and transfer
hydrogenation in cells†
Sonia Infante-Tadeo,
a
Vanessa Rodr´
ıguez-Fanjul,
a
Abraha Habtemariam
ab
and Ana M. Pizarro *
ac
Aquation is often acknowledged as a necessary step for metallodrug activity inside the cell. Hemilabile
ligands can be used for reversible metallodrug activation. We report a new family of osmium(II) arene
complexes of formula [Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(XY)Cl]
+/0
(1–13) bearing the hemilabile h
6
-bound arene 3-
phenylpropanol, where XY is a neutral N,N or an anionic N,O

bidentate chelating ligand. Os–Cl bond
cleavage in water leads to the formation of the hydroxido/aqua adduct, Os–OH(H). In spite of being
considered inert, the hydroxido adduct unexpectedly triggers rapid tether ring formation by attachment
of the pendant alcohol–oxygen to the osmium centre, resulting in the alkoxy tethered complex [Os(h
6
-
arene-O-k
1
)(XY)]
n+
. Complexes 1C–13C of formula [Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH/O)(XY)]
+
are fully
characterised, including the X-ray structure of cation 3C. Tether-ring formation is reversible and pH
dependent. Osmium complexes bearing picolinate N,O-chelates (9–12) catalyse the hydrogenation of
pyruvate to lactate. Intracellular lactate production upon co-incubation of complex 11 (XY ¼4-Me-
picolinate) with formate has been quantified inside MDA-MB-231 and MCF7 breast cancer cells. The
tether Os–arene complexes presented here can be exploited for the intracellular conversion of
metabolites that are essential in the intricate metabolism of the cancer cell.
Introduction
Cisplatin (cis-diamminedichloridoplatinum(II)) is one of the
most well-known anticancer drugs due to its widespread use in
the clinic. Its intracellular mechanism of action is believed to
involve Pt–Cl hydrolysis prior to target interaction.
1,2
Following
the success of Pt drugs, a number of new organometallic coor-
dination compounds with labile chlorido ligands have been
synthesized and tested for anticancer activity. New complexes of
Pt,
3,4
Au,
5
Ru,
6–8
Re,
9
Ir,
10,11
and Os
12–14
have been reported to be
highly cytotoxic, even circumventing cisplatin-resistance.
Additionally, a search for control over metallodrug activation
has resulted in a variety of interesting selectivity-seeking
approaches, such as photoactivation,
15–17
ligand redox-
mediated activation,
11,18–20
and pH-dependent activation,
21–26
among others.
We
25–28
and others
21–23,29,30
have seen an opportunity in
designing organometallic compounds bearing hemilabile
ligands for metallodrug controlled activation. Hemilabile
ligands are dened as bidentate non-symmetric ligands in
which one of the binding groups is rmly attached to the metal
centre. The other coordinated group is weakly bound and
therefore can easily be dissociated.
31
Since the displaced atom
with metal-binding capabilities stays in close proximity to the
metal, the process of opening and closing is reversible. Some
have described hemilabile ligands as “swinging gates”to enable
metal reactivity.
32,33
A signicant number of ruthenium(II) half-
sandwich complexes bearing tethered groups containing
C,
34–36
N,
23,25,37
O,
26,37–39
S,
40–42
and P
37,43–46
donors have been re-
ported. However, the only tethered h
6
-arene–osmium(II)
described to date in the literature is the [3 + 2] annulation
product of osmium hydrido alkenylcarbyne complex [OsH
{^CC(PPh
3
)]CHPh}(PPh
3
)
2
Cl
2
]
+
with a substituted allenoate,
which affords a typical three-legged piano-stool structure with
the h
6
-benzene, two PPh
3
, and an alkenyl carbon atom (pendant
from the arene) connected to the metal center.
47
In addition,
octahedral complexes of formula [OsCl
2
(N^P)
2
] have shown
some cis versus trans uxional conversion attributable to the
hemilability of the N^P ligand in solution.
48
Organometallic Os(II) complexes have similar, oen almost
identical, structures to the analogous ruthenium compounds,
yet they are oen more inert,
49
a useful feature vis-`
a-vis exerting
a
IMDEA Nanociencia, Faraday 9, 28049 Madrid, Spain. E-mail: ana.pizarro@imdea.
org
b
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4
7AL, UK
c
Unidad Asociada de Nanobiotecnolog´
ıa CNB-CSIC-IMDEA, 28049 Madrid, Spain
†Electronic supplementary information (ESI) available. CCDC 2026997–2027004.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/d1sc01939b
Cite this: Chem. Sci.,2021,12,9287
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Received 7th April 2021
Accepted 9th June 2021
DOI: 10.1039/d1sc01939b
rsc.li/chemical-science
© 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,9287–9297 | 9287
Chemical
Science
EDGE ARTICLE

control over metal reactivity. Os(II) half-sandwich compounds of
general formula [Os(h
6
-arene)(XY)Z]
n+
are less reactive towards
hydrolysis —the rate of aquation is oen ca. 100 times slower
than for Ru—
50–53
and once aquated the aqua ligand is more
acidic, ca. 1.5 pK
a
units.
49,51
Slowing down metal reactivity,
which can also be achieved by replacing chlorido by iodido
ligand, has indeed been related to high cytotoxic potency.
14,18,54
For iridium complexes we have recently reported that stabilis-
ing a closed tether structure in complexes [Ir(h
5
:k
1
-C
5
Me
4
CH
2
-
py)(C,N)]PF
6
, and thus delaying metal reactivity towards
substitution reactions, increases cytotoxicity by up to two orders
of magnitude.
28
Transfer hydrogenation (TH) by transition metal compounds
of Ru(II),
8,26,55–57
Rh(III),
58
and Ir(III),
8,10,27
are attracting much
attention in bioinorganic chemistry since they have demon-
strated the potential of catalytically consuming or producing
important metabolites, such as NADH, that are vital for
a number of cellular processes.
