Photocontrol of Antibacterial Activity: Shifting from UV to Red Light Activation

Article abstract of DOI:10.1021/jacs.7b09281

The field of photopharmacology aims to introduce smart drugs that, through the incorporation of molecular photoswitches, allow for the remote spatial and temporal control of bioactivity by light. This concept could be particularly beneficial in the treatment of bacterial infections, by reducing the systemic and environmental side effects of antibiotics. A major concern in the realization of such light-responsive drugs is the wavelength of the light that is applied. Studies on the photocontrol of biologically active agents mostly rely on UV light, which is cytotoxic and poorly suited for tissue penetration. In our efforts to develop photoswitchable antibiotics, we introduce here antibacterial agents whose activity can be controlled by visible light, while getting into the therapeutic window. For that purpose, a UV-light-responsive core structure based on diaminopyrimidines with suitable antibacterial properties was identified. Subsequent modification of an azobenzene photoswitch moiety led to structures that allowed us to control their activity against Escherichia coli in both directions with light in the visible region. For the first time, full in situ photocontrol of antibacterial activity in the presence of bacteria was attained with green and violet light. Most remarkably, one of the diaminopyrimidines revealed an at least 8-fold difference in activity before and after irradiation with red light at 652 nm, showcasing the effective "activation" of a biological agent otherwise inactive within the investigated concentration range, and doing so with red light in the therapeutic window.

Full text of DOI:10.1021/jacs.7b09281

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Article
Photocontrol of Antibacterial Activity: Shifting from UV to Red Light Activation
Michael Wegener, Mickel J. Hansen, Arnold J.M. Driessen, Wiktor Szymanski, and Ben L. Feringa
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Photocontrol of Antibacterial Activity: Shifting from UV to Red
Light Activation
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†,║, 1
†, 1
Michael Wegener,
Ben L. Feringa*,†
Mickel J. Hansen, Arnold J. M. Driessen,‡ Wiktor Szymanski,*,†,§ and
†Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG,
Groningen, The Netherlands.
‡Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7, 9747 AG,
Groningen, The Netherlands.
§Department of Radiology, University of Groningen, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ,
Groningen, The Netherlands.
ABSTRACT: The field of photopharmacology aims to introduce smart drugs that, through the incorporation of molecular
photoswitches, allow for the remote spatial and temporal control of bioactivity by light. This concept could be particularly
beneficial in the treatment of bacterial infections, by reducing the systemic and environmental side-effects of antibiotics.
A major concern in the realization of such light-responsive drugs is the wavelength of the light that is applied. Studies on
the photocontrol of biologically active agents mostly rely on UV light, which is cytotoxic and poorly suited for tissue pen-
etration. In our efforts to develop photoswitchable antibiotics, we introduce here antibacterial agents whose activity can
be controlled by visible light, while getting into the therapeutic window. For that purpose, a UV light-responsive core
structure based on diaminopyrimidines with suitable antibacterial properties was identified. Subsequent modification of
an azobenzene photoswitch moiety lead to structures that allowed us to control their activity against Escherichia coli in
both directions with light in the visible region. For the first time, full in situ photocontrol of antibacterial activity in the
presence of bacteria was attained with green and violet light. Most remarkably, one of the diaminopyrimidines revealed
an at least eight-fold difference in activity before and after irradiation with red light at 652 nm, showcasing the effective
“activation” of a biological agent otherwise inactive within the investigated concentration range, and doing so with red
light in the therapeutic window.
