Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

Article abstract of DOI:10.1002/anie.201800288

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and fo

Full text of DOI:10.1002/anie.201800288

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Accepted Article
Title: Bioorthogonal Catalytic Activation Of Pt And Ru Anticancer
Complexes By FAD And Flavoproteins
Authors: Silvia Alonso-de Castro, Aitziber Lopez Cortajarena,
Fernando Lopez-Gallego, and Luca Salassa
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Angewandte Chemie International Edition
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COMMUNICATION
Bioorthogonal Catalytic Activation Of Pt And Ru Anticancer
Complexes By FAD And Flavoproteins
Silvia Alonso-de Castro, Aitziber L. Cortajarena, Fernando López-Gallego,* Luca Salassa*
Abstract: Recent advances in bioorthogonal catalysis promise to
deliver new chemical tools for performing chemoselective
transformations in complex biological environments. Herein we
report how FAD (flavin adenine dinucleotide), FMN (flavin
mononucleotide) and four flavoproteins behave as unconventional
In this context, we recently reported a new bioorthogonal
IV
reaction in which riboflavin photoactivates a Pt prodrug with
extremely low doses of blue light through a catalytic mechanism
in the presence of zwitterionic electron donors. Light activation
of the riboflavin-prodrug pair triggers cisplatin-related
IV
II
[11]
photocatalysts capable of converting Pt and Ru complexes into
antiproliferative activity in PC3 cancer cells.
Unlike classic
II
II
potentially toxic Pt or Ru –OH
2
species. Using electron donors and
organometallic catalysis, where metals act as catalysts, in this
[
12]
low doses of visible light, the flavoproteins mini Singlet Oxygen
Generator (miniSOG) and NADH oxidase (NOX) catalytically
reaction the metal complex is an unconventional substrate
and the biocompatible riboflavin is the catalyst.
IV
activate Pt prodrugs with bioorthogonal selectivity. In the presence
IV
Herein, we report fundamental discoveries in this new type
of bioorthogonal chemistry by (i) investigating the catalytic
behavior of various flavin catalysts, including four flavoproteins
with diverse biological functions and flavin binding pockets, (ii)
of NADH, NOX catalyzes Pt activation in the dark as well,
indicating for the first time that flavoenzymes may contribute to
IV
initiate the activity of Pt chemotherapeutic agents.
[
1]
IV
The latest advancements in bioorthogonal chemistry
increasing the pool of inorganic reactions to different Pt and
II
demonstrate how organometallic compounds and inorganic
materials are capable of catalyzing the activation of
profluorescent substrates and prodrugs with remarkable
Ru prodrug complexes and (iii) evaluating the efficiency of
(bio)organic electron donors (Figure 1). Furthermore, our work
shows for the first time that certain flavoproteins may be directly
implicated in the activation of metallodrugs under biologically
relevant conditions in the absence of light.
[
2-10]
efficiency in biological environments.
These selective
catalysts carry out non-natural reactions dodging the
interference of biological molecules, using in some cases
[
3, 9]
endogenous cellular components as co-reactants.
Figure 1. Structures of catalysts, substrates and electron donors employed in this study and proposed catalysis mechanism.
Initially we investigated the capacity of FAD (flavin adenine
dinucleotide) as catalyst for the photoactivation of two classes of
[
*]
Prof. Luca Salassa
Donostia International Physics Center
Paseo Manuel de Lardizabal 4, Donostia, 20018 (Spain)
E-mail: lsalassa@dipc.org
IV
II
anticancer metal complexes, namely Pt octahedral and Ru -
arene piano-stool complexes. Complexes 1–3 are prodrugs of
[
13]
cisplatin and carboplatin,
photoactivatable scaffolds capable of generating reactive Ru–
OH species, which can bind to biomacromolecules (Figure
and complexes 4 and 5 are
S. Alonso-de Castro, Prof. A. L. Cortajarena, Prof. F. López-Gallego
CIC biomaGUNE
Paseo de Miramón 182, Donostia, 20014 (Spain)
E-mail: flopezgallego@unizar.es
2
[14-17]
IV
II
2
a).
Importantly, these Pt and Ru complexes have poor
Prof. Luca Salassa, Prof. A. L. Cortajarena
Ikerbasque, Basque Foundation for Science
Bilbao, 48011 (Spain)
absorption properties in the visible (Figure 2b) compared to
other photoactivatable complexes, e.g. Ru polypyridyls.
