Facile and scalable synthesis of topologically nanoengineered polypeptides with excellent antimicrobial activities

Article abstract of DOI:10.1039/c9cc08095c

A facile and scalable strategy for the quick library synthesis of linear-, hinged-, star-, and cyclic-polypeptides with broad-spectrum antimicrobial activity has been reported. The topologically nanoengineered polypeptides show superior antimicrobial activity against Gram-positive and Gram-negative bacteria and low toxicity, allowing screening of architectural polypeptides as mimics of host defense peptides for antimicrobials.

Full text of DOI:10.1039/c9cc08095c

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Facile and scalable synthesis of topologically
nanoengineered polypeptides with excellent
antimicrobial activities†
Cite this: Chem. Commun., 2020,
6, 356
5
Received 15th October 2019,
Accepted 3rd December 2019
a
a
b
c
c
Yu Zhang, Wenliang Song,
Shuwei Li, Dae-Kyoung Kim, Jae Ho Kim,
*
b
a
Jung Rae Kim
and Il Kim
DOI: 10.1039/c9cc08095c
rsc.li/chemcomm
A facile and scalable strategy for the quick library synthesis of linear-,
To address these hurdles, synthetic AMP-mimetic polypeptides
hinged-, star-, and cyclic-polypeptides with broad-spectrum (AMPPs) have recently been recognized as promising alternatives
antimicrobial activity has been reported. The topologically nanoen- for conventional antibiotics, especially via the strategy of controlled
gineered polypeptides show superior antimicrobial activity against ring-opening polymerization (ROP) of a-amino acid N-carboxyan-
Gram-positive and Gram-negative bacteria and low toxicity, allowing hydrides (NCAs), which makes it possible to synthesize AMPPs on
7
screening of architectural polypeptides as mimics of host defense a large scale and at low cost. Zhou et al. recently reported the
peptides for antimicrobials.
synthesis of a polypeptide-grafted chitosan-based nanocapsule
by transition metal complexes and hexylamine initiated ROP of
8
Bacterial infection is a serious global issue threatening human NCAs. However, all these synthetic procedures require a com-
1
health. Though conventional antibiotics can alleviate illness, promise between employing metal-free initiators and obtaining
the abuse of antibiotics has led to the emergence of multi-drug well-defined AMPPs. It is well-known that the conventional amine
2
resistant pathogens and even ‘‘superbugs’’. Antimicrobial peptides or amine-derivative induced ROPs usually need multiple days to
(AMPs) have emerged as a new concept for the next-generation reach completion and result in unavoidable side reactions due to
antibacterial agents due to their excellent broad-spectrum anti- the competitive mechanisms, hampering the achievement of
9
bacterial activity and the fact that they induce barely any antibiotic- well-defined polypeptides in a commercially tangible way. Metal
3
resistance. AMPs usually contain cationic moieties along their catalysts can accelerate the polymerization to obtain controlled
structures, which endow AMPs with a unique bactericidal mechanism AMPPs within a day, but also introduce some toxic impurities,
that the positively charged moieties destabilize and pierce through making the system essentially impractical for biomedical appli-
10
the lipophilic layer of the pathogen membranes by electrostatic cations. Other innovations like high vacuum or UV irradiation
interactions, resulting in membrane disintegration and cytoplasmic techniques have averted these issues; however, they still suffer
4
leakage. The pathogen eventually dies without developing resis- drawbacks such as a complex and expensive experimental setup
11
tance. Unfortunately, the pervasive application of AMP-based (Table S1, ESI†).
antimicrobial agents is hampered by their low profitability from
an industrial perspective and from severe toxicity in academia.
Recently, we reported a versatile, eco-friendly, and metal-free
imidazolium hydrogen carbonate (IHC)-mediated rapid NCA
5
Although solution-coupling and solid-phase synthesis techniques polymerization, which is suitable for high-throughput screening
1
2
have circumvented this problem, these strategies are usually sub- of polypeptide libraries for biomedical applications.
jected to a trade-off between obtaining precise control over the To expand the practical application of this approach in
chain compositions and utilizing multistep processes in a laborious, biomedicine, it is essential to synthesize topologically nanoengi-
6
time-consuming and expensive manner.