59–62
In particular, lactate and
pyruvate are key cell metabolites that the tumour cell requires to
promote cancer invasion as they help circulating tumour cells to
survive by enhancing their resistance against oxidative stress.
63
Osmium(II) arene [Os(h
6
-p-cymene)(impy-NMe
2
)Cl/I]PF
6
(impy ¼iminopyridine) can oxidize NADH to NAD
+
by Os-driven
hydride abstraction.
64
Importantly, chiral 16-electron complex
[Os(h
6
-p-cymene)(TsDPEN)], (TsDPEN ¼N-(p-toluenesulfonyl)-
1,2-diphenylethylenediamine), inspired by the Noyori-type of
catalysts for asymmetric transfer hydrogenation,
65
is capable of
catalysing asymmetric reduction of pro-chiral pyruvate to
lactate using non-toxic doses of sodium formate as a hydride
donor inside cells.
66,67
Herein we describe the synthesis of thirteen osmium(II)
complexes 1–13, of formula [Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(XY)Cl]
+/0
,
bearing the hemilabile h
6
-bound arene 3-phenylpropanol and
where XY is a neutral N,N or an anionic N,O

bidentate
chelating ligand. We report for the rst time Os(II) tethered half-
sandwich complexes in their closed-tether form, 1C–13C,
including the X-ray structure of cation 3C, where the hemilabile
arene ligand chelates the osmium centre by simultaneous h
6
-
C(arene) and k
1
-O(R) binding. We show how the high stability of
Os(II)h
6
-arene and the exceptional lability of alcohol oxygen as
a coordinating group appear as the ideal combination to allow
for control over metal reactivity in aqueous solution throughout
the biologically relevant pH range. Aiming to unravel speciation
in aqueous solution we have been surprised by the unexpected
reactivity of the hydroxido adduct (the Os–OH bond), and the
highly reversible open/close nature of the Os–O(tether) ring.
Finally, we evaluate the reactivity of these complexes toward
catalytic transfer hydrogenation of pyruvate using formate as
hydride-source, nding that compounds reported here are
capable of producing quantiable excess lactate inside cancer
cells.
Results and discussion
Synthesis and characterization
While a common synthetic method to introduce variations in
Ru(II) arenes involves arene-exchange reactions in [Ru(h
6
-Et/Me-
benzoate)Cl
2
]
2
or other electron withdrawing substituted arene
Ru(II) dimers,
25,34,43,45
this method proved ineffective in the case
of osmium, as our arene-exchange reactions repeatedly failed
when using [Os(h
6
-Et-benzoate)Cl
2
]
2
as a suitable dimer
precursor. It was thus deemed necessary to reduce the specied
arene ligand to the corresponding cyclohexadiene. To obtain 3-
(1,4-cyclohexadien-1-yl)-1-propanol, the Birch reduction of 3-
phenylpropanoic acid was carried out, following an experi-
mental procedure similar to that described by Habtemariam
et al.
68
The reduced cyclohexadiene carboxylic acid was further
reduced to the corresponding alcohol by the addition of LiAlH
4
.
Osmium(II) dimer [Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(m-Cl)Cl]
2
was then
synthesized by the reaction of 3-(1,4-cyclohexadien-1-yl)-1-
propanol with OsCl
3
$3H
2
O, as reported previously (Chart
S1A†).
49
Microwave synthetic methods reported by T¨
onnemann
et al.
69
were used to improve the yield and reaction times.
All chlorido complexes 1–13 were synthesized from the
reaction of the dimeric precursor, [Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(m-Cl)
Cl]
2
, with the corresponding chelating ligand using similar
procedures to those reported for other half-sandwich arene
complexes (Chart S1B†).
13,52,64
Chart 1 shows the compounds
obtained with general formula [Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(XY)Cl]
+/0
,
where XY ¼ethylenediamine, en (1), bipyridine, bipy (2), phe-
nanthroline, phen (3), 1-(2-pyridinyl)methanamine, ampy (4), N-
phenyl-1-(pyridin-2-yl)methanimine, Ph-impy (5), N-(4-(tert-
butyl)phenyl)-1-(pyridin-2-yl)methanimine, tBuPh-impy (6), N-
Chart 1 Open and closed tethered osmium(II) arene complexes
studied in this work of the general formulae [Os(h
6
-C
6
H
5
(CH
2
)
3
-
OH)(XY)Cl]
+/0
and [Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH/O)(XY)]
+
.
9288 |Chem. Sci.,2021,12,9287–9297 © 2021 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

(2-bromophenyl)-1-(pyridin-2-yl)methanimine, BrPh-impy (7),
N-phenethyl-1-(pyridin-2-yl)methanimine, PhEt-impy (8), pico-
linate, pico (9), 6-methylpicolinate, 6-Me-pico (10), 4-methyl-
picolinate, 4-Me-pico (11), 4-carboxy-2-pyridinecarboxylate, 4-
COOH-pico (12), and 8-quinolinate, quinol (13). Cationic
complexes 1–8were isolated as chloride salts while 9–13 as
neutral. All complexes were isolated in good yields (56–94%).
The corresponding closed tether complexes of formula
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH/O)(XY)]
+
(1C–13C; Chart 1) were syn-
thesised using silver nitrate to abstract the chlorido ligand of
the open-tether monomer in water or methanol (Chart S1B†). In
several cases it was necessary to modify the pH of the aqueous
solution for the intramolecular rearrangement (closure of the
tether-ring) to occur (vide infra). Closed-tether complexes 2C,
3C,4C,8C,10C,11C and 13C were synthesised and obtained as
nitrate salts in good yields (56–99%). Complexes 1C,5C,6C,7C,
9C and 12C were characterized in solution as isolation was
unsuccessful due to the fast interconversion dynamics with
their open-tether counterparts. The
1
H NMR spectra of open-
and close-tether complexes display in all cases distinct patterns
for the resonances of the h
6
-bound arene protons. In complexes
1–13,Dd(bound arene) spans 0.14–0.54 ppm, and in the closed-
tether counterparts, 1C–13C, spans 0.34–1.02 ppm (Table S1†),
that is, the increment of chemical shibetween the most
deshielded and most shielded h
6
-bond arene proton signals
was larger for closed-tether complexes than that of their open
tether counterparts, attributable to restricted h
6
-bound arene
rotation along the Os-centroid axis for closed tether complexes.
