Photosensitizer-peptoid conjugates for photoinactivation of Gram-negative bacteria: structure-activity relationship and mechanistic studies

Article abstract of DOI:10.1039/d1ob00926e

Multitarget engagement is considered an effective strategy to overcome the threat of bacterial infection, and antimicrobials with multiple mechanisms of action have been successful as natural chemical weaponry. Here, we synthesized a library of photosensitizer-peptoid conjugates (PsPCs) as novel antimicrobial photodynamic therapy (aPDT) agents. The peptoids, linkers, and photosensitizers were varied, and their structure-antimicrobial activity relationships againstEscherichia coliwere evaluated; PsPC9was indicated to be the most promising photoresponsive antimicrobial agent among the synthesized PsPCs. Spectroscopic analyses indicated that9generated singlet oxygen upon absorption of visible light (420 nm) while maintaining the weakly helical conformation of the peptoid. Mechanistic studies suggested that damage to the bacterial membrane and cleavage of DNA upon light irradiation were the main causes of bactericidal activity, which was supported by flow cytometry and DNA gel electrophoresis experiments. We demonstrated that the optimal combination of membrane-active peptoids and photosensitizers can generate an efficient aPDT agent that targets multiple sites of bacterial components and kills bacteria by membrane disruption and reactive oxygen species generation.

Full text of DOI:10.1039/d1ob00926e

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Photosensitizer–peptoid conjugates for
photoinactivation of Gram-negative bacteria:
structure–activity relationship and mechanistic
studies†
Cite this: Org. Biomol. Chem., 2021,
19, 6546
Woojin Yang,‡a Younggun Yoon,‡b Yunjee Lee,‡a Hyeongyeol Oh, ‡a Jieun Choi,a
a
Sujin Shin, b Seongsoo Lee,c Hohjai Lee, a Yunho Lee *b and Jiwon Seo
*
Multitarget engagement is considered an effective strategy to overcome the threat of bacterial infection,
and antimicrobials with multiple mechanisms of action have been successful as natural chemical weap-
onry. Here, we synthesized a library of photosensitizer-peptoid conjugates (PsPCs) as novel antimicrobial
photodynamic therapy (aPDT) agents. The peptoids, linkers, and photosensitizers were varied, and their
structure-antimicrobial activity relationships against Escherichia coli were evaluated; PsPC 9 was indicated
to be the most promising photoresponsive antimicrobial agent among the synthesized PsPCs.
Spectroscopic analyses indicated that 9 generated singlet oxygen upon absorption of visible light
(420 nm) while maintaining the weakly helical conformation of the peptoid. Mechanistic studies
suggested that damage to the bacterial membrane and cleavage of DNA upon light irradiation were the
main causes of bactericidal activity, which was supported by flow cytometry and DNA gel electrophoresis
experiments. We demonstrated that the optimal combination of membrane-active peptoids and photo-
sensitizers can generate an efficient aPDT agent that targets multiple sites of bacterial components and
kills bacteria by membrane disruption and reactive oxygen species generation.
Received 11th May 2021,
Accepted 9th July 2021
DOI: 10.1039/d1ob00926e
rsc.li/obc
and TD-1607 5), quinolone–oxazolidinone (cadazolid6 and
MCB3681 7), and rifamycin–quinolone (TNP-2092 8) are in
Introduction
Multitarget antimicrobials have been preferentially used in
nature as host-defense weaponry.1 This multitarget strategy
has been used to minimize resistance development and has
proven to be effective in the clinic, as exemplified by long-
established antibiotics such as penicillins2 and polymyxin B.3
More recently, a multitarget strategy has been adopted with
various investigational antibiotic drugs by conjugating
two known antibiotics with different mechanisms of action.
Stable hybrids of vancomycin–cephalosporin (cefilavancin4
clinical trials against antibiotic-resistant strains.
Antimicrobial peptides (AMPs) often take advantage of this
multitarget approach. As a molecule produced by the innate
immune system, nature has utilized AMPs as a first-line
defense against invading pathogens. More than 5000 AMPs
have been discovered to date, including peptides used in their
natural form and rationally designed artificial peptides.9 The
activity of AMPs originates from their structural characteristics
that can damage multiple components of a bacterial cell. For
example, cationic AMPs such as cathelicidin (LL-37),10 magai-
nin-2,11 and lactoferrin12 have both cationic and hydrophobic
residues, forming an amphipathic α-helical conformation.
This structural feature allows electrostatic interactions with the
negatively charged bacterial membrane components, including
teichoic acid (cell wall, Gram-positive bacteria) or the phospho-
lipid head groups of the bacterial membrane, leading to the
subsequent destruction of bacterial barriers.13 Recent studies
have also reported cationic AMPs that target intracellular
nucleic acids and proteins, demonstrating nature’s preference
for a multi-effective mechanism.14 However, despite their
potential, only seven AMPs have been approved by the FDA
thus far, with four of them being vancomycin or its derivatives
aDepartment of Chemistry, Gwangju Institute of Science and Technology,
123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, South Korea.
E-mail: jseo@gist.ac.kr
bSchool of Earth Sciences and Environmental Engineering, Gwangju Institute of
Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005,
South Korea. E-mail: yhlee42@gist.ac.kr
cGwangju Center, Korea Basic Science Institute (KBSI), 49 Dosicheomdansaneopro,
Nam-gu, Gwangju 61751, South Korea
†Electronic supplementary information (ESI) available: Synthetic procedure for
photosensitizers, peptoids, and PsPCs; HPLC data and ESI-MS data for 1–16; the
results of additional photoinactivation studies, circular dichroism spectra, and
flow cytometry experiments; and cytotoxicity of 9, 14, and 16. See DOI: 10.1039/
d1ob00926e
‡These authors contributed equally to this work.
