Controlling Antibacterial Activity Exclusively with Visible Light: Introducing a Tetra-ortho-Chloro-Azobenzene Amino Acid

Article abstract of DOI:10.1002/chem.202102370

The introduction of a novel tetra-ortho-chloroazobenzene amino acid (CEBA) has enabled photoswitching of the antimicrobial activity of tyrocidine A analogues by using exclusively visible light, granting spatiotemporal control under benign conditions. Compounds bearing this photoswitchable amino acid become active upon irradiation with red light, but quickly turn-off upon exposure to other visible light wavelengths. Critically, sunlight quickly triggers isomerisation of the red light-activated compounds into their original trans form, offering an ideal platform for self-deactivation upon release into the environment. Linear analogues of tyrocidine A were found to provide the best photocontrol of their antimicrobial activity, leading to compounds active against Acinetobacter baumannii upon isomerisation. Exploration of their N- and C-termini has provided insights into key elements of their structure and has allowed obtaining new antimicrobials displaying excellent strain selectivity and photocontrol.

Full text of DOI:10.1002/chem.202102370

Communication
doi.org/10.1002/chem.202102370
Chemistry—A European Journal
Controlling Antibacterial Activity Exclusively with Visible
Light: Introducing a Tetra-ortho-Chloro-Azobenzene Amino
Acid
Xavier Just-Baringo,*[a, b] Alejandro Yeste-Vázquez,[a] Javier Moreno-Morales,[c]
Clara Ballesté-Delpierre,[c] Jordi Vila,[c, d] and Ernest Giralt[a]
Dedicated to Prof. Mercedes Álvarez on her retirement
Abstract: The introduction of a novel tetra-ortho-chloroazo-
benzene amino acid (CEBA) has enabled photoswitching of
Antibiotic resistance is an increasing concern worldwide.
Accumulation of antibiotics in the environment increases the
the antimicrobial activity of tyrocidine A analogues by using
evolutionary pressure on microorganisms, which evolve and
exclusively visible light, granting spatiotemporal control
adapt at a great pace. Moreover, the number of new antibiotic
under benign conditions. Compounds bearing this photo-
classes entering the pipeline has been alarmingly low during
switchable amino acid become active upon irradiation with
the last decades.[1] Although new antimicrobials are still needed,
red light, but quickly turn-off upon exposure to other
a change of paradigm on how we fight bacterial infections is on
visible light wavelengths. Critically, sunlight quickly triggers
high demand as new drugs are bound to suffer from the
isomerisation of the red light-activated compounds into
emergence of resistances as well.[2] On this regard, gaining the
their original trans form, offering an ideal platform for self-
ability to switch drugs on and off offers a unique opportunity to
deactivation upon release into the environment. Linear
avoid the release of active drugs into the environment, thus
analogues of tyrocidine A were found to provide the best
reducing the chances of resistance arising and disseminating. In
photocontrol of their antimicrobial activity, leading to
particular, visible light can be used as a clean and harmless
compounds active against Acinetobacter baumannii upon
stimulus to modulate the structure of a molecule and thus, its
isomerisation. Exploration of their N- and C-termini has
provided insights into key elements of their structure and
biological profile.[3]
Natural antimicrobial peptides play a crucial role as first line
has allowed obtaining new antimicrobials displaying ex-
of defence against pathogenic bacteria.[4] These are produced
cellent strain selectivity and photocontrol.