This is in agreement with the observed differences of
1
H NMR
shis corresponding to closed vs. open Ru(II) tether
compounds.
25,26,37
Ruthenium open and closed tether complexes bearing bipy
and phen 2Ru,2CRu,3Ru and 3CRu and complex [Ru(h
6
:k
1
-
C
6
H
5
(CH
2
)
3
OH)Cl
2
] have been reported before,
37,70,71
and are
included for comparison purposes. The cations were isolated as
their chloride or BF
4

salts while the dichlorido complex is
neutral.
Details of the synthesis and full characterization of all
complexes are given in the ESI (Fig. S1–S23†).
Interestingly, while N,N-chelating ligands afforded Os
closed-tether complexes with an alkoxy group coordinatively
bound to the metal (deprotonated tethered alcohol), complexes
bearing N,O-chelating ligands produced complexes with the
alcohol attached to the metal. All our closed complexes are
therefore +1 cations. Conductivity measurements supported the
observation, giving values in the range ca. 100 S cm
2
mol
1
corresponding to 1 : 1 electrolytes.
72
The
1
H NMR signal of such
an alcohol proton can indeed be identied in the N,O closed
tether structures when the spectrum is recorded in DMSO-d
6
(see for example spectrum of 10C in Fig. S17†), contrariwise the
proton signal cannot be found in DMSO-d
6
solutions of N,N-
chelated complexes.
The X-ray crystal structures of chlorido complexes 5$PF
6
,
8$PF
6
,9,10,11,13, and closed tether complexes 3C$PF
6
and
dichlorido [Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)Cl
2
] (Fig. 1), unambigu-
ously supported our structural determination. All complexes
present the familiar pseudo-octahedral “three-legged piano-
stool”geometry, with osmium(II)p-bonded to the h
6
-arene
ligand. Open tether complexes are s-bonded to a chlorido and
a bidentate chelating ligand, while in closed tether complexes
one of the three “legs”is occupied by the s-bonded alcohol/
alkoxy pendant from the h
6
-arene, affording an h
6
-Os-k
1
six-
membered chelate ring in the structure (Fig. 1G and 1H).
The crystal structures presented in this work are the rst to
be reported with a pendant alcohol function attached to the h
6
-
bound arene in complexes of general formula [Os(h
6
-arene)(XY)
Z]
n+
. Moreover, no X-ray structures of osmium closed tether
complexes of formula [Os(h
6
-arene-O-k
1
)(XY)]
n+
, where a coor-
dinating tethered oxygen is bound to the metal centre, have
been previously reported. In fact, tethered h
6
-arene–osmium(II)
compounds are extremely rare; we only found complex [Os(h
6
-
benzene-C-k
1
)(PPh
3
)
2
]
+
, with a tethered alkenyl pendant from
the derivatised arene,
47
in the Cambridge Database.
Fig. 1 ORTEP diagrams and atom numbering schemes for
compounds: (A) 5$PF
6
, (B) 8$PF
6
, (C) 9, (D) 10, (E) 11, (F) 13, (G) 3C$PF
6
,
and (H) dichlorido complex [Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)Cl
2
] (50% prob-
ability ellipsoids). The H atoms (with the exception of the alcohol
hydrogens), the solvent molecules, and the counterions have been
omitted for clarity. Complex 10 in (D) shows disorder of the tethered
oxygen occupying two different positions, resulting in a lower quality
of structural determination.
© 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,9287–9297 | 9289
Edge Article Chemical Science

Closed-tether structures feature a strong offset of C7 (rst
carbon in the tether chain attached to the arene) toward the
osmium atom with regard to the plane that contains the h
6
-
bound arene, with calculated values of 0.138 and 0.193 
A,
respectively, for both 3C$PF
6
and dichlorido [Os(h
6
:k
1
-
C
6
H
5
(CH
2
)
3
OH)Cl
2
] (Table S2†). Another interesting feature in
all the X-ray structures is the extensive hydrogen bonding of the
oxygen pendant from the arene, especially strong in the case of
cation 3C between the tethered O-alkoxy and a water molecule
of crystallization (Fig. S24 and Table S3†).
Full crystallographic analysis, selected bonds and angles
(Table S2†), p–pintermolecular interactions, directionality,
angles and distances of hydrogen-bonding interactions
(Fig. S24 and Table S3†), and detailed crystallographic data
(Table S4†), are included in the ESI†of this manuscript. CCDC
2026997–2027004 contain the ESI crystallographic data for this
paper.
Aqueous solution studies
Metallodrug activity inside the cell is believed to be triggered by
aquation. The hydrolysis rate of Os(II) arene compounds of the
type [Os(h
6
-arene)(XY)Cl]
n+
is highly affected by the nature of
the chelating ligand.
13,49,52,53
pK*
aIt has been proven that
compounds with N,O-chelated ligands display aqueous behav-
iour intermediate between those bearing N,N- and O,O-chelates,
the former being too slow for the complex to have an impact on
cell survival, and the latter being too fast, resulting in the loss of
the XY ligand and leading to the formation of inert di-osmium
OH-bridged species.
53
Following Os–Cl cleavage, the aqua
adduct is formed. Above the pK*
acorresponding to the Os–OH
2
/
OH equilibria, the hydroxido Os–OH species will predominate.
This is believed to render the complex inactive towards substi-
tution reactions,
49
including potential intracellular targets, and
thus negatively impacting their biological effectivity.