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and the other three being gramicidin, daptomycin, and colis- cationic amphipathic peptoids that mimic the structural fea-
tin.15 Therefore, researchers have focused on various strategies tures of the natural AMP magainin-2, invented by Barron and
to overcome the limitations of AMPs (e.g., a short plasma half- coworkers.43 These α-helical peptoids showed broad-spectrum
life)15 by incorporating unnatural amino acids,16 modulating antimicrobial activity against both Gram-positive and Gram-
the length of the AMP,17 or cyclizing the sequence.18
negative bacteria in the low micromolar range.44,45 Follow-up
Antimicrobial photodynamic therapy (aPDT) was intro- studies have reported the preparation of various antimicrobial
duced in the early 20th century19 and has been rediscovered as peptoids through tuning the cationic charge and
a promising multitarget strategy to fight against recent wide- hydrophobicity,46,47 introducing heterocyclic rings,48,49 as well
spread multidrug resistant pathogens.20 aPDT utilizes reactive as adopting different scaffolds such as peptide–peptoid
oxygen species (ROS) generated by a photosensitizer during hybrids50 and cyclic peptoids.51 The effects of the confor-
•
light irradiation. Different types of radical species (i.e., OH, mational flexibility of peptoids on their antimicrobial activity
O2•−) or singlet oxygen (1O2) are produced during this process were investigated by a method called helicity modulation.52
through the type I and type II pathways, respectively.21 The Therein, a peptoid with moderate helicity showed the most
generated ROS can cause fatal damage to multiple sites in bac- dramatic increase in selectivity, maintaining its antimicrobial
teria, including membrane components22 and intracellular activity. Unlike peptides, peptoid scaffolds offer a unique
biomass (e.g., nucleic acids and proteins).23,24 Therefore, advantage of evaluating the structure–activity relationship by
various types of photosensitizers have been developed, includ- modulating their secondary structure while maintaining the
ing phenothiazinium,25 tetrapyrroles (i.e., porphyrin,26 physicochemical properties (e.g., cationic charge and hydro-
chlorin,27 and phthalocyanine28), and riboflavin.29 Researchers phobicity). Thus, introduction of peptoids with varying degree
have focused on increasing the efficiency of aPDT by introdu- of helicity (e.g., fully helical, non-helical, and moderately
cing photosensitizers into biomaterials, such as celluloses,30 helical) into the aPDT system would facilitate the discovery of
cyclodextrins,31 and aptamers.32
optimal conformation that has strong interaction with bac-
The combination of an AMP and a photosensitizer has terial membrane and has a synergistic antimicrobial activity
demonstrated strong synergistic effects caused by targeting the upon conjugation with a photosensitizer.
bacterial membrane while simultaneously generating toxic
ROS. The potential of this approach was actively demonstrated based aPDT agent:
In the present study, we introduce a new type of peptoid-
photosensitizer–peptoid conjugate
a
by Gobbo and coworkers with their porphyrin–AMP (PsPC). Several types of porphyrins, chlorins, and helicity-
conjugates.33,34 AMPs such as apidaecin 1b, magainin, and modulated peptoids were adopted for the design of our PsPC
buforin were linked to porphyrinoids, and these photosensiti- library. The PsPCs were expected to localize on the bacterial
zer–peptide conjugates induced the death of bacterial cells membrane or penetrate the membrane, followed by the gene-
upon light irradiation. Sol and coworkers expanded the appli- ration of singlet oxygen during visible light irradiation, which
cations of porphyrin–peptide conjugates using cationic por- consequently leads to the death of the target bacteria.
phyrin and the macrocyclic antibiotic polymyxin B, which is Photosensitizers with either carboxylic acid or primary amine
active against only Gram-negative bacteria.35,36 The conjugate functional groups have been successfully conjugated to anti-
cationic porphyrin–polymyxin
B
exhibited antimicrobial microbial peptoids, whose helical integrity is controlled by the
activity against both Gram-positive and Gram-negative strains, helix-inducing peptoid monomers. The antimicrobial activities
overcoming the limitations of the peptide acting alone. of the prepared set of PsPCs were screened by counting the
Photosensitizers conjugated with other peptides, such as lipo- colony-forming units (CFUs) and determining the minimum
polysaccharide-binding peptide,37 oligoarginine,38 and tat inhibitory concentration (MIC) of each PsPC. Further investi-
peptide,39 have also demonstrated the effectiveness of this gation was conducted by spectroscopic and bioanalytical
approach. Nevertheless, this strategy needs to be further methods, including UV-vis, fluorescence, phosphorescence,
explored by employing various types of membrane-active pep- circular dichroism (CD), flow cytometry, and gel electrophor-
tides and peptidomimetics to take full advantage of this esis, to determine the origin of the photoresponsive activity of
strategy.
selected PsPCs. PsPCs with unique photophysical and confor-
Among peptidomimetics, peptoids or N-substituted glycine mational characteristics could provide insight for ongoing
oligomers have attracted attention as promising antimicrobial research on antimicrobial peptoid development and aPDT
agents. Antimicrobial peptoids have several advantages that agent design.
originate from their structural features and synthetic pro-
cesses. Peptoid structures are composed of a repeating tertiary
amide backbone that provides increased stability towards pro-
Results and discussion
Synthesis of PsPCs
teolytic degradation.40 The peptoid sequence can be elongated
via a submonomer method, which enables various side chain
incorporations in a sequence-specific manner.41,42 Cationic PsPCs were designed to have each of the components, namely
and hydrophobic residues can be precisely located at specific photosensitizer and peptoid, connected by a linker (Fig. 1 and
positions covering a wide spectrum of physicochemical and Scheme S1†). Two porphyrins (meso-tetra(4-N-methylpyridyl)
conformational properties. Representative examples are the porphine (TMPyP), tetraphenylporphyrin (TPP)) and three
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Fig. 1 Structures of photosensitizers, linkers, and peptoids.