neered antimicrobial polypeptides (TNAPs), including linear- (l-),
hinged- (h-), star- (s-), and cyclic- (c-)poly(L-lysine) -random-
poly(g-benzyl-L-glutamate) (K -r-BE ) with different K to BE
x
a
y
x
y
BK21 PLUS Center for Advanced Chemical Technology, Department of Polymer
ratios (Fig. 1) and to compare the effect of architectural differences
on their antimicrobial efficacy. The pendent cationic K moieties
are expected to adhere to the bacteria membrane, which gives
the hydrophobic BE group an opportunity to penetrate the cell
membrane. Linear-, hinged-, and star-TNAPs are easily accessible by
IHC-organocatalyzed fast ROP of Ne-tert-butyloxycarbonyl-L-lysine
NCA (BLL NCA) and g-benzyl-L-glutamate NCA (BLG NCA) in the
Science and Engineering, Pusan National University, Busan 609-735,
Republic of Korea. E-mail: ilkim@pusan.ac.kr
b
School of Chemical and Biomolecular Engineering, Pusan National University,
Busan, 46241, Republic of Korea
c
Department of Physiology, School of Medicine, Pusan National University,
Yangsan 626-870, Gyeongsangnam-do, Republic of Korea
†
Electronic supplementary information (ESI) available: Detailed experimental
methods and additional data. See DOI: 10.1039/c9cc08095c
356 | Chem. Commun., 2020, 56, 356--359
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1
4
and narrow Ð. In contrast, IHC-mediated polymerization of
BLG NCAs in the presence of ANI, at [M] /[I] ratio of 90: 1,
0
0
obtained 92% conversion and Ð = 1.29 in 80 min of polymeriza-
tion (Table 1, entry 2). The sterically hindered tertiary amine of
TREN activated NCA and facilitated the three primary amines to
1
5
bring about the living ROP of NCAs. Intriguingly, our findings
not only reinforce the advantages of TREN initiated polymeriza-
tions relative to those induced by primary amines, but also prove
that IHC can act cooperatively with TREN and in synergy to trigger
a faster polymerization of NCAs. For example, TREN initiated
Fig. 1 Illustration of the tailored TNAPs as antimicrobial agents for combating
bacteria.
polymerization ([M] /[I] = 90 : 1) catalyzed by IHC achieved a 98%
presence of aniline (ANI), ethylenediamine (EDA), and tris-(2-
aminoethyl)amine (TREN), respectively, while the cyclic-TNAPs
are prepared in the absence of amine initiators. Notably, this
work is a proof-of-concept study of large-scale and quick library
synthesis of architectural amphiphilic polypeptides as mimics of
host defence peptides for antimicrobial activity screening.
A systematic investigation into the polymerization behaviours
of IHC-catalysed ROP is necessary to understand the mechanistic
characters of this organocatalysis machinery, and thus guide the
synthesis of TNAPs with high chain-end fidelity. We found that
the four series of polymerizations performed by controlling the
monomer/initiator ([M] /[I] ) ratios from 30 to 200 proceeded very
0
0
BLG NCA conversion in 20 min (Table 1, entry 10), whereas solely
TREN induced ROP at a similar feed ratio needed 96 min for
complete conversion.
Size exclusion chromatography (SEC) analysis of the as-obtained
BEs revealed symmetrical and monomodal traces and M
shifted monotonously toward the higher M sides with increasing
feeding [M] /[I] ratios (Fig. S2, ESI†). To evaluate the operability of
n
n
0
0
scalable synthesis by this approach, IHC-catalysed polymerization
was performed at the gram scale in the presence of a TREN
initiator. As illustrated in Fig. S3 (ESI†), the well-defined s-BE with
À1
M
n
= 21.4 kg mol and Ð = 1.27 was accessible in 25 min of
0
0
polymerization.
rapidly to achieve a high BLG NCA conversion, as summarized in
Table 1. For reactions involving a [M] /[I] ratio of 30 : 1, the ANI,
EDA, TREN, and IHC induced polymerizations reached 27%, 59%,
6%, and 78% BLG NCA conversions in 5 min, respectively
Fig. S1, ESI†). It is worth mentioning that ANI is a significantly
‘poor’’ nucleophile compared to aliphatic primary amines, leading
to slow and uncontrolled polymerization. Although the frustrated
Lewis pairs of bulky borane Lewis acid and aromatic amine
have previously been employed to initiate the ROP of NCAs to
afford controlled polymerization, it takes longer reaction time to
The kinetic study of IHC-mediated polymerizations of BLG
NCA in the presence of ANI, EDA and TREN initiators and with
no initiator at different feed ratios showed that the rates of
polymerization with TREN initiator and with no initiator were
faster than those with EDA and ANI initiators (Fig. S4, ESI†).
0
0
7
(
‘
1
3
The plots of ln([M] /[M]) versus time fit well to a series of linear
0
theoretical lines, implicating that the polymerization rates are
first-order dependent on NCA concentration with negligible
termination and/or chain transfer reactions.