53
Hydrolysis of the Os–Cl bond. The Os–Cl bond hydrolyses in
all cases at 310 K within 24 h to different extents (Fig. 2), as
determined by
1
H NMR. Although speciation evolves over
several days, the main changes occur within 24 h. Fig. 2 shows
that the extent of the Os–Cl bond cleavage in water is indeed
highly dependent on the nature of the XY ligand.
13,49,52,53
Complexes 1–8, bearing N,N-chelating ligands, barely undergo
hydrolysis of the Os–Cl bond within the rst 24 h (hydrolysis
ranges between 0–19%) in agreement with previous
literature.
53,73
However, the chlorido ligand of complexes 9–12
dissociates readily in water, surpassing ca. 40% Os–Cl cleavage
in the rst 24 h. Quinolinate complex 13 fully hydrolyses within
the rst minutes of dissolution in water, ruling out hydrolysis
rate determination by
1
H NMR, which mirrors the hydrolysis of
reported quinolinate compound [Os(h
6
-p-cym)(oxine)Cl].
53
The relatively faster hydrolysis of the Os–Cl species in
complexes 9–12, containing picolinate ligands, is in agreement
with previous reports.
13,49,53
It may be explained on the basis of
an increased electron density at the metal centre due to the
anionic chelating ligand, possibly aided by the stabilising
interaction between hydrogen atoms of the coordinated water
in the aqua species and the oxygen atom of the tethered alcohol
group. Indeed, the X-ray structures of complexes 9,11 and 13
indicate strong hydrogen bonding of the tethered alcohol
oxygen with neighbouring Os(II) molecules with strong covalent
character (Fig. S24 and Table S3†).
74
The rate of hydrolysis in D
2
O of picolinate compounds 9–12
was monitored by
1
H NMR at 300, 310 and 320 K at various time
intervals. The percentage of disappearance of chlorido adduct
for 9–12 (based on peak integration) was plotted against time
and tted to pseudo-rst order kinetics, and their half-life times
were calculated. For all four complexes hydrolysis reaches
equilibrium within the rst hour even at the lowest temperature
(Table 1 and Fig. S25–S27†).
Arrhenius activation energies (E
a
) and activation enthalpies
(DH
‡
) of hydrolysis of compounds 9,10 and 12 (Table 1 and
Fig. S26–S27†) are lower than those previously found for similar
picolinate Os–arenes, which are reported to be ca. 90 kJ mol
1
for both parameters.
13,53
However, activation entropies (DS
‡
) are
similar to those determined for similar complexes varying from
70 to 50 J K
1
mol
1
.
13,53
The large negative activation
entropies (DS
‡
) found for the hydrolysis of 9,10,11 and 12
indicate that entropy decreases on forming the transition state,
which suggests that an associative mechanism is involved, in
which two reaction partners form a single activated complex.
The fastest hydrolysing compound, 11, presents the most
negative entropy, 131.7 J K
1
mol
1
, a value more than two-
fold of its analogue [Os(h
6
-C
6
H
5
C
6
H
5
)(4-Me-pico)Cl]
+
, with an
entropy of 55.6 1.6 J K
1
mol
1
.
13
Complex 11 also requires
the lowest DH
‡
towards hydrolysis.
pH-dependent speciation. Hydrolysis of the osmium–chlor-
ido bond in complexes 1–13 is not an isolated event. As depicted
in Chart 2, speciation in aqueous solution of the complexes
reported here involves: (i) hydrolysis of the Os–Cl bond to form
the Os-aqua adduct, that is, the chlorido ligand is substituted by
a water molecule forming an Os–OH(H) bond; and (ii) intra-
molecular rearrangement, i.e., a further substitution reaction of
the aqua/hydroxido ligand by the tethered alcohol/alkoxy,
prompting tether ring closure through formation of an Os–
O(H)R s-bond.
Such a three-way speciation of complexes 1–13 was rst
detected by
1
H NMR in unbuffered D
2
O (Fig. S28†). Addition of
excess sodium chloride to the mixture of species at equilibrium
conrmed the assignment of the aqua adduct by
1
H NMR (by
conversion of the aqua to the chlorido species). Addition of
AgNO
3
to solutions of complexes 2,3,8,10 and 11 aided the
Fig. 2 Hydrolysis of the Os–Cl bond for complexes 1–13. The bars
represent the percentage of remaining open-tether chlorido
complexes 1–13 over time in unbuffered D
2
O as determined by
1
H
NMR. Equilibrium is mostly reached in the first 24 h. Complex 13 is fully
hydrolysed from the first data recording (t#15 min upon dissolution).
9290 |Chem. Sci.,2021,12,9287–9297 © 2021 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

unambiguous characterization of the closed tether species.
Furthermore, Ag
+
-mediated choride sequestration indicated
that Os–Cl bond cleavage favours tether-ring closure.
Peacock et al. have shown pH to be important in the aqueous
behaviour and thus the biological impact of hydrolysable Os–
arenes as potential anticancer agents.
13,49,52,53,75
We aimed to
assess the effect of pH on interconversion of species in aqueous
solution of the Os(II) half-sandwich OH-tethered complexes.
Selected compounds 1,2,5,8,9,10,11 and 13 were dissolved in
D
2
O (at [Os] ¼5 mM) in four unbuffered pH solutions (pH 1, 4, 7
and 10). Fig. 3 shows how our complexes undergo speciation,
whereby the aqua (or hydroxido) species seemingly evolve to the
closed tether complex until reaching equilibria where coexis-
tence of the three species (chlorido, aqua/hydroxido and closed
tether species) occurred upon 24 h of incubation.