by a plethora of microorganisms, but also by complex animals,
including humans.[5]
Previous works have shown how the introduction of photo-
switches into small molecules can allow control over the activity
of antimicrobial compounds, but only recently has the control
of antibacterial activity with visible light been reported.[6] In this
context, a photoswitchable amino acid fully operated under
[a] Dr. X. Just-Baringo, A. Yeste-Vázquez, Prof. E. Giralt
Institute for Research in Biomedicine (IRB Barcelona)
Barcelona Institute of Science and Technology (BIST)
Baldiri Reixac 10, 08028 Barcelona (Spain)
visible light is a coveted goal in order to gain control over the
structure of peptides under harmless conditions.[7,8] Tetra-ortho-
[b] Dr. X. Just-Baringo
chloro-azobenzenes can be isomerised efficiently under red or
green light into their cis form and will quickly return to their
trans isomer under other visible light sources unless kept in the
dark. Importantly, the use of harmful UV light is avoided. This
feature offers a unique platform for safe in situ activation of
drugs, also through living tissue, and subsequent deactivation
upon release into the environment after their therapeutic use,
once exposed to sunlight.
The pioneering work of Feringa and co-workers showed
that photocontrol of antibacterial activity to fight the appear-
ance of resistances is possible.[6a] Short after, Ulrich and
Komarov reported the use of visible light to trigger the
Laboratori de Química Orgànica
Facultat de Farmàcia
IBUB, Universitat de Barcelona
08028 Barcelona (Spain)
E-mail: xavier.just@ub.edu
[c] J. Moreno-Morales, Dr. C. Ballesté-Delpierre, Prof. J. Vila
Institute for Global Health (ISGlobal)
Hospital Clínic - Universitat de Barcelona
Barcelona (Spain)
[d] Prof. J. Vila
Department of Clinical Microbiology – CDB
Hospital Clínic - University of Barcelona
Barcelona (Spain)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/chem.202102370
antimicrobial activity of
a gramicidin S photoswitchable
© 2021 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution Non-Com-
mercial NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and
no modifications or adaptations are made.
peptidomimetic, which could be deactivated upon irradiation
with UV light thanks to a diaryl-ethane photoswitch (Fig-
ure 1A).[6b,f,i] Other works have also used gramicidin S as a
platform to study the photocontrol of antibacterial activity in
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Chemistry—A European Journal
Figure 1. Photoswitchable building blocks used to photocontrol the anti-
bacterial activity of tyrocidines’ and gramicidins’ analogues.
cyclic peptidomimetics (Figure 1B).[6g] However, all reports on
the photocontrol of peptidomimetic’s antibacterial activity
require the use of UV light for one of the isomerisations. In this
context, we embarked on the design of a photoswitchable
amino acid building block that could be operated with visible
light for both trans!cis and cis!trans isomerisations, resulting
in CEBA (tetra-ortho-chloro-diethylelene-azobenzene Amino
acid) (Figure 1C). Importantly, it was crucial to achieve fast cis!
trans upon exposure to sunlight and other white light sources
in order to achieve self-deactivation of the compounds upon
release into the environment.
Figure 2. Tyrocidine A and examples of cyclic and linear visible light active
photoswitchable analogues described in this work.
Tyrocidine A is a cyclic decapeptide with antimicrobial
activity originally found along gramicidin in tyrothricin, a
mixture of peptide antibiotics isolated from Brevibacillus brevis.
Like gramicidin, tyrocidine is primarily active against Gram-
positive bacteria. Modification of its structure has granted
access to analogues with improved biological profiles such as
an expanded panel of susceptible bacterial strains and higher
therapeutic indexes.[9] However, its precise mechanism of action
has remained elusive.[10]
We hypothesized that modification of tyrocidine A’s
structure by introducing our tetra-ortho-chloro-azobenzene
amino acid (CEBA) in substitution of . Gln6 and Tyr7 would
distort the natural peptide’s β-hairpin secondary structure while
in its trans configuration. However, upon isomerisation under
red light, the cis form would become the major isomer and re-
establish the biologically active secondary structure of the
natural product (Figure 2).
Scheme 1. Synthesis of tetra-ortho-chloro-azobenzene amino acid 3 (Tfa-
CEBA-OH). Reaction conditions: a) Zn powder (2.5 equiv), NH4Cl (1.5 equiv),
°
H2O, 2-methoxyethanol, 0 C to rt, 17 h; then FeCl3 (3.0 equiv), EtOH, H2O,
°
0 C, 1.5 h. b) 6 (1 equiv), AcOH, rt, 18 h, 54% (2 steps). c) Pd(OAc)2, NCS
°
(6 equiv), AcOH, 140 C, 19 h, 85%. Tfa=trifluoroacetyl; NCS=N-chlorosucci-
nimide.