chlorins (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a phenylethylamine, Nspe).56,57 The degree of helicity can be
(HPPH), verteporfin, chlorin e6) were selected as photosensiti- modulated by the substitution of chiral Nspe monomers with
zers, each of which is known to generate singlet oxygen upon achiral monomers (i.e., benzylamine, Npm).52,58 Depending on
visible light irradiation (Fig. 1, red box).53 Cationic TMPyP and the degree of unfolding, moderately helical peptoids could
hydrophobic TPP are meso-substituted porphyrins that are better distinguish between bacterial and eukaryotic mem-
functionalized with a carboxylic acid or a primary amine, branes, leading to more selective antimicrobial peptoids.52 All
respectively, for peptoid conjugation. The other three com- four peptoids A–D have a fixed number of cationic residues
pounds, the chlorins, have been investigated in the clinic for (i.e., 1,4-diaminobutane, NLys), but the position-specific incor-
anticancer photodynamic therapy. HPPH has been evaluated poration of helix-inducing Nspe monomers resulted in
for multiple tumor types, including esophageal cancers.54 different degrees of helicity. Peptoids A and D have fully
Verteporfin has been used for the treatment of age-related α-helical and nonhelical conformations, respectively.43 The
macular degeneration.54 Chlorin e6 was formulated with poly- degree of folding of peptoids B and C are between the fully
vinylpyrrolidone (PVP), and chlorin e6-PVP induces apoptosis helical and nonhelical, with peptoid B showing moderate heli-
of malignant tumors after localization in the mitochondria city and peptoid C being weakly helical.52
and lysozymes.55 HPPH, verteporfin, and chlorin e6 were pre-
pared as NHS esters to facilitate peptoid conjugation.
The photosensitizers and peptoids were conjugated using
different linkers, glycine (Gly) and polyethylene glycol (PEG4
Peptoids A–D were used as membrane-active antimicrobial and PEG8) (Fig. 1, black box). The linkers were attached to the
agents (Fig. 1, blue box).41,43 Previous studies have revealed N-terminus of each resin-bound peptoid and conjugated with
that the unique conformational feature, a polyproline type I the photosensitizer through an amide bond (Scheme S1†).
(PPI)-like helix with three residues per turn, is a result of the Diglycine (Gly–Gly) and 6-aminohexyl linkers were also tested;
incorporation of chiral aromatic side chains (i.e., (S)-(−)-1- however, these linkers were not optimal, and poor anti-
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Table 1 Antimicrobial activity of 1–16 against S. aureus (ATCC 25923) and E. coli (ATCC 25922)
S. aureus
E. coli
MICb (μM)
MICb (μM)
Dark
Lightc
MBCb (μM)
MBCb (μM)
Compd
PSa
Linker
Peptoid
Dark
Lightc
Lightc
Lightc
1
2
3
4
5
6
7
8
TMPyP
TPP
Gly
Gly
Gly
Gly
Gly
PEG4
PEG8
Gly
Gly
Gly
PEG4
—
A
A
A
A
A
A
A
B
C
D
C
A
B
C
D
—
6.3
>25
>25
>25
>25
3.1
6.3
6.3
6.3
12.5
ndd
1.6
3.1
6.3
6.3
>25
6.3
>25
>25
>25
>25
6.3
6.3
6.3
6.3
6.3
ndd
1.6
3.1
6.3
6.3
>25
6.3
>25
>25
>25
>25
6.3
12.5
12.5
6.3
12.5
>25
>25
>25
>25
6.3
12.5
6.3
6.3
6.3
3.1
3.1
6.3
12.5
>25
>25
>25
>25
6.3
12.5
6.3
1.6
3.1
3.1
3.1
6.3
12.5
>25
>25
>25
>25
6.3
12.5
6.3
1.6
3.1
3.1
3.1
6.3
HPPH
Verteporfin
Chlorin e6
TMPyP
TMPyP
TMPyP
TMPyP
TMPyP
TMPyP
—
—
—
—
TMPyP
9
10
11
12
13
14
15
16
25
ndd
3.1
—
—
—
—
12.5
12.5
25
12.5
12.5
>25
12.5
12.5
>25
6.3
12.5
>25
>25
a Photosensitizer. b The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) data represent the mean
values of three independent experiments. c The samples were irradiated under blue light for 15 min (390–460 nm, 420 nm max, 15.1 J cm−2).
d Not determined.
microbial activity was observed (data not shown). In peptoids accounted for by the presence of a neutral photosensitizer.
A–D, three linkers and five photosensitizers were combined to Considering that the N- or C-terminus of the peptoid helix is
find the PsPC with optimal biological activity (1–11 in inserted into the phospholipid membrane,43 the interaction
Table 1). Peptoids A–D, without photosensitizer conjugation between the peptoid and bacterial membrane, with a net nega-
(12–15 in Table 1), were prepared for comparison. Each com- tive surface charge, was hampered by the presence of a bulky
pound was purified by preparative HPLC (>97%) and character- neutral photosensitizer. However, TMPyP with four permanent
ized using ESI-MS (Fig. S1, S2 and Table S1†).
cationic charges appears to have a better interaction with the
bacterial membrane, leading to increased antimicrobial
activity. Thus, TMPyP was selected among the five photosensi-
Antimicrobial activity of PsPCs
The photoresponsive antimicrobial activity of PsPCs was evalu- tizers, and the effect of changing the linker and peptoid was
ated by measuring the minimum inhibitory concentration subsequently investigated. A PEG linker (or spacer) was intro-
(MIC) and minimum bactericidal concentration (MBC). Blue duced to increase the distance between the bulky photosensiti-
light irradiation was applied for 15 min with a homemade zer and the antimicrobial peptoid. The MICs and MBCs of 6
LED array (390–460 nm, 420 nm max, 16.8 mW cm−2). It was and 7 were not dramatically different from those of 1 (i.e.,
confirmed that the growth of bacterial cells in the absence of 12.5 μM), indicating that the linker may not play a key role in
PsPCs was not affected under these conditions (Fig. S3†). the optimal PsPC.