The percentage of hydrolysis-triggered species is similar at
pH 1 and 4, i.e., pH-variation when the proton concentration is
high has no impact in the speciation of these complexes. The
predominant species are chlorido and aqua species (Chart 2). At
pH 10, however, most complexes show a remarkable preference
for intramolecular rearrangement, as seen by the evolution over
time towards the closed tether species. All but complexes 2and
8show more than 60% of closed tether structure at basic pH
aer 24 h. Even fully aquated complex 13 shows a clear pref-
erence for the closed tether species 13C at pH 10 even at t¼
15 min.
pK*
adetermination. Following hydrolysis rate determination
and nding reversible interconversion of open and closed
tether complexes in a pH-dependent manner, we endeavour to
determine the pK
a
s of the species in solution. Both aqua (1A–
13A) and closed tether (1C–13C) species are subjected to acid–
base equilibria (Chart 2). Alcohol coordination to osmium(II)in
Table 1 Rate data for the aquation of complexes 9,10,11 and 12 at 300, 310 and 320 K
T(K) k
hyd
(min
1
)t
1/2
(min) E
a
(kJ mol
1
)DH
‡
(kJ mol
1
)DS
‡
(J K
1
mol
1
)
9300 0.08 8.6 67.0 0.9 64.4 1.0 51.5 3.1
310 0.19 3.7
320 0.48 1.4
10 300 0.06 11.9 63.9 15.5 61.4 15.5 65.0 49.9
310 0.09 7.5
320 0.29 2.4
11 300 0.20 3.5 40.8 11.1 38.3 11.0 131.7 35.6
310 0.26 2.7
320 0.54 1.3
12 300 0.05 13.2 67.0 4.7 64.4 4.7 55.0 15.1
310 0.11 6.1
320 0.28 2.5
Chart 2 Aqueous speciation of open and closed tether complexes,
including acid–base equilibria (n¼1 for complexes 1–8;n¼0 for 9–
13).
Fig. 3 Speciation of chlorido complexes (A) 1, (B) 2, (C) 5, (D) 8, (E) 9,
(F) 10, (G) 11, and (H) 13 at time ca. 15 min and after 24 h in unbuffered
aqueous solutions at pH 1, 4, 7 and 10. The arrow represents time
forward, from t¼15 min to t¼24 h.
© 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,9287–9297 | 9291
Edge Article Chemical Science

the closed tether complexes renders the s-bonded alcohol
proton highly acidic, favouring deprotonation of an otherwise
largely basic group (estimated pK
a
of 3-phenyl-1-propanol ca.
16).
Complexes 1–13 were treated with AgNO
3
in water or alco-
holic aqueous solutions to ensure the formation of both the
aqua and subsequently the closed tether species. Compounds
2Ru and 3Ru were synthesised
37,70
and included for comparison
purposes. The pK*
a1 values for the aqua/hydroxido (OH
2
/OH)
adduct of formula [M(h
6
-C
6
H
5
(CH
2
)
3
OH)(XY)(OH
2
/OH)]
n+
, for
complexes 1A,2A,2ARu,3A,3ARu,8A,12A and 13A, and the
pK*
a2 values for the coordinated alcohol/alkoxy group (ROH/RO)
in closed tether complexes of formula [M(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH/
O)(XY)]
n+
, for complexes 1C,2C,2CRu,3C,3CRu,8C and 12C,
were determined and are presented in Table 2 and Fig. S29.†
1
H
NMR peaks corresponding to coordinated arene protons in
closed and aqua adducts 1C–3C,3CRu,1A–3A and 3ARu,or
chelating ligand protons in 8A,8C,12A,12C and 13A, were
followed as they shied to high eld on increasing pH*
(Fig. S30†contains the titration of a mixture containing 3,3A
and 3C, as an example). Changes in the
1
H NMR chemical shis
of closed tether and aqua species were recorded at 298 K over
the 0–12 pH range by the addition of NaOD or DNO
3
as
appropriate. The chemical shi-versus-pH plots and pK*
avalues
obtained from the titrations are compiled in Fig. S31.†
The nature of the chelating ligand (N,N, N,O or O,O) shows
a great inuence on the pK*
avalues of the M–OH
2
group (basicity
increasing N,N < N,O < O,O, with better p-acceptors, such as
phen, affording more acidic metal centres, which resulted in
lower pK*
as for the aqua adduct).
13,26,49,51–53,76
The high acidity of
osmium arene aqua complexes has been previously attributed
to increased mixing of the ds*(Os) /s(OH

) orbitals.
77,78
The
higher acidity of Os versus Ru can also explain the acidity of the
coordinated hemilabile alcohol/alkoxy (Fig. S29†).
23,49,77
Closed-tether and aqua species identication was conrmed
by the
1
H NMR pH titration associated with both pK*
as:The
chlorido species are devoid of chemical shivariation due to
the lack of (de)protonatable positions in the structure, with the
exception of complex 12, which bears a carboxylic acid in the
picolinate ligand (Fig. S31F†).
Since the pK*
a1 data is below 7.4 (physiological pH) for all Os–
arenes, in a live cell environment, the complexes are expected to
be present in the hydroxido form rather than the aqua species.
Yet in our complexes the hydroxido species readily evolves to
the closed tether complex. Contrary to previous reports on Os–
arene aquation,
49,53,75
inert hydroxido-bridged dimers have not
been detected throughout this work. In fact, the species re-
ported here are interconvertible in a reversible manner over the
pH range. The formation of closed tether species presents a very
exciting turn of events due to their strong predominance at
pH .pK*
a1;preventing deactivation of the Os(II) compound.
Speciation proved indeed highly dependent on pH (Fig. 3),
clearly favouring tether ring closure even at pH .pK*
a2;that is,
the pK*
aof the Os-bound alcohol. We postulate a concerted
associative mechanism for intramolecular rearrangement
resulting in tether ring formation by which an incoming ligand,
the tethered alcohol, likely assisted by H-bonding, transfers its
proton to the hydroxido ligand (favoured by pK*
a1 .pK*
a2),
thereby facilitating the release of a water molecule from the
osmium rst coordination sphere (Chart 3). This is supported
by extensive hydrogen-bonding by the pendant oxygen as
observed upon X-ray analysis, especially strong for closed tether
cation 3C (Fig. S24 and Table S3†).