The emerging fields of photopharmacology10 and pho-
INTRODUCTION
tochemotherapy11 offer a promising approach to deal with
The often life-saving treatment of infectious diseases is
both systemic and environmental side-effects resulting
increasingly compromised by the emergence of antimi-
from chemotherapy (Figure 1). Rendering a drug pho-
crobial resistance,1 which can be traced back to a build-up
toswitchable allows for the spatial and temporal control
of antibiotics in the environment due to their excessive
over its biological activity with light. Light is, in principle,
application in both human health care and animal hus-
not harmful and easily delivered with high precision,
bandry.2 Another issue that plagues especially long-term
which has already lead to its application in antibacterial
photodynamic therapy12 for the formation of singlet oxy-
antibiotic treatment is the common occurrence of severe
adverse effects. A highly relevant case can be found in the
gen. Examples of the successful utilization of photophar-
fluoroquinolone antibiotics.3 Quinolones are a class of
macology, which is singlet oxygen independent, include
photoswitchable antimicrobials13 and anti-tumor thera-
synthetic, broad-spectrum antibiotics widely relied upon
in the clinic since their discovery in the 1960s;4 however,
safety concerns regarding the treatment with fluoroquin-
olones are still being raised, with the FDA issuing the
latest of numerous warnings as recently as 20165 (risks
include selection of resistant pathogens,6 tendon rupture7
and nerve damage8). Such a decline in the reliability of
long-established classes of antibiotics is particularly trou-
bling in the face of resistant pathogenic bacteria and the
slow pace of innovation in antibacterial drug discovery
over the last five decades.9
py,14 control of neuronal networks,15 and vision restora-
tion.16 However, so far, the application of photopharma-
cology in a reversible manner (on/off switching of bioac-
tivity with light) is often limited by the need of UV light
to effect photoisomerization. UV light is toxic to healthy
cells17 and has a limited tissue penetration depth.18 To
ultimately realize their full potential by achieving reversi-
ble control of activity in vivo (Figure 1C), photopharmaco-
logical agents should ideally be responsive to light in the
therapeutic window between 650 and 900 nm.18
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Here we report the control of antibiotic activity with
red light for the first time and show in situ photocontrol
of antibacterial activity in the presence of bacteria. In our
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Figure 1. Potential scenarios for the application of photopharmacology compared to conventional pharmacotherapy: (A) Classic
chemotherapeutic treatment without control over drug activity leads to systemic side-effects and environmental build-up that is
particularly damaging in the case of antibiotics, as this constitutes a major cause for the emergence of antimicrobial resistance.
(B) Photopharmacological agents responsive to UV light may offer only a partial solution to these issues, as a drug with suitable
properties may possibly be activated before administration by UV light irradiation, with the potentially unstable active state
losing its activity over time, thus counteracting the environmental build-up. (C) Full control of photopharmacological agents
outside as well as inside the human body to address both systemic and environmental side-effects can only be realized if the
photoswitchable drug is responsive to visible or near-IR light, both not harmful to cells and, particularly in the near-IR range,
better suited to penetrate tissue.
effort to create photoswitchable antimicrobials while
addressing the challenge to use visible or red light, we
focused on antifolates, particularly in the antibiotic trime-
thoprim as a starting point for structural design. Antifo-
lates are a class of drugs that interfere with the biosynthe-
sis of folate, by inhibition of dihydrofolate reductase
(DHFR) which catalyzes the reduction of dihydrofolate to
the active cofactor tetrahydrofolate.19 The latter plays a
vital role in the biosynthesis of essential bacterial metabo-
lites, such as the amino acids glycine and methionine, as
well as purines and thymidine triphosphate. As a result,
tetrahydrofolate depletion sets off a complex cascade that
ultimately leads to the cessation of DNA, RNA and pro-
tein synthesis.20 Antifolates include antibacterial,19,21 anti-
protozoal22 and anticancer agents.23 Among these, the
antibiotic trimethoprim24 shows a high selectivity towards
bacterial DHFR compared to mammalian DHFR.25 It is
active against a broad spectrum of Gram-positive and
Gram-negative bacteria (including Staphylococcus aureus,
Streptococcus pneumoniae, Klebsiella pneumoniae, Esche-
richia coli and Hemophilus infIuenzae) and widely used in
the clinic (especially against urinary and respiratory tract
infections).26 However, treatment with trimethoprim is
plagued by the emergence of bacterial resistance.27 This
makes it a particularly interesting candidate for photo-
pharmacology, i.e. the development of photoresponsive
analogues, as the spatial and temporal control over their
activity could reduce excessive and unwanted exposure of
bacteria to the active form of the drug, and thus drastical-
ly limit the number of bacteria with engaged resistance
mechanisms.