Azobenzene amino acid 3 possesses the required features
and its synthesis proved to be reliable and easily scalable,
offering a useful route to this building block (Scheme 1). First,
3-(4-nitrophenyl)propanoic acid 4 was turned into the corre-
sponding nitroso derivative 5 and upon condensation with
aniline 6, the azobenzene scaffold was set, yielding amino acid
7. Subsequent palladium-catalysed chlorination gave the de-
sired amino acid photoswitch 3 in very good yield.[11–13] Overall,
Tfa-CEBA-OH (3) was obtained in 46% yield in just three steps,
needing only one chromatographic purification at the end of
the synthesis.
At this stage, we were ready to synthesize a photoswitch-
able analogue of tyrocidine A using solid phase peptide
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synthesis (SPPS) starting from Gln5 anchored from its amide
side chain to the resin (Scheme 2). Elongation of the peptide
was performed uneventfully and TFA-CEBA-OH (3) was intro-
duced last, prior to acidic cleavage to produce 8. Subsequent
basic hydrolysis in solution did not yield the expected N- and C-
termini unprotected peptide, but a product with the same m/z
as the target photoswitchable cyclic tyrocidine A analogue.
Further analysis and mechanistic studies revealed that basic
conditions had triggered cyclisation of the Asn residue side
chain onto the allyl ester to form succinimide 9, whose
unanticipated structure prompted us to study it further and
carry on with the remaining deprotection step.[14] Thus, the
azide group in 9 was reduced to yield the corresponding amine
2 via a mild Staudinger reduction.[15] Alternatively, and in order
to obtain the desired cyclic analogue 1, The photoswitch was
introduced as its allyl carbamate analogue Alloc-CEBA-OH,
which was obtained quantitatively from 3 in a two-step, one-
pot operation.[14] Once peptide 10 was ready, both termini were
deprotected under Pd catalysis to yield 11. At this stage,
conditions were screened to optimise cyclisation of the peptide,
which proved to be very reagent and solvent dependent.[14] In
the end, the cyclised product was most efficiently obtained in
solution, and a one-pot Staudinger reduction was performed
last to reveal the free amine in 1.
A kinetic profile of trans to cis isomerisation under red light
(650 nm) was recorded and in both cases a virtually identical
behaviour was observed.[14] Upon exposure to red light, the
amount of cis isomer reaches a plateau after ca. 40 min,
indicating the minimum time required to achieve the highest
ratio of cis isomer under these conditions. The reverse process
was also studied using daylight to irradiate the samples, as it
would be the ideal source of light to trigger deactivation of the
compounds.[14] To our delight, less than three minutes were
required for the opposite isomerisation, highlighting the
viability of the proposed idea of self-deactivation upon release
into the environment (Figure 3). Importantly, the same behav-
iour was observed for all compounds described in this work.
With 1 and 2 in our hands, we carried out some preliminary
antibacterial activity assays, which showed a more interesting
biological profile for 2 (see below). This finding prompted us to
investigate further the role of the N- and C-termini moieties
present in this linear analogue. Thus, using the same synthetic
approach, we prepared a small library of linear peptides with
modifications on both termini. These included substitution of
the photoswitchable amino acid CEBA for a photoswitchable
carboxylic acid analogue without the 2-aminoethyl tail (12) and
substitution of the Gln-derived succinimide moiety for the
corresponding glutamine carboxylic acid or glutamine amide
(Figure 4).
Photoisomerisation of the compounds was studied using 1
and 2 as models for cyclic and linear compounds, respectively.