Therefore, novel strategies to prompt their photochemistry at
longer wavelengths are pivotal for use in photochemotherapy.
Complexes 1–5 are stable towards hydrolysis in the dark, and
have either no or poor photoreactivity under blue light
Dr. F. López-Gallego
ARAID Foundation
Zaragoza, Spain
[
11, 17-18]
Supporting information for this article is given via a link at the end of
the document.
excitation.
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COMMUNICATION
FAD photocatalysis towards 1–5 was performed employing
catalytic reaction between FAD and the complex. Conversely,
reduction of 1 at 20 mM NADH took place readily in the dark
when FAD was present, or under light irradiation when the flavin
was absent (Figure S1–8).
1
0 μM of catalyst and 200 μM of metal substrate (5% catalyst
load). In all irradiation experiments, we used an LED light source
–
2
1
(
6 mW·cm ) with an emission maximum at 460 nm and H NMR
to monitor and quantify the evolution of the photoreactions.
Description of experimental methods and a complete set of dark
and light-irradiation experiments are provided in the Supporting
Information (Figure S1–S76).
On this basis, we used 20 mM MES and 2 mM NADH to
[
23]
determine FAD photocatalytic activity towards 1–5 (Figure 2).
All complexes underwent photochemical activation in the
presence of catalytic quantities of FAD (Figure S1–38).
Consistently with their redox chemistry in the biological
[
24]
context,
FAD photoactivation of 1–4 with NADH was
approximately twice as fast as MES. The only exception was 5,
towards which FAD displayed 3.4 times lower turnover
frequency (TOF) using NADH than using MES (Table 1).
The kinetics of these catalytic reactions showed clear
dependency on the substrate nature. Complexes 1, 2 and 4
were the best substrates, having the highest TOFs and total
turnover numbers (TTNs). Remarkably, FAD was able to
complete the conversion of 1 and 2 into its corresponding
photoproducts regardless of the electron donor used.
–
1
Table 1. Turnover frequency (TOF, min ) and total turnover number (TTN) for
the FAD- and flavoprotein-catalyzed photoactivation of complexes 1–5 in the
presence of MES and NADH.
[
a]
MES (20 mM)
NADH (2 mM)
Complex
TOF
TTN
% Conv.
FAD
TOF
TTN
% Conv.
1
2
3
2.3 ± 0.2
4.0 ± 0.5
0.6 ± 0.1
20
20
11
100
100
55
5.0 ± 1.7
7.1 ± 1.8
2.3 ± 0.6
20
20
14
100
100
70
Figure 2. (a) Flavin-mediated photoactivation reactions of complexes 1–5; (b)
absorption spectra of FAD and 1–5.
4
5
4.5 ± 0.6
2.2 ± 0.5
16
16
80
80
9.0 ± 2.3
0.6 ± 0.1
20
14
100
70
In first instance, we evaluated the effect of electron donors
on the catalytic process using complex 1, with the aim of
optimizing reaction conditions. Three concentrations (0.2, 2 and
miniSOG
2
0 mM) of MES (as buffer, pH 6) or NADH (pH 7, 100 mM PB,
i.e. phosphate buffer) were employed for this purpose. MES was
selected as electron donor to follow up our previous work on
1
2
1.0 ± 0.2
1.2 ± 0.1
20
20
100
100
7.1 ± 0.4
8.6 ± 2.2
20
20
100
100
[
11]
riboflavin, while NADH for its relevance as biological cofactor
[
19]
in numerous reactions catalyzed by flavoenzymes. Moreover,
metal-based catalytic drugs have been recently shown to kill
NOX
+
[b]
1
2
0.62 ± 0.01
4.7 ± 1.2
20
20
100
100
4.3 ± 1.6
8.3 ± 1.6
20
20
100
100
cancer cells by interfering with the cellular NAD /NADH
[
20-22]
homeostasis.