RESULTS AND DISCUSSION
The potential to modify the structure of trimethoprim21,28
without losing affinity towards bacterial DHFR further
encouraged us to use it as a basis for photoswitchable
antibiotics. Initially we sought to employ a simple, modu-
lar approach to attach regular, UV light-responsive azo-
benzene photoswitches to the trimethoprim core struc-
ture in different ways, thus providing quick, synthetically
facile access to a small collection of compounds that ex-
hibit high structural diversity. In this way, we expected to
identify a promising structural platform with the desired
photopharmacological properties. Subsequent exchange
of the azobenzene switch with a red-shifted analogue
would enable photoisomerization with visible light, ideal-
ly with retention of the antibacterial properties.
The first stage of this approach is illustrated in Scheme
1: Photoresponsive diaminopyrimidines of the general
structure 2 were generated by attachment of largely un-
substituted azobenzenes to the core structure of trime-
thoprim (1) (see also SI). Switches were either directly
connected to the 2, 3 and 4 positions of the drug’s meth-
oxyphenyl unit by Suzuki cross-coupling (2a-f), or by
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alkylation of the free hydroxy group in the 4 position via
different linkers (2g-j). Before starting with biological
experiments, basic photochemistry of compounds 2a-j
was studied in DMSO to ensure satisfactory
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2
3
Scheme 1. Structurally diverse set of UV light-responsive diaminopyrimidines
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5
6
7
NH2
NH2
NH2
N
2
2
MeO
MeO
(MeO)
(MeO)
(MeO)
(MeO)
3
1
3
1
N
N
UV
4
4
N
N
∆
Vis/ T
N
NH2
N
N
NH2
N
N
NH2
OMe
trimethoprim (1)
OMe
OMe
8
trans-2
cis-2
9
MeO
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MeO
1
1
N
N
3
1
4
4
N
N
N
N
N
N
3
1
N
OMe
OMe
MeO
N
OMe
2d
2f
2a: R = H
2b: R = OMe
2c
MeO
2e
R
MeO
O
OMe
1
MeO
O
1
4
2
MeO
H
MeO
MeO
1
1
4
N
4
N
N
OMe
N
O
N
N
O
OMe
N
OMe
2h: R = H
2i: R = OMe
OMe
R
2j
2g
introduction of a methoxy group in 4’-position, resulting
in compound 2i.
With this small library of structurally diverse com-
pounds 2 in hand, subsequently their antibacterial activity
was investigated against E. coli in two-fold dilution series,
both before and after irradiation with UV light at 365 nm
(see Figure S43). With the exception of 2f and 2j, which
proved to be mostly inactive up to a concentration of
20 μM, all compounds effectively inhibited bacterial
growth with various degrees of potency (see Figures S21-
S29). When comparing inhibition of growth by irradiated
and non-irradiated samples, in most cases no significant
difference was observed. However, 2i stood out in that
respect, as a considerable change in potency was evident
upon trans-cis isomerization (see growth curves in Figure
2, which translate into a MIC50 of 20 μM before and 10 μM
after irradiation). Fortunately, the cis isomer was found to
be the more active one, setting the stage for targeted
structural modifications of the photoswitch to specifically
red-shift the wavelength for trans-cis isomerization (vide
infra). In addition, the fact that the active cis isomer usu-
ally has a limited half-life and thermally isomerizes to the
more stable – but less active – trans isomer, enables an
intrinsic autodeactivation pathway that would help pre-
vent the build-up of active drugs in the environment (cf.
Figure 1B).13a
Prior to structural modification of 2i to achieve the de-
sired red-shift of its activating wavelength, we further
investigated the actual nature of its observed effect on
bacterial growth upon photoisomerization. From chemi-
cal actinometry and quantum yield determination, (see
Figure S43 and S45) we obtained ɸ(trans-cis) = 0.18 and ɸ(cis-
trans) = 0.02 for 2i, which showed that switching efficiencies
are preserved when the azobenzene core was substituted
with the trimethoprim motif.UV-Vis analysis of 2i in
aqueous medium at 30 μM showed some form of aggrega-
tion of the compound in its trans state (Figure S11A). Up-
on irradiation with 365 nm, the aggregates appear to dis-
Figure 2. Bacterial growth curves of E. coli CS1562 at increas-
ing concentrations of 2i. (A) Samples after thermal adapta-
tion. (B) Samples after irradiation with UV light at
λ = 365 nm. Error bars show s.d. calculated from measure-
ments in triplicate.