Scheme 2. Synthesis of linear and cyclic Visible Light photoswitchable tyrocidine A analogues 1 and 2. Reaction conditions: a) see Supporting Information for
details on manual and microwave-assisted SPPS methods. b) Alloc-CEBA-OH or Tfa-CEBA-OH (2.3 equiv), DIC (2.5 equiv), oxyma (2.5 equiv), DMF, rt, 16 h. c)
°
TFA/CH2Cl2 (95:5), rt, 5×45 min. d) NaOH (3.5 equiv), THF, MeOH, H2O, rt. e) PMe3 (3 equiv), H2O, THF, 0 C to rt. f) Pd(PPh3)4 (10 mol%), PhSiH3 (20 equiv),
°
CH2Cl2, rt, 15 min. g) HATU (2 equiv), DIPEA (6 equiv), DMF, rt, 16 h; then PMe3 (5 equiv), H2O, 0 C to rt. Alloc=Allyloxycarbonyl; DIC=N,N’-
diisopropylcarbodiimide; oxyma=ethyl cyanohydroxyiminoacetate; DMF=dimethylformamide; TFA=trifluoroacetic acid; THF=tetrahydrofuran HATU=1-[bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIPEA=diisopropylethylamine.
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baumannii, showing lower MICs for analogue 13 (8–16 μgmLÀ 1)
than analogue 14 (>64 μgmLÀ 1) in either photostationary state,
whereas they behaved very similarly against the rest of the
panel. Removal of the 2-aminoethyl tail from CEBA (analogues
15–17) drastically changed the biological profile of the
peptides, as they lost activity against most strains, including
Gram-positive bacteria. However, upon irradiation at 650 nm,
an increased susceptibility was observed for compounds 16 and
17 against S. pyogenes. In particular, 17 displayed great
selectivity against this strain, whereas it remained inactive
against all the other ones tested during this study.
Figure 3. Photoisomerisation of 2. UPLC analysis of sequential trans!cis and
cis!trans isomerisation of 2.
Depending on the compound, differences were also
observed in terms of their haemolytic effect.[14] TA, 2, 13 and 15
were less haemolytic in their non-irradiated state compared to
the irradiated one, whereas compounds 1, 14, 16 and 17
showed the opposite effect. In the case of compound 17, which
showed good antibacterial activity against S. pyogenes in the
irradiated state, a high IC50 value (173 μgmLÀ 1) was also
observed (low haemolytic effect), which can likely be attributed
to its lack of a net positive charge under the assay conditions
and is an optimistic starting point for therapeutic purposes.
In conclusion, we have developed an efficient synthesis of
the novel tetra-ortho-chloro-photoswitchable azobenzene
amino acid (CEBA) that can be incorporated into peptidic
sequences to grant photocontrol using exclusively visible light.
This has led to the development of 2, a photoswitchable
tyrocidine A linear analogue with a C-terminal succinimide
moiety that can be activated under red light to become active
against a clinically relevant Gram-negative strain such as A.
baumannii. Importantly, the compound quickly becomes in-
active against this pathogenic strain upon exposure to sunlight,
enabling self-immolation upon release to the environment.
Taking into consideration the available data for the linear
analogue 17, selectivity and photocontrol have been observed
against S. pyogenes, achieving the highest IC50 value in both
photostationary states out of all the analogues tested. To
confirm their antimicrobial effect against this species, further
studies increasing the number of S. pyogenes strains should be
conducted. In addition, the decreased toxicity (haemolytic
assay) observed in some of the non-irradiated compounds is an
important added value of this strategy that may also contribute
to preserving the ecosystem and should be considered in
further studies. We hope that these findings will help advancing
in the development of more efficient strain selective and self-
Figure 4. Library of linear photoswitchable analogues of tyrocidine A.