1
13
H and C NMR spectra of the resultant BEs show all signals due
to the proton resonance of the repeating units and corresponding
initiators (Fig. S15–S22, ESI†). To further substantiate the chain-end
fidelity of polypeptides, a matrix-assisted laser desorption/ionization-
time of flight mass spectrometry (MALDI-TOF MS) image was
recorded. As shown in Fig. 2, the molar mass distributions of l-, h-,
n
achieve polypeptides with controllable molecular weights (M )
Table 1 Results of IHC-organocatalyzed polymerizations of BLG NCA
À1
M
n
(kg mol )
a
b
b
c
c
Entry Initiator [M]
0
/[I]
0
Time (min) Conv. (%) Calc. SEC
Ð
s-, and c-BEs appeared narrow with a peak-to-peak mass increment of
À1
2
19.09 g mol corresponding to the molar mass of a BLG NCA unit.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
ANI
30 : 1
90 : 1
150 : 1
200 : 1
30 : 1
30
80
160
250
20
40
60
83
10
20
30
42
10
20
50
80
93
92
6.28
6.43 1.34
Additionally, the m/z values of discrete peaks are consistent with the
18.27 16.31 1.29
90
29.67 33.02 1.23 theoretical M
of BEs possessing the respective ANI, EDA, TREN, and
IHC termini. No cyclic analogues contaminate the l-, h- and s-BEs. All
these observations indicate that the IHC-catalysed polymerizations of
n
d
d
d
d
499
98
43.91 47.15 1.25
6.41 5.70 1.21
18.90 22.04 1.13
EDA
TREN
IHC
90 : 1
96
150 : 1
200 : 1
30 : 1
93
30.73 35.11 1.08 BLG NCA in the presence of ANI, EDA and TREN initiators and with
499
97
43.88 51.32 1.19
6.49
19.43 17.65 1.12
n
no initiators result in excellent control over M with narrow Ð and
high chain-end fidelity, which enables the scalable synthesis of
31.26 27.57 1.18 tailored polypeptides for biomedical applications.
As guided by the above systematic investigations, a library
x y
of TNAPs, including l-, h-, s-, and c-K -r-BE copolymers with
30.39 32.54 1.09 various K to BE ratios, was synthesized from the IHC-mediated
6.71 1.07
0
1
2
3
4
5
6
90 : 1
98
150 : 1
200 : 1
30 : 1
90 : 1
150 : 1
200 : 1
95
499
96
43.96 38.36 1.24
6.50
18.77 19.41 1.22
7.13 1.14
95
92
499
43.53 49.22 1.27
ring-opening copolymerization of BLL NCA and BLG NCA in the
M] /[I] represents the initial molar ratio of monomer to initiator. Note presence of ANI, EDA and TREN initiators and in the absence of
0 0
a
[
that IHC was used as a catalyst in entries 1–12 with the initial [I]
0
/[IHC]
were calculated by
H NMR spectra. Determined by SEC calibrated with polystyrene stan-
0
any initiators. The resultant TNAPs with an average degree of
polymerization (DP) of 33 residues per initiating site have narrow
Ð and controllable M that are consistent with the feed ratios
n
b
ratio of 1 : 0.3. Conversion and experimental M
n
1
c
d
dards. Polymerization time was determined for 99% conversion.
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are lower than those of commonly used antibiotics, such as
1
7
kanamycin and ampicillin, and also lower than those of a lot of
antimicrobial polymers consisting of K subunits, as summarized
in Table S4 (ESI†). In addition, the increase in relative amount of
BE in the TNAPs by keeping DP constant significantly lowered
the MICs, suggesting that the hydrophobic moieties have a great
impact on their antibacterial effect. Nevertheless, an excess
amount of BE component reduced the electrostatic interaction
between the TNAPs and the bacterial membrane due to the
decrease of the cationic densities, and thus elevated MICs of
the TNAPs.
It is noteworthy that s-TNAPs consistently displayed the
strongest antimicrobial effect, as compared to those of l-, h- or
c-TNAPs at similar K to BE ratios. Qiao et al. reported that the
polymer with a star architecture exhibited considerably higher
antibacterial activities than their linear analogues, while Coelho
et al. reported that the polymer architecture played no key role in
3
,18
the bactericidal efficacy.