[b]
IV
Upon 460-nm light excitation, FAD photoconverted the Pt
GOX
Not active
<0.2
substrate and showed a catalytic efficiency which increased
linearly alongside the MES concentration. FAD was fully inactive
in the dark at any tested MES concentration. In the absence of
light, 0.2 and 2 mM NADH did not induce any reaction for 1,
whereas light irradiation switched on the generation of
photoproducts at 2 mM when FAD was present. At 2 mM NADH,
FAD photocatalyzed the full conversion of 1 in only 2.5 min
against the 5–10 min required by 20 mM MES. At the lowest
1
2
<0.1
<0.1
5
20
50
7.4
37
[
a]
GR
1
2
10
0.42 ± 0.07
1.2 ± 0.3
20
20
100
100
Not active
concentration (0.2 mM), NADH was instantaneously
+
photooxidized to NAD by molecular oxygen (O
2
), precluding any
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Angewandte Chemie International Edition
10.1002/anie.201800288
COMMUNICATION
2
00 μM 1 or 2 and either 20 mM MES or 2 mM NADH, in order
[
a] = experiments for GR were run using NADPH; [b] = in the dark
to directly compare activities with the corresponding free flavin.
Concentrations of flavins bound to proteins were calibrated by
optical methods using FAD and FMN (for miniSOG) absorbance
at 460 nm. Catalysis results for flavoproteins are summarized in
Table 1.
FAD allowed employing a convenient excitation wavelength
460 nm) and an extremely low light dose for the photoactivation
(
–
2
of 1–5. In the case of 1, 2 and 4, a light dose of ca. 1 J·cm was
sufficient to fully convert the complexes in their activated
As anticipated from inspecting their flavin active site, GOX
II
photoproducts. Ru -arene derivatives such as 4 and 5 typically
IV
and GR showed the lowest catalytic activity towards the Pt
require irradiation times exceeding 1 hour to reach ca. 50%
substrates. GOX presented no catalytic activity towards 1 under
none of the experimental conditions tested. Lack of activity was
also found when glucose (20 mM), a natural substrate for the
enzyme, was employed as source of electrons instead of MES
or NADH. Conversion of 2 by GOX occurred in the presence of
both electron donors, however reactions were slow and did not
reach completion after 1 h of light irradiation (conversion < 40%).
In MES, GR was poorly or no active towards 1 and 2. On the
contrary, NADPH prompted significantly higher TOF values and
complete substrate conversion within few minutes of light
exposure.
[
14-17]
conversion (Figure S24 and S32).
Herein, we show that less
than 15 min are sufficient to achieve comparable effects in 4 and
5
when FAD was used as photocatalyst.
In the cell milieu, flavins are bound to proteins through
[
19]
covalent and non-covalent interactions,
which control their
[
24]
(
photo)redox properties.
Exploiting flavoproteins as selective
catalysts is therefore an exciting prospect for the design of
bioorthogonal activation strategies for metal-based prodrugs.
Accordingly, we selected four flavoproteins for their diverse
flavin-binding pockets and explored their capacity to catalyze the
photoreduction of 1 and 2 as substrates. This part of the study
was limited to these derivatives for their relevance as anticancer
In MES, miniSOG and NOX converted 1 and 2 into their
photoproducts exclusively upon blue light excitation. Whereas
miniSOG showed no preference between the two substrates,
NOX was ca. 7 times more efficient towards 2 than 1. Light
irradiation also switched on the catalytic activity of miniSOG in
PB/NADH. The flavoprotein achieved full conversion of 200 μM
[
13, 25]
II
compounds
with respect to the Ru complexes 4 and 5.
IV
Furthermore, FAD had superior activity towards these Pt
substrates with respect to 3. The use of 2 was also aimed at
gauging the role played by the charge of the substrates on the
catalysis, having this complex a neutral alkyl chain at the axial
position compared to the negatively charged succinate of 1.
1
and 2 in ca. 4 min. In the case of 1, this is approximately 5
-1
times less efficient than free FMN (TOF 35.6 ± 4.3 min , Figure
S50).
The flavoprotein catalysts tested were miniSOG (mini Singlet
[
26]
Oxygen Generator),
NOX (NADH oxidase from Thermus
To our surprise, NOX behaved differently, activating 1 and 2
in the dark when co-incubated with 2 mM NADH. Under such
conditions, 10 μM NOX completely converted 1 in less than 7.5
min, while free FAD did not give any reaction with 1 over 3 h
[
27]
thermophilus),
GOX (glucose oxidase from Aspergillus
[
28]
[29]
niger)
and GR (glutathione reductase from S. cerevisiae).