Several PsPCs (e.g., 6, 8–11) exhibited potent antimicrobial
Variation in the peptoids resulted in a more significant
activity against S. aureus and E. coli (Table 1). Notably, PsPC 9 enhancement in antimicrobial activity. The MICs and MBCs of
and 10 showed more potent antimicrobial activity against conjugates 8–10 were 6.3 μM or lower, whereas those of the
Gram-negative E. coli than Gram-positive S. aureus. For Gram- corresponding peptoids themselves (13–15) were 6.3 μM or
positive bacteria, none of PsPCs (1–10) showed better anti- higher. Interestingly, PsPC 9 and 10 showed further enhance-
microbial activity than peptoid alone (12), and no benefit of ment in antimicrobial activity in response to light irradiation.
photosensitizer conjugation was found. Thus, further analysis PsPC 9 exhibited the largest increase in photoresponsive
and discussion were focused on using PsPC as an aPDT agent activity and the most potent aPDT effect (MIC and MBC of
against E. coli.
1.6 μM). The Gly linker in PsPC 9 was changed to PEG4 to
The antimicrobial activities of PsPCs based on peptoid A examine whether activity enhancement could be observed, as
(1–7) were lower than those of peptoid alone (12) under both seen in PsPC 1 and 6. Compared to PsPC 9, PEG4-linked PsPC
dark and light conditions (Table 1). Among the five photosen- 11 exhibited stronger antimicrobial activity in dark conditions,
sitizers, only cationic TMPyP appeared to be effective when but no photoresponsive activity increase was observed.
conjugated with a peptoid. The MICs and MBCs of 2–5 were
The singlet oxygen generated upon light irradiation
larger than 25 μM, while those of 1 were both 12.5 μM. The damages vital membrane components of bacteria only if
decreased antimicrobial activity in compounds 2–5 can be sufficient amounts are present near the bacterial outer mem-
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Table 2 Photophysical properties of 9 and TMPyP (16) in deuterium
oxide
Considering that TMPyP (16) itself does not have enough
affinity for the bacterial membrane without peptoids and that
the singlet oxygen lifetime is short (i.e., ∼3 μs),59 it is reason-
ably speculated that the singlet oxygen generated by TMPyP
(16) was quenched before reaching the membrane. Peptoids
12, 14, and 15 showed a decrease in their activity similar to
that reported,52 indicating that the activity was not signifi-
cantly affected by light irradiation. However, PsPCs 1, 9, and
10 showed complete killing at a much lower concentration
than both TMPyP (16) and the corresponding peptoids (12, 14,
and 15), which exist independently. This result implies that
the conjugation provided a synergistic effect, which induced
targeting to the bacterial membrane followed by damage to
b
c
Compound
ε
a (M−1 cm−1
)
ΦF
ΦΔ
τF d (ns)
τΔ e (ns)
9
1.8×105
2.6×105
0.07
0.06
0.85
0.90
11.9
6.1
67.9
69.9
TMPyP (16)
a ε: extinction coefficients at 426 nm (9) and 421 nm (16). b ΦF: relative
fluorescence quantum yield (TPP in DMF was used as a standard; Φ =
0.11).63 c ΦΔ: relative singlet oxygen quantum yield (TMPyP in D2O was
used as a standard; Φ = 0.90).53 d τF: fluorescence emission lifetime at
715 nm. e τΔ: singlet oxygen phosphorescence emission lifetimes at
1275 nm.
brane.22 Due to the short half-life of singlet oxygen in aqueous the structure by generated singlet oxygen.
media (Table 2),59 the decrease in cytotoxicity is generally
The relationship between PsPC and the degree of mem-
observed as the distance between the singlet oxygen source brane association was confirmed by washing the cells before
and the targeting site of bacteria. It can be inferred that 9 and light irradiation. According to Gobbo and coworkers, the CFU
10 were localized on the bacterial membrane, and subsequent counts recovered when the photosensitizer–AMP conjugates
singlet oxygen generation induced damage to the bacteria. weakly bound to the bacterial membrane were removed during
Since the helical antimicrobial peptoid can penetrate the the washing step.33 The same experiment was performed with
membrane and induce intracellular biomass flocculation,60 it PsPCs, which showed a recovery of CFU counts after the
is also reasonable to assume that 9 and 10 can damage the washing step (Fig. 2b). PsPC 1 showed a recovery of CFU
intracellular components. Further experiments were conducted counts of less than one log scale, whereas PsPCs 9 and 10
to clarify the origin of their photoresponsive antimicrobial resulted in 3 and 1.5 log scale recoveries of CFU counts,
activity.
respectively. These results suggest that PsPCs 9 and 10 were
The concentration-dependent photoresponsive anti- weakly associated with the bacterial membrane, which greatly
microbial activity of PsPC (1, 9, and 10) was more rapidly affects their photoinduced antimicrobial activity.
increased than those of TMPyP (16) and peptoids (12, 14, and
Spectroscopic studies of PsPCs
15; Fig. 2a). The growth of bacteria was not disturbed under
the given conditions unless the compounds were treated,
showing 5 log cell counts. The antimicrobial activity of TMPyP
(16) barely changed depending on its concentration.
The photophysical and conformational properties of the pre-
pared compounds were investigated by UV-vis absorption, fluo-
rescence emission, phosphorescence and CD spectroscopy
Fig. 2 (a) Concentration-dependent growth inhibition of E. coli (ATCC 25922). After blue light irradiation for 15 min (390–460 nm, 420 nm max,
15.1 J cm−2), the cells were cultured overnight at 37 °C. 1, 9 and 10 are PsPCs. 12, 14 and 15 are peptoids alone. 16 is TMPyP. (b) Effect of the
washing step on antimicrobial photoinactivation against E. coli (ATCC 25922). After preincubation of the cells with compounds (1.6 μM) in the dark
for 2 h, the cells were washed with MHB2 media (pH 7.4) three times and irradiated following the above conditions in (a). “w/o wash” indicates no
buffer washing step between preincubation and light irradiation.