n
In our study, the M value increases
in multiples from l- and c-, to h-, and to s-TNAPs with the same K
to BE ratio due to the increased number of initiating sites, leading
to the increase of K moieties and equating to the increase of local
concentration of cationic charges. The elevated local concentration
Fig. 2 MALDI-TOF MS spectra of (a) linear-, (b) hinged-, (c) star-, and
(
d) cyclic-BEs possessing the respective ANI, EDA, TREN, and IHC termini. of cationic charges may result in a greater cell membrane-induced
polypeptide aggregation and a stronger electrostatic interaction
between the TNAPs and the bacterial membranes, enabling the
(
Table S2 and Fig. S23–S46, ESI†). We have noticed that the TNAPs to more easily achieve the high threshold concentrations
19
hydrodynamic diameters of TNAPs are around 7–25 nm and needed for membrane disruption. Finally, the MIC value of the
slightly increase with the increase of Mn value, as judged by TNAPs at the same K to BE ratios enhances in the following order:
dynamic light scattering (Table S2 and Fig. S9, ESI†). The zeta l- o c- o h- o s-TNAP. Interestingly, compared to l-TNAPs, the
n
potential (z) measurements for TNAPs disclosed the positive c-analogues with a similar M value showed higher activity against
charges on the surface of the TNAPs, which is expected to both bacterial strains. The disparity in the antimicrobial activity
promote the electrostatic interactions between TNAPs and the might be ascribed to the polymer aggregation-promoted initial
negatively charged membrane of bacteria, probably endowing adsorption/binding interaction between bacteria and agent, which
20
the antimicrobial activities of TNAPs. The increase of relative is stronger for c-TNAPs compared to l-TNAPs. Other studies have
composition of BE to K lowered the cationic density, leading indicated that the cyclic architecture of antibiotics named man-
to the decrease of z value (Table S2, ESI†).
nopeptimycins plays an important role in the antimicrobial
1
6
To assess the antimicrobial activity of various TNAPs with activity. Therefore, it is also valuable to investigate the quick
different architectures and compositions against both Gram- library synthesis of cyclic polypeptides for antibacterial activity
positive (B. subtilis) and Gram-negative (E. coli) bacteria, the screening. Circular dichroism (CD) measurements were per-
minimal inhibitory concentrations (MICs) were measured based formed to evaluate whether the secondary structure of TNAPs
on a microdilution assay performed in Luria-Bertani (LB) broth might contribute to their antibacterial efficacy. It was found that
(Table S3, ESI†). According to the bacterial inhibition growth homo- and amphiphilic-TNAPs with lower content of BE showed
curves (Fig. S11 and S12, ESI†), bacterial proliferations were a typical random coil conformation with negative bands around
highly visible in the control groups with the broth-containing 200 nm, while the TNAPs with higher content of BE displayed an
bacteria only, whereas the presence of TNAPs restrained the a-helical conformation, as evident from the characteristic double
proliferations of both bacterial strains. More specifically, upon minima at 208 nm and 222 nm in the CD spectra (Fig. S10, ESI†).
incubation of both bacteria with s-K
sedimentation phenomena were observed within 1 h, indicating parameter for TNAPs to exert their antimicrobial activity.
the fast kill-kinetics (Fig. S13, ESI†). Meanwhile, the growth of To get visual insights into the interaction between TNAPs
1
-r-BE0.2 at 3 Â MIC value, Thereby, the secondary structure may not be an important
21
bacteria exhibited a typical dose-dependent antimicrobial effect. and bacteria, the cellular morphology changes of E. coli and
Nutrient/ion-rich medium usually contains anionic peptides and B. subtilis exposed to representative s-K -r-BE at 3 Â MIC
1
0.2
protein fragments, which might bind non-specifically to the value were detected by scanning electron microscopy (SEM).
cationic antimicrobial agents, reducing their antimicrobial Fig. 3a1 and a3 show the SEM images of pristine E. coli and
activity. However, the superior antimicrobial efficacy of TNAPs B. subtilis with a smooth surface and intact cell wall, while the
tested in nutrient/ion-rich LB broth eliminates the concern of s-TNAP-treated bacteria have a wrinkled surface, deep craters
3
,16
latent protein/ion binding.
Notably, the MICs of the TNAPs and lysed cellular morphologies (Fig. 3a2 and a4). Therefore, we
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bacteria and displayed low toxicity. The antimicrobial activity
was positive charge-dependent, related to the hydrophobicity
and architecture of the TNAPs, and, to a lesser extent to the
secondary structure. We believe that TNAPs hold considerable
promise for efficient antibacterial applications in future, and
this versatile synthetic strategy can be broadly applied for the
high-throughput functional screening of polypeptide libraries
for biomedical applications, such as antibiofilm and antifouling.
The Basic Science Research Program through the National
Research Foundation of Korea (2018R1D1A1A09081809) supported
this work. The authors also thank the BK21 PLUS Program for
partial financial support.
Conflicts of interest
There are no conflicts to declare.
Notes and references
Fig. 3 (a) SEM micrographs and (b) confocal fluorescence microscopic
images of control and TNAP-treated groups of E. coli and B. subtilis.
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