MiniSOG is an FMN-containing (flavin mononucleotide) small
protein investigated as a genetically encodable photosensitizer
(
vide supra). The TOF values of 1 and 2 for NOX were
[
30-32]
for the selective generation of singlet O
enzyme generates hydrogen peroxide (H
NADH, while the eukaryotic GOX naturally oxidizes glucose to
and D-glucono-δ-lactone. NOX and GOX have been both
2
.
The bacterial NOX
-1
-1
estimated to be 4.3 ± 1.6 min and 8.3 ± 1.6 min respectively,
using less than 2 μM of flavoprotein to allow monitoring of the
reaction by NMR.
2
O
2
) from O oxidizing
2
2 2
H O
[
27, 33]
widely exploited in biocatalysis.
GR is a NADPH-dependent
oxidoreductase exerting a central role in glutathione metabolism
[
34]
for most aerobic organisms.
Conversely from the other
flavoproteins, GR was selected because does not generate
reactive oxygen metabolites.
Different chemical environments surround the flavin binding-
pocket in these four flavoproteins, controlling solvent and
substrate accessibility to the active site. As shown in Figure 3,
[
35]
miniSOG and NOX, have more exposed flavins than GOX and
GR, in which FAD is deeply buried into the protein scaffold.
2
Solvent accessible surface areas of the flavins are 45.50 Å for
2
2
miniSOG, 67.92 Å for NOX, while only 2.39 Å for GOX and
2
4
.01 Å for GR. Moreover, they display different electrostatic
surfaces in the proximity of the flavin binding-pocket (Figure
S39–43). At pH 6–7, the NOX and GR active sites are neutral,
whereas miniSOG and GOX display positive and negative
electrostatic charges respectively.
Figure 3. Electrostatic surface potential of the binding sites of (a) miniSOG,
(
b) NOX, (c) GOX and (d) GR (calculated using Bluues server). Red and blue
[36]
colors represent anionic and cationic residues, respectively.
The discovery of NOX catalytic activity in the dark has broad
Unless otherwise stated, photocatalysis experiments (Figure
S44–76) were performed employing 10 μM flavoprotein catalysts,
IV
relevance for understanding the mechanism of action of Pt
IV
anticancer agents. It is common assumption that Pt complexes
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Angewandte Chemie International Edition
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COMMUNICATION
are converted into active species by biological molecular
reductants, such as glutathione or ascorbic acid, under
(Figure 4a) when 1 mM 1 was incubated with equimolar NADH.
Under these conditions, the enzyme worked faster because it
had access to higher concentrations of electron acceptors (1
[
37]
physiological conditions.
Nevertheless, NOX-catalyzed
activation of 1 and 2 in the presence of 2 mM NADH is
significantly rapid and suggests that flavoproteins can provide
alternative and highly efficient activation pathways for
and O
2
), and produced lower amounts of H
2
O
2
because the
and the metal
hydride of NADH ought to be shared between O
2
complex reduction reactions. Conversely, miniSOG production
of H is independent of the presence of 1 (Table S1), as it is
likely occurring via photosensitization.
[
38]
metallodrugs.
2 2
O
[
40]
Considering that the cellular concentration of NADH is in the
[
39]
0
.1–0.2 mM range,
convert 200 μM 1 with an equimolar quantity of NADH in PB
Figure S64). NOX naturally uses O as electron acceptor to
generate H in the presence of NADH.
we evaluated the capacity of NOX to
The capacity of miniSOG and NOX to act as bioorthogonal
IV
catalysts towards Pt prodrugs was investigated using 1 in cell
(
2
culture medium, where components such as proteins, vitamins
and salts can interfere with the activation process. Reactions in
the presence of NADH (2 mM) showed that miniSOG converted
1 in the biological environment only under light irradiation, while
the same reaction occurred already in the dark with NOX (Figure
4b and Figure S75). The flavoproteins retained practically the
same selectivity and efficiency of free FAD under the same
conditions (Figure S76).
[
27]
2
O
2
At such low
concentration, the enzyme consumed NADH too rapidly,
precluding any catalytic conversion of 1. For this reason, the
reaction was studied under N
determined that NOX could activate approximately a third of 1 in
the absence of O , revealing that indeed flavoenzymes may turn
2
atmosphere. Accordingly, we
2
on Pt drug activity under certain cellular conditions, i.e. hypoxia.