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Fig. 3 (a) UV-vis absorption spectra obtained for PsPC 9 and TMPyP 16 (D2O, 10 μM, 20 °C). The inset represents the Q-band absorption region
(475–715 nm). (b) Fluorescence emission spectra of PsPC 9 (0.75 μM) and TMPyP 16 (0.50 μM; D2O, 20 °C, λex = 405 nm). (c) Phosphorescence emis-
sion spectra of singlet oxygen generated by PsPC 9 (0.75 μM) and TMPyP 16 (0.50 μM; D2O, 20 °C, λex = 425 nm). (d) CD spectra obtained for PsPCs
(1, 9, and 10) and peptoids (12, 14, and 15) in 10 mM Tris-HCl buffer (pH 7.0; 50 μM, 20 °C).
(Fig. 3). The absorption properties of PsPC 9 were similar to ways in TMPyP, thereby forming
those of TMPyP (16), aside from a decrease in the extinction complex with different fluorescence characteristics.
coefficient (ε) and a slight redshift of peaks (Fig. 3a and The phosphorescence emission spectra showed that PsPC 9
a new photosensitizer
Table 2). The UV-vis spectrum of 9 showed a sharp Soret band generated singlet oxygen (Fig. 3c). PsPC 9 and TMPyP (16) were
(λmax = 426 nm) and four distinctive Q-bands (480–700 nm), dissolved in deuterium oxide for direct observation of singlet
which is a distinctive feature of a porphyrin.61 No J- or H-type oxygen through their characteristic emission (∼1275 nm) in an
aggregation was observed in the Soret region under the con- aqueous environment. The excitation wavelength was 425 nm,
ditions used.
at which point porphyrin was dominantly excited. Both 9 and
Fluorescence emission spectroscopy was conducted to TMPyP (16) exhibited characteristic emission at approximately
measure the characteristics upon excitation (Fig. 3b and 1275 nm, indicating radiative relaxation of singlet oxygen (i.e.,
Table 2). The emission spectrum of 9 was clearly resolved into a1Δg → X3Σg−).59 The singlet oxygen lifetime (τΔ) of TMPyP
two hills with peaks at 661 nm and 723 nm, while that of (16) matched that of a previous report,53 and the singlet
TMPyP (16) showed a single peak at 721 nm with a shoulder oxygen lifetime of 9 was similar to that of TMPyP (Table 2).
near 665 nm (Fig. 3b), a peak shape reminiscent of previously The quantum yield of singlet oxygen generation (ΦΔ) was
reported supramolecular assemblies with TMPyP.61,62 The fluo- slightly reduced in 9 compared to that in TMPyP (Table 2),53
rescence lifetime (τF) of 9 was approximately twice as high as which might be the result of quenching due to the nearby
that of TMPyP (16), and the fluorescence quantum yield (ΦF) peptoid. Taken together, these results show that 9 is a complex
of 9 was slightly higher (Table 2). Conjugation of TMPyP to the with different fluorescence characteristics from those of
peptoid may hamper the assembly of the dyes in aqueous TMPyP (16) but generates singlet oxygen as much as TMPyP
media and modulate the intramolecular charge transfer path- (16) does upon excitation.
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The conformation of PsPCs was analyzed by CD spec- polygon (Fig. S5†). It could be inferred that the integrity of
troscopy (Fig. 3d). Peptoids composed of α-chiral aromatic side cells was not severely damaged (e.g., cell lysis) under the
chains (e.g., Nspe) show polyproline type I (PPI)-like helical measurement conditions. Experiments with the same concen-
structures.56,57 The characteristic peaks are observed by their tration (i.e., 1.6 μM) of peptoid C (14) or TMPyP (16) produced
carbonyl π → π* (∼192 nm and ∼202 nm) and n → π* similar results (Fig. S7†).
(∼220 nm) transitions in the CD spectra.56,57 Peptoid 12, com-
Bacterial membrane damage caused by 9 under light
posed of 8 Nspes, indicates a fully helical conformation irradiation was observed through ICC using SGI/PI conditions
(Fig. 3d). As previously reported, peptoids 14 and 15 exhibit (Fig. 4). Bacterial cells were observed inside the polygon when
weakly helical and nonhelical conformations.52,58 The helicity the bacterial membrane was intact, as appeared when measur-
of the conjugates was not significantly affected by TMPyP con- ing TCCs (Fig. 4 and S5†). However, the cells were observed
jugation. All PsPCs showed a degree of helicity similar to that outside the polygon (i.e., a decrease in green fluorescence and
of the corresponding peptoids (i.e., fully helical (1), weakly an increase in red fluorescence) when the bacterial membrane
helical (9), and nonhelical (10)), without significant peak was impaired and stained by PI. The ICC was maintained when
shifts (Fig. 3d). It was confirmed that the conjugation of the cells were treated with 9 and incubated in the dark but dra-
TMPyP at the N-terminus of the peptoids did not induce a dra- matically decreased when the cells were irradiated (Fig. 4 and
matic change in the conformational characteristics of the S7†). These results imply that the membrane was damaged by
resulting complexes. The features were further investigated in oxidative stress caused by singlet oxygen generated during
the presence of lipid vesicles representing negatively charged irradiation. Membrane-damaged cells were not observed with
bilayer models of bacterial membranes by mixing 1-palmitoyl- either peptoid C (14) or TMPyP (16) treatment (Fig. S7†),
2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-pal- suggesting that the conjugation provided a strong synergistic
mitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)
effect. The number of ICCs decreased with an increasing con-
(POPG).52,64,65 PsPC 9 did not exhibit the characteristic CD sig- centration of 9 regardless of the washing step (Fig. 4). An
nature, while the other conjugates exhibited the same CD sig- increase in the ICC was observed at the same concentration
nature (Fig. S4†). According to our previous studies, a higher- when the cells were washed (Fig. S6†), indicating that a weak
order self-assembly was predicted for peptoid C (14), whereas association existed between the bacterial membrane and 9.