With all the analogues in hand, we next tested them against
a panel of Gram-positive and Gram-negative bacterial strains
(see the complete table in the Supporting Information). To our
surprise, cyclic analogue 1 displayed a lack of photocontrol
against all strains tested and its biological profile was very
similar to TA, with the exception of A. baumannii (Table 1). Very
interestingly, linear analogue 2 also had a similar profile,
although photoswitchability against A. baumannii (both a
susceptible -ATCC 19606- and a colistin-resistance strain -CR17-)
was now observed and at least a 3-fold decrease in the MIC of
the compound was observed upon activation with red light.
Substitution of the Gln-derived succinimide for the correspond-
ing glutamine amide (13) or carboxylic acid (14) had completely
opposite effects on the compounds’ activity against A.
Table 1. Selected in vitro antibacterial activities of TA and its photoswitchable analogues.[a]
TA
1
2
13
14
15
16
17
NoIrr Irr[b]
NoIrr Irr[b]
NoIrr Irr[b] NoIrr Irr[b] NoIrr Irr[b]
NoIrr Irr[b]
NoIrr Irr[b]
NoIrr Irr[b]
Bacillus subtilis[c]
4
4
4
4
4
4
8
4
2
4
2
4
1
4
2
4
4
8
16
4
>64 >64 64
>64 64 >64 16
64
>64 >64
>64 16
Streptococcus pyogenes 016[c]
Escherichia coli ATCC 25922[d]
Acinetobacter baumannii ATCC
19606[d]
>64 >64 >64 >64 >64 64
16
>64 32
8
>64 >64 >64 >64 >64 >64 >64 >64
>64 >64 >64 >64 >64 >64 >64 >64
16
>64 >64 64
8
8
Acinetobacter baumannii CR17[d]
32
16
>64 >64 >64 32
16
8
>64 >64 >64 >64 >64 >64 >64 >64
[a] Measured as minimum inhibitory concentration (MIC) in μgmLÀ 1. [b] Plates were irradiated at 650 nm for 40 min before incubation in the dark. [c] Gram-
positive strain. [d] Gram-negative strain.
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Acknowledgements
We thank AGAUR and Generalitat de Catalunya for a Beatriu de
Pinós fellowship to X.J.B. We acknowledge support from the
Spanish Ministry of Science and Innovation through the “Centro
de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-
000806-S), and support from the Generalitat de Catalunya
through the CERCA Program. Supported by Plan Nacional de I+
D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección
General de Redes y Centros de Investigación Cooperativa,
Ministerio de Economía, Industria y Competitividad, Spanish
Network for Research in Infectious Diseases (REIPI RD16/0016/
0010) - co-financed by European Development Regional Fund
“A way to achieve Europe”, Operative program Intelligent
Growth 2014–2020. We would also like to thank Prof. Pau
Gorostiza and Dr. Rossella Castagna for lending us their 650 nm
LED lamp.
[7] To the best of our knowledge, only one visible light operated
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: antibiotics
·
azobenes
·
peptidomimetics
·
photochemistry · tyrocidine A
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Manuscript received: July 1, 2021
Accepted manuscript online: July 6, 2021
Version of record online: ■■■, ■■■■
Chem. Eur. J. 2021, 27, 1–6
www.chemeurj.org
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© 2021 The Authors. Published by Wiley-VCH GmbH
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COMMUNICATION
Dr. X. Just-Baringo*, A. Yeste-Vázquez,
J. Moreno-Morales, Dr. C. Ballesté-
Delpierre, Prof. J. Vila, Prof. E. Giralt
1 – 6
Controlling Antibacterial Activity Ex-
clusively with Visible Light: Introduc-
ing a Tetra-ortho-Chloro-Azobenzene
Amino Acid
A novel photoswitchable amino acid
has provided access to tyrocidine A
analogues fully operated under visible
light. This has led to linear analogues
that have shown good photoswitch-
ability and strain selectivity against A.
baumannii and S. pyogenes clinical
strains by using different analogues.
Activated cis compounds turn back to
their deactivated trans form upon
short exposure times to sunlight,
paving the way to self-deactivating
antibacterials with minor evolutionary
pressure on the environment.