IV
Although the Pt conversion did not reach completion, the
A molecular description of the catalytic mechanism through
concentration of activated 1 should reasonably be sufficient to
IV
II
which free flavins and flavoproteins activate Pt and Ru
induce cell death in cancer tissues.
complexes requires further investigations and is out of the scope
of this manuscript. Our previous study suggests the catalysis is
linked to the generation of the reduced forms of FAD and FMN
(
2 2
e.g. FADH and FMNH ), either through photoinduced electron
transfer (MES) or by hydride transfer (NADH/NADPH). High
levels of electron donors and light help increasing the catalytic
efficiency of the flavins by stabilizing their active species and
consequently enhancing reaction rates. Metal complexes may
form transient adducts with the reduced flavins and undergo
[
11]
chemical and photochemical transformations.
Nevertheless, it is clear from the results of this study that
protein scaffolds play a crucial role in governing the accessibility
of the metal substrate to the flavin catalytic site. So, the negative
electrostatic surface of GOX and the shielded channel in which
its FAD is bound prevent any interaction with the negatively
charged 1. Consistently, we observed that the consumption of
glucose by GOX was not affected by the presence of equimolar
1
(Table S2). Although with poor efficiency, GOX activated 2 in
agreement with the absence of charged chemical groups in the
[
41]
complex and its lower reduction potential compared to 1.
In the case of miniSOG and NOX, however, the protein
scaffold enables the artificial catalysis by facilitating the
formation of reduced FMN/FAD and favoring its stabilization for
IV
the subsequent electron transfer interaction with the Pt
Figure 4. (a) NOX catalytic consumption rate of NADH (magenta) and
substrates. Actually, miniSOG and NOX have more solvent
exposed flavins and suitable electrostatic surfaces to allow metal
substrates to access the active site. The role played by the
protein scaffold is dramatic for NOX, which is an enzyme
optimized by nature to transfer hydrides from NADH to electron
acceptors. Consistently, NOX activity towards 1 and 2 is
observed almost instantaneously in the dark with NADH. On the
contrary, miniSOG, a protein derived from phototropin 2, which
naturally does not use NADH as cofactor, requires light
activation. Similarly to NOX and in contrast to GOX, GR has a
mostly neutral FAD binding pocket and slightly positive surface
2 2
generation of H O (blue) measured employing a 1:1 ration of 1 and NADH at
a concentration of 1 mM; (b) Dark catalytic activity of NOX in cell culture
1
medium (pH 7) in the presence of NADH. H NMR spectra were recorded for
1
solutions of 200 µM 1 and 10 µM NOX and 2 mM NADH. H NMR signal
–
–
–
labelling:
p
Pt–OCOCH
2
CH
2
CO
2
,
p
Pt–OCOCH
2
CH
2
CO
2
, ! free
–
O
2
CCH CH CO .
2
2 2
At higher concentrations of electron donor in aerated
solution, the enzyme reaction pathway is altered by 1, which
effectively competes with O and intercepts electrons from the
reduced flavoenzyme. In fact, the activity of NOX was increased
by 2.3 fold while the production of H simultaneously lowered
2
charge, which allow catalytic activation of the
1 and 2.
2
O
2
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Angewandte Chemie International Edition
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COMMUNICATION
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[
[
[
21]
22]
23]
IV
II
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operating either in the dark or upon light excitation. Some of
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activated metal-based prodrugs, whose biological effects could
be triggered endogenously by bioorthogonal flavoprotein
catalysts.
S. Bose, A. H. Ngo, L. H. Do, J. Am. Chem. Soc. 2017, 139, 8792-
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Figure S30 and 38).
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Keywords: photocatalysis, flavoproteins, metal-based
prodrugs, bioorthogonal, photochemotherapy
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Silvia Alonso-de Castro, Aitziber L. Cortajarena,
Fernando López-Gallego,* Luca Salassa,*
Bioorthogonal inorganic
photochemistry: Flavins
function as selective
Page No. – Page No.
photocatalysts for the
activation of metal-based
prodrugs in the biological
environment. Differently
from typical catalysis, metal
complexes are substrates
and not catalysts in these
reactions!
Bioorthogonal Catalytic Activation Of Pt And
Ru Anticancer Complexes By FAD and
Flavoproteins
(
(Insert TOC Graphic here))
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