a monomeric form was observed for peptoid A (12), both of
Photoinduced DNA damage by PsPC
which were obtained in an aqueous environment.52 The
similar CD signatures of the PsPCs and peptoids imply that Gel electrophoresis was performed to investigate the effect of
the conformational properties were maintained. Thus, it is PsPC on nucleic acids (Fig. 5). The experiment focused on the
speculated that the local concentration of 9 was increased due fate of plasmid DNA (e.g., pUC19) that could be transferred to
to aggregate formation, and a subsequent increase in the other bacteria, causing one of the major forms of antibiotic re-
singlet oxygen concentration induced photoresponsive damage sistance.67 The marker loaded on the gel confirmed that intact
to the bacteria.
pUC19 has a band located below 2 kb and is known to have a
supercoiled form. A larger band showing its original length
(i.e., 2686 bp) appeared near 3 kb when the DNA was linearized
Photoinduced membrane damage by PsPCs
Flow cytometry (FCM) experiments were conducted to evaluate by a restriction enzyme, EcoRI. Distinct band locations result
the interaction between bacterial membranes and 9. Bacterial from conformational differences, which induce different
samples were prepared via preincubation of the cells for 2 h migration speeds during electrophoresis.
with 9 in the dark, light irradiation for 15 min, washing with
Plasmid DNA was affected by photoresponsive damage gen-
MHB2 media (pH 7.4), and then treatment under two different erated by photosensitizers. A supercoiled form of pUC19 was
conditions of FCM analyses. Under the first condition, bac- sustained when the compounds were treated in the dark
terial cells were stained with only SYBR Green I (SGI), which (Fig. 5a). However, the results changed dramatically after
emits strong green fluorescence when bound to DNA, to count irradiation (Fig. 5b). pUC19 treated with 9 resulted in band dis-
the total number of cells (= total cell count, TCC). Under the appearance, which implies extensive fragmentation of DNA.
second condition, cells were stained with a combination of SGI The DNA either maintained its conformation or linearized
and propidium iodide (PI). Cells can be stained with PI only after EcoRI treatment when treated with peptoid C (14) or
when their membrane is damaged because PI is a cell- TMPyP (16), respectively.68
impermeable nucleic acid-binding dye.66 The number of intact
The results of electrophoresis suggest that 9 is a potential
cells was counted for the latter condition (= intact cell count, multitarget agent. It is well documented that positively
ICC). The difference relative to the concentration of 9 and the charged porphyrin (e.g., TMPyP) intercalates into negatively
difference between washed and unwashed cells before the charged DNA, which induces DNA lesions by photoresponsive
measurements were compared.
damage.69 However, long periods of irradiation were required
The TCC did not significantly differ as the concentration of for this mechanism.70 PsPC 9 resulted in DNA fragmentation
9 increased or with washing of the cells before irradiation with concomitant membrane damage under the same con-
(Fig. S5 and S6†). In all cells stained with only SGI, a TCC of ditions (Fig. 4 and 5). It could be reasoned that the conju-
∼3 × 105 cells per mL was determined within the region of the gation of cationic porphyrin with a cationic amphipathic
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Fig. 4 Flow cytometric density plots (530 nm vs. 670 nm) for the ICC changes during aPDT against E. coli (ATCC 25922) as a function of the mole-
cular concentration of 9 from zero to 1.6 μM. The samples were prepared with (a–e) or without (f–j) an additional washing step and stained with SGI
(10 μL mL−1)/PI (0.6 mM) to assess membrane damage. The polygons illustrate gates depicting the region where membrane-intact cells appear.
against Gram-negative E. coli. A weakly helical peptoid exhibi-
ted optimal photoresponsive antimicrobial activity when con-
jugated with a cationic photosensitizer (i.e., TMPyP), while a
fully helical peptoid was less effective when used as a conju-
gate. PsPC 9 could damage multiple sites on E. coli. Bacterial
membrane damage was observed when the cells were treated
with 9 followed by light irradiation, which demonstrated that
the oxidative damage was due to the generated singlet oxygen.
The weak association between 9 and the bacterial membrane
can explain the photoresponsive inactivation of bacteria. The
weakly helical peptoid can be considered “sticky tape” with
cationic amphipathic and conformationally flexible properties,
which result in an optimal association of PsPC with bacterial
membranes. In addition, the decrease in membrane accessibil-
ity of peptoids after N-terminal porphyrin conjugation can be
compensated for by using flexible peptoids rather than using
fully structured peptoids. Similar to membrane damage,
plasmid DNA damage occurred after treatment with 9 under
light irradiation. The fragmented DNA demonstrated that 9 led
to oxidative damage to intracellular targets, which had been
difficult to achieve with a peptoid or photosensitizer alone.
The combined effect of the physicochemical and confor-
mational properties of 9 was crucial for this multitargeting
bacterial killing approach.
Fig. 5 Agarose gel electrophoresis of extracellular pUC19 plasmids
treated with 9 (1.6 μM), peptoid C (14; 1.6 μM), and TMPyP (16; 1.6 μM)
incubated (a) in the dark or (b) under irradiation with blue light for
15 min (390–460 nm, 420 nm max, 15.1 J cm−2). The first column shows
the standard DNA ladder. The last column shows the linearized pUC19
after restriction digestion with EcoRI.
peptoid (e.g., peptoid C) enhanced the interaction between the
conjugate and biomolecules (e.g., membrane and nucleic
acids) and the subsequent photoresponsive damage at mul-
tiple sites on bacteria with lower conjugate concentrations.