photoswitching behavior (see Figures S1-S10). In case of
2h, NMR analysis revealed degradation of the compound
upon irradiation with UV light, which was remedied by
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solve during trans-cis isomerization, providing a possible behavior upon photoisomerization in aqueous medium
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3
4
explanation for the difference in potency. However, this
effect was no longer observed at a concentration of 5 μM
(Figure S11B), whereas a difference in antibacterial activity
was still evident at that concentration. Furthermore, oth-
er diaminopyrimidines 2 exhibited similar aggregation
(Figure S13), without any significant difference in activity
between the respective photoisomers observed during
antibacterial experiments. This encouraged us to proceed
under
the
premise
that
the
difference
in
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6
Scheme 2. Synthesis of red-shifted diaminopyrimidines 12 and 13
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9
Br
X
N
1 equiv n-BuLi
1 equiv 5 or 6
1 equiv NBS
5 mol% AIBN
X
N
X
N
X
X
N
X
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CCl4
85 °C
THF
X
ꢀ
-78 °C
rt
X
X
X
NH2
N
7
3: X = F
: X = F, 72%
9: X = F
10
X
MeO
O
4
8: X = Cl, 75%
: X = Cl
: X = Cl
N2
BF4
X
N
1.1 equiv t-BuOK
X
N
NH2
X
DMSO
rt
N
X
OMe
5: X = F
: X = Cl
X
6
12
: X = F, 46% over two steps
13: X = Cl, 34% over two steps
NH2
N
NH2
N
MeO
MeO
MeO
HO
aq. HBr (48%)
100 °C
66%
N
NH2
N
NH2
OMe
OMe
11
1
antibacterial activity of 2i effected by photoisomerization
does indeed directly originate from structural changes on
a molecular level, and is not caused by differences in sol-
ubility in the bacterial growth medium. We envisioned
that further modification of the basic structure of 2i
would not only bring forth the desired red-shift of the
Figure 3. Bacterial growth curves of E. coli CS1562 at increas-
ing concentrations of 12. (A) Samples after thermal adapta-
tion. (B) Samples after irradiation with green light at
λ = 527 nm. Error bars show s.d. calculated from measure-
ments in triplicate.
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Figure 4. Bacterial growth curves of E. coli CS1562 at increas-
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2
3
4
ing concentrations of 12. (A) Samples after thermal adapta-
tion. (B) Samples after in situ irradiation with green light at
λ = 527 nm in the presence of bacteria. Error bars show s.d.
calculated from measurements in triplicate.
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6
7
8
activating wavelength, but will also resolve any issue of
possible aggregation in aqueous medium.
Among ongoing efforts to realize azobenzene pho-
toswitches that can be addressed with visible light,29 the
groups of Hecht30 and Woolley31 made significant progress
with the design and synthesis of azobenzenes bearing
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1
Figure 5. (A) UV-Vis absorption spectra of 13 before and after irradiation with various light sources in DMSO. (B) Partial H
NMR spectra of 13 before and after irradiation with various light sources in DMSO-d6 (C) Bacterial growth curves of E. coli
CS1562 at increasing concentrations of 13 after thermal adaptation. (D) Bacterial growth curves of E. coli CS1562 at increasing
concentrations of 13 after irradiation with red light. Irradiation was performed on a solution of 13 in DMSO for 2.5 h with red
light at λ = 652 nm. Error bars show s.d. calculated from measurements in triplicate.
fluoro-, chloro- or methoxy-substituents in all positions
ortho to the azo moiety. The influence of these substitu-
ents allows for the effective use of lower-energy n-π*
excitation to trigger trans-cis photoisomerization. Taking
advantage of this approach in the creation of red-shifted
analogues of structure 2i, we were particularly interested
in the tetra-fluoro-substituted azobenzenes, since such a
modification would provide us with a structure closest to
2i in terms of size. In order to synthetically access the
modified structure 12 (Scheme 2), we utilized the meth-
odology, recently introduced by our group, for the facile
preparation of ortho-substituted azobenzenes:32 Lithiation
of 3,5-difluorotoluene (3) and subsequent conversion with
the corresponding diazonium salt 5 gave azobenzene 7 in
good yield. After radical bromination, bromide 9 was used
in an alkylation of phenol 11, which in turn could be di-
rectly obtained from trimethoprim (1) by selective ether
cleavage.28b Thus, photoresponsive diaminopyrimidine 12
was readily prepared by direct modification of the com-
mercially available drug.