Hydrolysis of the Os–O bond for the closed tether complexes.
Solutions of closed complexes 2C,10C and 13C were prepared
in D
2
O and their speciation was evaluated by
1
H NMR in three
different solutions at pH 1, 7, 12 (Fig. S32†). The three
complexes favour the aqua species at equilibrium at pH 1.
However, at pH 7 and more signicantly at pH 12, even complex
10C, which initially undergoes major cleavage of the Os–O bond
(up to 70%), reverts to the closed tether complex over time (ca.
80% closed tether aer 24 h) showing a clear preference for the
tether-ring formation at basic equilibrium highlighting the
reversibility of the process. The pH in all the samples dropped
aer 24 h, the samples prepared in unbuffered pH 12 solutions
showed the most signicant change. There is a clear correlation
between these data and those of speciation of chlorido
complexes 2,10 and 13 as described in Fig. 3.
Table 2 pK*
avalues of the aqua ligand in complexes 1A,2A,2ARu,3A,
3ARu,8A,12A and 13A, and the tethered alcohol in complexes 1C,2C,
2CRu,3C,3CRu,8C and 12C
pK*
a1 pK*
a2
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(en)(OH
2
)]
2+
(1A) 5.83 —
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(en)]
2+
(1C)—5.46
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(bipy)(OH
2
)]
2+
(2A) 5.81 —
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(bipy)]
2+
(2C)—4.23
[Ru(h
6
-C
6
H
5
(CH
2
)
3
OH)(bipy)(OH
2
)]
2+
(2ARu) 7.28 —
[Ru(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(bipy)]
2+
(2CRu)—5.78
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(phen)(OH
2
)]
2+
(3A) 5.62 —
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(phen)]
2+
(3C)—4.26
[Ru(h
6
-C
6
H
5
(CH
2
)
3
OH)(phen)(OH
2
)]
2+
(3ARu) 7.18 —
[Ru(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(phen)]
2+
(3CRu)—6.39
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(PhEt-impy)(OH
2
)]
2+
(8A) 5.69 —
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(PhEt-impy)]
2+
(8C)—4.41
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(4-COOH-pico)(OH
2
)]
+
(12A) 6.64 —
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)(4-COOH-pico)]
+
(12C)—5.63
[Os(h
6
-C
6
H
5
(CH
2
)
3
OH)(quinol)(OH
2
)]
+
(13A) 7.01 —
Chart 3 Suggested mechanism of tether-ring closure as dependent
on pH, given that pK*
a1 .pK*
a2:
9292 |Chem. Sci.,2021,12,9287–9297 © 2021 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

Speciation of closed tether complexes 2C,10C and 13C in the
presence of chloride ions was also investigated. Since water-
mediated speciation is highly dependent on pH, the behav-
iour of 2C,10C and 13C was evaluated in 0.1 M DCl and 0.1 M
DClO
4
solutions (Fig. S33†) at 310 K over 24 h. These solutions
were selected because the concentration of protons is equiva-
lent in both, yet the chloride concentration varies from 100 mM
Cl

in DCl (similar to that in plasma) to nil in DClO
4
. The
results indicate that the tether-ring opening occurs to approxi-
mately the same extent, clarifying that pH dominates speciation
over chloride concentration.
Catalytic reduction of pyruvate to lactate
Osmium(II) arenes have been reported to carry out catalytic
transfer hydrogenation reactions.
64,66
A screening of selected
complexes 1,3,5,6,9,10,11,12,13 was carried out to evaluate
the conversion of pyruvate (pyr) to lactate using formate (for) as
the hydride source, at ratios 1 : 2 : 100 (Os catalyst/pyr/for) at
pH 4 and 310 K, by means of
1
H NMR.
Complexes 1,3,5and 6with N,N ligands did not transform
pyruvate to lactate over 48 h, even aer formation of the Os-
formate adduct for complexes 1and 5was observed by
1
H
NMR. The formate adduct is believed to precede hydride
abstraction by the metal centre during the catalytic cycle.
55,56,66,79
It
has been reported that chlorido removal increases the activity of
catalytic metal complexes by generating species that can bind to
formate.
56
Hydrolysis of complexes 9–13, bearing N,O-chelating
ligands, could thus be in their favour to mediate the trans-
formation of pyruvate to lactate. In the
1
H NMR spectra of the
hydrogenation reactions by 9,11,12 and 13 two new sets of peaks
were attributed to the Os–aqua (Os–OH
2
)andtheOs–formate (Os–
OOCH) adducts. A sharp singlet at ca. 7.9 ppm was attributed to
the Os-bound formate (free formate appears at ca. 8.4 ppm), in
accordance with analogous Ru(II) arene formate complexes
(Fig. S34†).
26,80
Although the species (h
6
-arene)Os–HinTHcatal-
ysis has been reported to appear around 4ppmby
1
HNMR,
64
in
our experiments the hydrido species was elusive.
26,81
The intensity
of the signals of free pyruvate (3H, singlet, 2.36 ppm) decreased
and a new peak assignable to lactate (3H, doublet, 1.33 ppm)
appeared. Compound 11 mediated total reduction of pyruvate to
lactate aer 18 h (Fig. S35†)whereas13 barely achieved stoi-
chiometric conversion aer 2 days (Fig. S36A†). Since more than
one mol equiv. of pyruvate was reduced per mol equiv. of osmium
complexes 9,11 and 12, a catalytic mechanism is implied.
We further explored the catalytic capabilities of complex 9,
bearing the unsubstituted picolinate, by varying the pH as well
as increasing the amount of H-source and the temperature.
TON
t
and TOF
max
data are compiled in Fig. S36B.†The most
favourable pH in the range 3–7 was 5 (reaction almost 4faster
than at pH 3), which could be rationalized by considering that
the pK
a
of formic acid is 3.75 and that the formation of the
formate adduct holds a central role in the catalytic trans-
formation.