Although promising antimicrobial agents, PsPCs pose a
limitation to overcome. The effective association with the bac-
terial membrane and subsequent generation of singlet oxygen
upon visible light irradiation could provide advantages when
Conclusion
In summary, we synthesized photosensitizer–peptoid conju- utilized for biofilm prevention and irradication,26 inhibition of
gates (PsPCs) as aPDT agents and investigated their activity fungal spore germination,71 and wastewater sterilization.72 The
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utility of PsPCs for the above applications certainly calls for
Fluorescence emission from PsPCs was measured by a
further studies. For PsPC to be used in the clinic, we found the FLS980 fluorescence spectrometer (Edinburgh Instruments,
current irradiation conditions (i.e., 420 nm, 15.1 J cm−2), the Livingston, UK) with UV-vis PMT (R928P, Hamamatsu, Japan).
long interval between sample treatment and irradiation (i.e., The steady-state fluorescence spectra were recorded with
2
h
preincubation), and cytotoxicity are suboptimal 405 nm excitation, a 2 nm spectral bandwidth for both exci-
(Table S2†).22 Thus, our ongoing studies are focused on exam- tation and emission, and a 0.1 s dwell time. Ten replicate
ining whether selectivity acceptable for clinical use can be spectra were obtained and averaged. Time-resolved fluo-
attained by expanding the PsPC library and changing the rescence profiles of PsPCs were measured by using the time-
sample treatment conditions, eventually reducing the toxicity. correlated single-photon counting (TCSPC) module in FLS980
Otherwise, the potential usage of PsPCs will be limited to at an emission wavelength of 715 nm with a 405 nm pico-
environmental or device sterilization.
second pulsed excitation (LDH405, PicoQuant, Germany).
Phosphorescence emission from singlet oxygen was
The multitarget bacterial inactivation of PsPC was optimal
with conjugation to weakly helical antimicrobial peptoids and measured by a FLS980 system with a liquid N2-cooled NIR
cationic photosensitizers. The molecular design and biological PMT (R5509-72, Hamamatsu, Japan). Steady-state phosphor-
activity of PsPCs reported herein are expected to provide escence spectra were recorded with 425 nm excitation, a 0.2 s
insight into the development of next-generation peptoid-based dwell time, and 4 nm and 15 nm spectral bandwidths for exci-
aPDT conjugates.
tation and emission, respectively. Fifty replicate emission
spectra were recorded and averaged. Time-resolved phosphor-
escence emission profiles of singlet oxygen were measured by
a multichannel scaling (MCS) module in the FLS980 at an
emission wavelength of 1275 nm, synchronized with 425 nm
nanosecond excitation pulses from an optical parametric oscil-
lator (MagicPRISM VIR, Opotek, USA) pumped by a Nd:YAG
laser (Q-smart 450, Quantel, USA). The typical pulse width was
7 ns, and the repetition rate was 10 Hz. All the emission decay
curves were fitted with a single exponential function using
MATLAB software.
CD spectra were recorded on a Jasco model 810 spectropo-
larimeter (Jasco, Easton, MD, USA) in a quartz cell with a
0.2 mm path length. The response time was set to 1 s with a
1.0 nm data pitch and 1.0 nm bandwidth for all spectra. The
spectra were acquired in the 190–260 nm range with a 100 nm
min−1 scanning speed at 20 °C, measured 40 times and aver-
aged. The samples were prepared in either 10 mM Tris-HCl
buffer (pH 7.0) or 5 mM lipid vesicles (POPE : POPG = 7 : 3) in
10 mM Tris-HCl buffer (pH 7.0).57,70 The results were
expressed in terms of the per-residue molar ellipticity, [θ] (deg
cm2 dmol−1). ([θ] = θ/(n × c × l), where θ is the ellipticity of the
polarization, n is the number of amide groups present, c is the
molar concentration, and l is the optical path length).
Experimental section
General information
Reactions were performed in oven-dried (110 °C) glassware
under
a nitrogen atmosphere unless otherwise noted.
Chemicals and solvents were purchased from Sigma-Aldrich
(St Louis, MO, USA), Acros Organics (Geel, Belgium), Alfa
Aesar (Haverhill, MA, USA), TCI (Tokyo, Japan), or Daejung
(Siheung, South Korea) and were used without further purifi-
cation. Fmoc-Rink Amide MBHA resin (100–200 mesh,
0.52 mmol g−1
)
was acquired from Merck Millipore
(Burlington, MA, USA). Fmoc-N-amido-dPEG®4-acid was pur-
chased from Quanta BioDesign (Plain City, OH, USA).
Photochlor, verteporfin, and chlorin e6 were acquired from
MedKoo Biosciences (Chapel Hill, NC, USA). 1-Palmitoyl-2-
oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmi-
toyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) were
purchased from Avanti Polar Lipids (Alabaster, AL, USA). Flash
column chromatography was conducted on silica gel 60
(230–400 mesh, Merck Millipore). The bands were monitored
by thin-layer chromatography (TLC) on aluminum-backed TLC
sheets (silica gel 60 F254, Merck Millipore). 1H NMR spectra
were recorded on a Jeol ECS400 spectrometer (400 MHz). The
spectra were referenced to trimethyl silane (0.00 ppm) or
residual chloroform (7.26 ppm). Chemical shifts are reported
in ppm, and multiplicities are indicated by s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). The coup-
ling constants, J, are reported in Hertz. Synthetic procedures
for photosensitizers, peptoids, and PsPCs were described in
the ESI.†
Photoinactivation of bacteria
The antimicrobial activity of the compounds was evaluated
against both E. coli (ATCC 25922) and S. aureus (ATCC 25923)
and reported as the MIC, which is the lowest concentration of
the antimicrobial agent that inhibits the growth of a microor-
ganism after overnight incubation at 37 °C. The minimum bac-
tericidal concentration (MBC), the lowest concentration of the
compound that is required to kill bacteria, is also reported.