analysis, see Figure S14D), while irradiation with violet
light at 400 nm (see Figure S42 and SI for specifications)
can be applied to switch back to the trans state. Pho-
toisomerization could also be performed selectively in
aqueous LB broth (used herein as medium for bacterial
growth), with no sign of fatigue evident over numerous
cycles at 37 °C (Figures S14B and C).
With a photoswitchable aminopyrimidine in hand that
can be photoisomerized in either direction with visible
light, its antimicrobial activity was investigated. Interest-
ingly, 12 showed increased activity against E. coli when
compared to 2i in both isomeric forms.
Furthermore, we noted a significant difference in anti-
bacterial activity between the two forms remained at
concentrations of 4–7 μM (Figure 3), with a genuine “acti-
vation” effect evident upon trans-cis isomerization with
green light (translating into a change of MIC50 from 10 μM
to 5 μM, see Figure S31). While these results were ob-
tained for pre-irradiation of the samples in the absence of
bacteria, analogous experiments with true in situ irradia-
tion after inoculation of bacteria yielded a similar effect
(Figure 4), as photoisomerization with non-toxic green
light could be achieved without influencing the bacterial
Efficient trans-cis isomerization of 12 in DMSO was ef-
fected with green light at 527 nm (see S44 and SI for spec-
ifications, for PSS of cis:trans = 89:11 established by NMR
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to the inactive trans form can be performed within the
growth with the applied light. This marks an important
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next step towards the reversible photocontrol of an anti-
bacterial agent in vivo with biocompatible wavelengths of
light.
visible light range, experiments were conducted with
violet light at 400 nm (cf. Figures 5A and 5B). This al-
lowed us to treat bacteria with a red-light-irradiated,
“activated” sample of 13 in two two-fold dilution series,
before irradiating half of the inoculated bacteria with
400 nm light, evidently without harming the bacteria in
the process (Figure 6B). We were pleased to find that,
indeed, samples irradiated with violet light were effective-
ly “deactivated” again, with a MIC50 > 80 μM, just as pre-
viously observed for the trans isomer, properly restored
(compared once again to the MIC50 = 10 μM of the active
cis isomer protected from violet light irradiation, Figure
6A).
Having successfully realized control over bacterial
growth with green light using photoresponsive aminopy-
rimidine 12, we sought to take our approach a step further
by red-shifting the wavelength of activation even more.
For this purpose, diaminopyrimidine 13 (as a structural
analogue of 2i and 12) was synthesized, featuring four
chloro substituents in ortho positions of the azobenzene
(see Scheme 2). Tetra-ortho-chloroazobenzenes have
been reported to engage in trans-cis isomerization upon
irradiation with red light between 630 and 660 nm.31,33
Indeed, irradiation of 13 with red light at 652 nm (see SI
for specifications) effected photoisomerization to a PSS of
cis:trans = 87:13 (in DMSO, Figures 5A and 5B), albeit
after prolonged irradiation times of 2–3 h. Faster isomeri-
zation was observed for irradiation with green light at
527 nm; however, this only resulted in a PSS cis:trans
ratio of about 1:1. A compromise between irradiation time
and PSS for the cis-trans isomerization of 13 could be
reached by using a strong white light source in combina-
tion with a filter cutting off wavelengths below 530 nm:
Using this light source, a PSS of cis:trans = 69:31 could be
reached within 10 min. Photoisomerization of 13 also
proceeded cleanly in aqueous medium and without fa-
tigue over numerous cycles (Figure S17).
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Due to our interest in the farthest possible shift of our
photoswitchable agents’ response towards the red, we
immediately focused on the influence of red light irradia-
tion on the antibacterial activity of 13. In that regard,
initial experiments performed analogously to the other
compounds in this study (two-fold dilution series in 96-
wells plates) yielded disappointing results, with a notice-
able but unsatisfactory difference in growth evident (Fig-
ure S34). However, we imagined that this lack of differ-
ence in activity before and after irradiation might well be
due to ineffective photoswitching in the 96-wells plates,
as the light source was usually positioned at a distance of
11.5 cm from the plate to ensure maximum coverage. To
verify this assumption, one half of a divided stock solu-
tion of 13 in DMSO was irradiated for 2.5 h with red light
at 652 nm at a distance < 1 cm, before treating bacteria
with the two separate samples in two-fold dilution series.