55,56
Lack of stability of pyruvate at low pH
82,83
could
be affecting the efficiency of our catalysts in acidic solutions.
Both an increase in the amount of formate and of temperature
resulted in higher TON
t
and TOF values as anticipated.
Complex 11, selected on the basis of its fast hydrolysis and
catalytic performance was probed as a catalyst at three different
temperatures (300, 310, and 320 K) and concentrations of
hydride source (100, 200 and 400 mM sodium formate; Table 3).
TOF values increased with temperature. For example, an
increment of 20 K resulted in ca. 11-fold increase in TH rate.
This is a similar increment to that reported for other Ru–arene
complexes bearing N,O-chelates, in which a similar variation of
temperature (increase of 23 K) resulted in an increment of 7-fold
in the TOF value.
84
Compound 11 has a maximum TOF value of
0.20 h
1
at pH 4 (Os/pyr/for; 1 : 2 : 100), which is comparable to
pyruvate reduction by the reported 16-electron species [Os(h
6
-p-
cymene)(TsDPEN)], for which the TOF
max
in PBS is 1.5 h
1
(for
Os/pyr/for, 1 : 200 : 400).
85
The percentage of conversion from
pyruvate to lactate aer 1 h incubation shows that an increment
of 10 K doubled the percentage of conversion (Fig. S37†).
Complexes 9–13 are, to the best of our knowledge, the rst
18-electron Os arene complexes to achieve catalytic TH of
pyruvate. Such an activity, in conjunction with the high solu-
bility in aqueous media and the protective nature of the
dynamic tether ring formation, prompted us to investigate the
potential of carrying out TH inside cells by selected complex 11.
Production of lactate inside cells
The majority of the Os(II) complexes reported here had no
impact on cell survival of MDA-MB-231 and HCT116 cells up to
a concentration of 200 mM (Fig. S38 and Table S5†), largely in
agreement with data reported for similar Os–arenes in other
cancer cell lines.
12,49,52,64
Only complexes 11 and 11C appear to
be an exception regarding cytotoxic potential, presenting the
lowest IC
50
value in the series in colon cancer cells HCT116 (IC
50
values 30.5 3.3 and 19.5 0.6 mM, respectively). Intracellular
accumulation experiments in MDA-MB-231 and HCT116 cells
showed that uptake for 11C was high, as determined by ICP-MS
(Table S6†). These results, together with the catalytic capabil-
ities of 11 for transfer hydrogenation, encouraged us to probe
this complex for osmium intracellular reactivity as a potential
metabolite-modulating tool.
We investigated whether direct detection of Os-mediated
lactate production was possible inside cancer cells. One
concern was the lack of structural compromise that could
promote enantiomeric enrichment in the reduction of pro-
Table 3 Catalytic data on the reduction of pyruvate (2 mM) to lactate
of 1 mM 9,11–13 in D
2
O at pH 4 as determined by
1
HNMR
spectroscopy
Compound T(K)
HCOONa
(mM) TOF
max
(h
1
)R
2
9310 100 0.049 0.992
11 300 100 0.037 0.989
310 100 0.200 0.990
320 100 0.417 0.985
300 200 0.052 0.998
300 400 0.061 0.983
12 310 100 0.050 0.967
13 310 100 0.140 0.970
© 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,9287–9297 | 9293
Edge Article Chemical Science

chiral pyruvate by 11. Our complex is synthesized as a racemic
mixture, and it is only reasonable to think that the product of
the reaction, lactate, would also be obtained as a racemic
mixture.
We rst optimized a UV-vis-based assay for lactate determi-
nation, examining whether complex 11 is capable of producing
a valid substrate for enzymatic quantication from pro-chiral
pyruvate (Fig. S39†). We used in this experiment L-lactate
deshydrogenase (L-LDH) as L-lactate is produced from pyruvate
during anaerobic glycolysis and is present in humans at
concentrations 100 times greater than D-lactate. L-Lactate
generated by 11 was conrmed by reaction with L-LDH enzyme,
a process accompanied by concomitant production of UV-vis-
active NADH (Fig. S39†). As anticipated, osmium-produced
lactate increased with increase in reaction time from 0 to 4 h
in the presence of formate (Fig. S39B†).
Next we carried out lactate determination in cells. The choice
of cell line to demonstrate whether it would be possible for 11 to
produce measurable lactate inside cancer cells was carefully
considered. MDA-MB-231 and MCF7 were selected on the basis
of their growth media composition (non-pyruvate and pyruvate
containing, respectively). The low cytotoxic effect of 11 in these
cell lines (IC
50
90.6 4.0 and 105.1 10.6 mM in MDA-MB-231
and MCF7, respectively) was also advantageous as it allowed us
to expose the cells to a relatively high osmium concentration
(300 mM). Pyruvate (2 mM) and formate (10 mM) were added to
the MDA-MB-231 cells media whereas only formate (10 mM) was
added to the MCF7 cells (media from commercial sources
already contain 1 mM pyruvate). Controls were stimulated only
with the osmium compound (we also included double-negative
controls; detailed protocol in the ESI†). Upon exposing the
drugs to 11 for either 12 or 24 h, the cells were washed, lysed,
deproteinated and centrifuged. The lysate supernatants were
then tested for L-lactate determination and quantication. The
results are shown in Fig. 4. Lactate increases when cells are
exposed to 11 and formate, in comparison to those exposed to
osmium alone. The impact of formate and Os in lactate gener-
ation in MCF7 at 24 h is the most noticeable and the differences
are signicate (p-value < 0.01) when compared to the same
experiment lacking formate, and despite being exposed to half
of the amount of exogenous pyruvate (1 mM in MCF7 and 2 mM
in MDA-MB-231). We tentatively attribute the different results in
both cell lines to the readiness of MCF7 cells to internalize
pyruvate, since it is a media component.