The MIC values of each compound were determined using a
broth dilution assay. For each assay, primary cultures of either
Spectroscopic measurement and analysis
UV-vis spectra were recorded on an Ultrospec 2100 Pro UV-vis E. coli or S. aureus were grown overnight in Mueller Hinton
spectrophotometer (GE Healthcare, Buckinghamshire, UK) Broth 2 (MHB2, cation-adjusted) medium at 37 °C. A second-
over a range of 300–800 nm in a quartz cell with a 1 cm path ary culture was started the next day and grown for 3 to 4 h. The
length at 20 °C. Baseline correction for each spectrum was bacteria used in the experiments were collected from cultures
carried out with a solvent blank.
in the stationary phase of growth. This culture was then
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diluted in MHB2 medium corresponding to approximately manually using the supplied FCM software. The cell quantifi-
(2–5) × 105 CFU mL−1 bacteria. Using polypropylene 96-well cation limits of the FCM methods in this study were ∼1000
plates (Corning, Corning, NY, USA), 100 μL of (2–5) × 105 CFU cells per mL, and the standard deviation of the measurement
mL−1 bacteria was added in triplicate for each compound. was below 5%.74
Each compound was assayed at six different concentrations in
2-fold serial dilutions. The plates were wrapped with alumi-
Gel electrophoresis
pUC19 plasmids (1 μg mL−1) treated with the compounds
num foil to block ambient light and placed at 37 °C for 2 h.
After incubation in the dark, the suspensions were directly
exposed to light (no washing) or centrifuged to wash the
unbound compounds left in the suspension. For the washing
experiment, the mixture was centrifuged (10 000g) for 3 min,
and the supernatant was removed. The cells were resuspended
in 1 mL of MHB2 medium, and the steps were repeated three
times. For light irradiation, aliquots of bacterial samples were
transferred into 96-well plates (100 μL per well). Irradiation
was performed with blue light (390–460 nm, 420 nm max,
16.8 mW cm−2) emitted by a homemade LED array. The
fluence rate was measured with a light meter (FieldMax-II,
Coherent, Santa Clara, CA, USA) with an optical sensor (OP-2
UV, Coherent). After irradiation for 15 min, MIC data were
recorded by measuring the optical density (OD) at 600 nm on a
microplate reader (BioTek Instrument, Winooski, VT, USA). All
bacterial solutions remaining on the plates after the MIC assay
were spread on a separate agar plate to determine the MBCs.
The plates were incubated at 37 °C overnight to confirm bac-
terial growth. The MBC was determined as the minimum con-
centration required to prevent bacterial growth on the agar
plates. For the colony count assay, bacterial suspensions
treated with each compound were serially diluted 10-fold
in MHB2 media, and aliquots of the appropriate dilutions
(100 μL) were spread in triplicate onto MHB2 agar. The
agar plates were further incubated for 24 h at 37 °C to
determine CFUs. Pictures of the plates were uploaded to
OpenCFU software to count the number of colonies grown on
the plates.
under different irradiation conditions were analyzed by gel
electrophoresis to assess the structural changes in plasmid
DNA (i.e., supercoiled, linearized, or open-circular formed).
Linearized pUC19 was prepared for use as a reference by incu-
bating the plasmid with the type II restriction enzyme EcoRI
(NEB, USA) at 37 °C for 1 h, followed by enzyme inactivation at
65 °C for 20 min. After irradiation, the samples and a 1 kb
DNA ladder (Enzynomics, Daejeon, South Korea) were loaded
on 0.8% agarose gels at 4 V cm−2 for 35 min. The bands were
visualized by ethidium bromide staining. Gel images were cap-
tured on a UV transilluminator (Universal mutation detection
system, UVP, Upland, CA, USA).
Author contributions
W. Yang, Y. Yoon, Y. Lee, and H. Oh contributed equally to
this work. Y. Lee and J. Seo conceived and directed the
project. W. Yang and Y. Lee synthesized and characterized the
compounds. W. Yang and J. Choi conducted the biological
assays. W. Yang and H. Oh performed spectroscopic
analyses. Y. Yoon, J. Choi and S. Shin conducted the flow cyto-
metry experiments and DNA gel electrophoresis. S. Lee pro-
vided the homemade LED array. J. Seo, Y. Lee, H. Lee,
W. Yang, and Y. Yoon wrote the manuscript with contribution
from all authors.
Conflicts of interest
Flow cytometry analysis
There are no conflicts to declare.
Flow cytometry (FCM) analysis was performed as described
previously.66,73,74 Briefly, SYBR Green
I (SGI, Invitrogen,
Carlsbad, CA, USA) was prepared by diluting SGI 100-fold with
DMSO. A mixture of SGI and PI (Invitrogen) was prepared by
mixing SGI and 30 mM PI at a final PI concentration of
0.6 mM. The working solutions (SGI and SGI/PI) were kept at
−20 °C until use. The sample (1 mL) was stained with SGI or
SGI/PI at 10 μL mL−1 and incubated in the dark for 12 min at
36 °C. Before FCM analysis, samples were diluted with auto-
claved water filtered through a 0.2 μm syringe filter (Whatman,
Sigma-Aldrich) to 1% or 10% v/v of the initial concentration.
FCM analyses were performed using a CyFlow® Cube 6 flow
cytometer (Sysmex Korea, Seoul, South Korea) equipped with a
488 nm blue laser. Fluorescence was detected at 530 nm
(green) and 670 nm (red). Data were arranged in density plots
of 530 nm versus 670 nm. Individual dots in the plots reflected
the signals for each particle that was a bacterial cell. Bacterial
cells were separated from the instrument or sample back-
ground noise by electronic gates, which were constructed
Acknowledgements
This work was financially supported by the National
Research Foundation of Korea (NRF-2018R1A2B6007535,
2018M3D1A1052659, 2021R1A2C2014421 to J. S.), Korea Basic
Science Institute (D110720 to S. L.) and by the GIST Research
Institute (GRI) GIST-CNUH Research Collaboration grant
funded by GIST in 2021.
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