To our delight, this experiment revealed a dramatic pho-
toactivation effect: Whereas non-irradiated 13 remained
largely inactive with a MIC50 > 80 μM (Figure 5C), red
light-irradiated 13 induced bacteriostasis down to 20 μM,
with an observed MIC50 of 10 μM (Figure 5D). It is worth
noting at this point that photoisomerization of 13 with red
light at close proximity also works effectively in aqueous
medium (Figure S18).
Figure 6. Two series of bacterial growth curves of E. coli
CS1562 at increasing concentrations of 13 irradiated with red
light at λ = 652 nm for 3 h in DMSO before treatment of
bacteria. One series of bacteria (A) was protected from fur-
ther irradiation. The other series (B) was irradiated with
violet light at λ = 400 nm after inoculation. Error bars show
s.d. calculated from measurements in triplicate.
While the inefficient photoisomerization of 13 with red
light prevented us from conducting red light irradiation
experiments in the presence of bacteria in our model in
vitro system, the results presented herein mark a signifi-
cant development in our ongoing efforts to create pho-
toresponsive antibiotics, as we were able, for the first
time, to control the activity (eight-fold difference) of an
antibacterial agent with light beyond the 650 nm mark,
To exclude the possibility that factors such as changes
in solubility upon photoisomerization have an undesired
influence on the antibacterial activity of 13 in our experi-
ments, we considered it vital to demonstrate that the
photocontrol of its activity is reversible. Taking advantage
of the fact that switching 13 back from its active cis form
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Research Council (Advanced Investigator Grant no. 694345
to B. L. F.).
which constitutes the lower margin of therapeutic win-
dow,18 bringing us an important step closer to future in
vivo control of photoswitchable antibiotics.
1
2
3
ABBREVIATIONS
4
CONCLUSIONS
s.d., standard deviation; PSS, photostationary state.
5
6
7
8
We successfully developed photoresponsive, antibacterial
diaminopyrimidines bearing azobenzene photoswitches,
whose activity can be controlled by light of various wave-
lengths. Identification of a UV light-responsive core struc-
ture with suitable antibacterial properties and subse-
quent, targeted modification of the photoswitch moiety
lead us to structures whose activity could be increased
upon irradiation with green and red light. Remarkably,
for the first time, these compounds allowed for the full in
situ photocontrol of antibacterial activity with green and
violet light, making it possible to trigger both the “activa-
tion” as well as “deactivation” of the antibacterial agent in
the presence of bacteria. Most significantly, diaminopy-
rimidine 13 revealed an at least eight-fold difference in
activity before and after irradiation with red light (previ-
ous efforts in our group show that much lower differences
are already sufficient for spatiotemporal patterning of
bacteria).13a,b More importantly, apart from showcasing
the effective “activation” of a biological agent otherwise
inactive within the investigated concentration range, we
were able to do so while also shifting the wavelength of
activation from the UV range towards red light in the
therapeutic window.
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* w.szymanski@umcg.nl
* b.l.feringa@rug.nl
Present Addresses
║M. W.: Helmholtz Centre for Infection Research, Medicinal
Chemistry, Inhoffenstraße 7, 38124 Braunschweig, Germany.
Author Contributions
1
These authors contributed equally.
All authors have given approval to the final version of the
manuscript.
ACKNOWLEDGMENT
M. W. gratefully acknowledges a postdoctoral fellowship of
the German Research Foundation (WE 5922/1). We gratefully
acknowledge generous support from NanoNed, The Nether-
lands Organization for Scientific Research (NWO-CW, Top
grant to B. L. F. and NWO VIDI grant no. 723.014.001 for W.
S.), the Royal Netherlands Academy of Arts and Sciences
(KNAW), the Ministry of Education, Culture and Science
(Gravitation programme 024.001.035) and the European
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