Our results indicate that the tether Os–arene complexes
presented here can be exploited for the conversion of metabo-
lites that are essential in the demanding metabolism of the
cancer cell. Depletion of the enantiomeric excess of the hydro-
genated product (racemic lactate as opposed to L-lactate) might
also have an impact on intracellular activity and warrants
further exploration. Additionally, articial non-enzymatic TH of
pyruvate inside cells can affect the activity of lactate dehydro-
genase (LDH) in converting pyruvate to lactate and vice versa,
which in turn is intimately ligated to the generation of NAD
+
and NADH for a number of intracellular processes. An organo-
metallic complex capable of modulating metabolites pyruvate/
lactate (and consequently NADH/NAD
+
) can be a powerful
intracellular tool to target the metabolic plasticity of the cancer
cell.
Conclusions
Water-mediated processes in Os–arenes (such as conversion of
the Os–chlorido bond into the Os–aqua bond) are either inef-
cient or tend to result in species with low reactivity. We have
overcome this issue by introducing a pendant arm primed with
a terminal alcohol functionality attached to the arene ligand.
The h
6
-bound arene behaves now as hemilabile, with a weakly
k
1
-bound alcohol group. Strikingly, this ligand furnishes the Os
complex with an entirely new reactivity prole. Osmium–arenes
oen generate Os–OH inactive species due to the acidity of the
metal.
52,53
In our compounds, the hydroxido adduct (Os–OH)
triggers intramolecular rearrangement culminating in the
binding of the pendant oxygen to the metal centre (formation of
a closed tether complex), protecting the complex towards irre-
versible inactivation. The ligand exchange uxionality of the
hemilabile ligand thus ensures the kinetic reactivation of the
metal centre. We reported here reversible formation of Os–
arene tether complexes in water. Our results imply the
unprecedented reactivation of the otherwise inert hydroxido
Os(II)–arene complexes.
Finding metallodrugs that can exert catalytic activity inside
cells is a challenging task. Alcohol-tethered Os(II)–arene
complexes have demonstrated to carry out transfer hydrogena-
tion reactions inside cells. The combination of tether-ring
protection towards excessive metal reactivity in the intracel-
lular nucleophile-rich microenvironment (protection towards
catalyst poisoning), their high-water solubility, and the readi-
ness towards catalytic transformations when exposed to the
appropriate sacricial agent, makes them strong contenders as
intracellular catalysts. Tumour cell metabolism is considered
an exploitable vulnerability in cancer therapy,
86
where lactate
Fig. 4 Lactate generated (nmol) per million cells determined in MDA-
MB-231 and MCF7 breast cancer cell lines at 12 and 24 h. In yellow,
lactate in cells exposed to 11 (lactate background level resulting from
Os-stimulated cell metabolism only). In grey, lactate produced upon
co-incubation of osmium, formate and pyruvate (0.3, 10, and 2 mM,
respectively) in MDA-MB-231 cells. In green, lactate determined upon
co-incubation of osmium, formate and pyruvate (0.3, 10, and 1 mM,
respectively) in MCF7 cells.
9294 |Chem. Sci.,2021,12,9287–9297 © 2021 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

and pyruvate have central, yet not completely unveiled, roles.
87
We have demonstrated that catalytic tethered Os–arenes can be
extraordinary candidates for modulating such an aspect of
cancer progression in foreseeable therapies.
As whether the pH-dependent aqueous dynamics of this new
class of Os compounds will have an impact on the anticancer
capabilities of these complexes, further work is necessary to
understand such subtleties. Internal pH (pH
i
) in heterogeneous
cancer cells varies in comparison to normal cells as a result of
different stages of metabolic reprogramming, and also varies
from tumour to tumour depending on malignity.
63,88
We
hypothesize that the cancer cell exceptional metabolism, which
results in cytosolic and intra-organelle pH disruption will
impact metallodrug speciation and thus tune its anticancer
effect. In other words, different drugs will exert differing
degrees of lethality to pre-selected tumours based on their pH-
responsiveness, highlighting an additional structural advantage
by the Os-tether design presented here.
Data availability
Crystallographic data for chlorido complexes 5$PF
6
,8$PF
6
,9,
10,11,13, and closed tether complexes 3C$PF
6
and dichlorido
[Os(h
6
:k
1
-C
6
H
5
(CH
2
)
3
OH)Cl
2
] has been deposited at the Cam-
bridge Crystallographic Data Centre under the accession
numbers CCDC 2026997–2027004 and can be obtained from
https://ccdc.cam.ac.uk. Additional data for this paper,
including NMR, UV-vis and ICP-MS datasets, are available at
IMDEA Nanociencia Repository at http://hdl.handle.net/
20.500.12614/2629.
Author contributions
A. M. P. conceived and designed the study. S. I.-T. and A. H.
developed the synthetic methodology. S. I.-T. synthesized the
complexes and carried out the aqueous studies. S. I.-T. and V.
R.-F. carried out the experiments in cells. S. I.-T, V. R.-F. and
A. M. P. analysed the data. S. I.-T., V. R.-F., A. H and A. M. P
discussed the ndings and contributed towards writing the
manuscript.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We thank Dr J. Perles and Dr M. Ram´
ırez (Universidad
Aut´
onoma de Madrid) for the analysis of X-ray data. Dr Z. Pardo
and Dr A. Arn´
aiz and Ms C. Leis (IMDEA Nanociencia) are
gratefully acknowledged for assistance with NMR experiments
and cell culture techniques, respectively. We acknowledge
funding from the EC (FP7-PEOPLE-2013-CIG, no. 631396), from
the Spanish MINECO (RYC-2012-11231, CTQ2014-60100-R, SEV-
2016-0686, and CTQ2017-84932-P), and the Comunidad
Aut´
onoma de Madrid (Scholarship PEJD-2016/IND-2608). Dr A.
Habtemariam was supported by the Comunidad Aut´
onoma de
Madrid (Professorship of Excellence 2016-T3/IND-2054).
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