Identification of New Nitric Oxide-Donating Peptides with Dual Biofilm Eradication and Antibacterial Activities for Intervention of Device-Related Infections

Article abstract of DOI:10.1021/acs.jmedchem.9b01832

Implantable medical device-related infections with biofilms have become a significant challenge in clinics. Based on the potential bacteria biofilm dispersing effect of nitric oxide (NO) and the unique antibacterial activity of antimicrobial peptides (AMP), we synthesized five peptides and selected the most potent one to conjugate its N-terminal with a furoxan moiety to offer a hitherto unknown NO-donating antimicrobial peptide (FOTyr-AMP), which exhibited Staphylococcus aureus and Escherichia coli biofilm dispersion and eradication, and potent antibacterial activities in vitro. In an implanted biofilm infection mice model, topical subcutaneous injection of FOTyr-AMP allowed synergetic eradication of bacterial biofilms and potent antibacterial activity, superior to the antibiotic cephalosporin C. Given the low hemolysis effect, little influence on the blood pressure, and potent in vivo efficacy of FOTyr-AMP, it is clear that subcutaneous administration of FOTyr-AMP could be a promising approach for the intervention of medical device-related biofilm infections with desirable safety.

Full text of DOI:10.1021/acs.jmedchem.9b01832

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Article
Identification of New Nitric Oxide-donating Peptides
with Dual Biofilms Eradication and Antibacterial
Activities for Intervention of Device-related Infections
Yue Fei, Jianbing Wu, Hong-Wei An, Kai Zhu, Bo Peng, Junquan
Cai, Yihua Zhang, Li-Li Li, Hao Wang, and Zhangjian Huang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01832 • Publication Date (Web): 05 Aug 2020
Downloaded from pubs.acs.org on August 5, 2020
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Identification of New Nitric Oxide-donating
Peptides with Dual Biofilms Eradication and
Antibacterial Activities for Intervention of Device-
related Infections
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Yue Fei, †, ‡ Jianbing Wu, Hong-Wei An, Kai Zhu, Bo Peng, Junquan Cai, Yihua
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‡
‡
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Zhang, Li-Li Li, Hao Wang, *, ‡ and Zhangjian Huang*, †
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*, ‡
† Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for
Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, China.
‡ CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical
Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and
Technology (NCNST), Center of Materials Science and Optoelec-tronics Engineering,
University of Chinese Academy of Sciences, Beiyitiao No.11, Haidian District, Beijing,
100190, China.
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Institute of High Energy Physics, Chinese Academy of Sciences (CAS), Yuquan
Road, Beijing 100049, China.
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ABSTRACT: Implantable medical device-related infections with biofilms have become a
significant challenge in clinic. Based on the potential bacteria biofilm dispersing effect of nitric
oxide (NO) and the unique antibacterial activity of antimicrobial peptides (AMP), we
synthesized five peptides and selected the most potent one to conjugate its N-terminal with a
furoxan moiety to offer a hitherto-unknown NO-donating antimicrobial peptide (FOTyr-AMP),
which exhibited S. aureus and E. coli biofilms dispersing and eradication, and potent
antibacterial activities in vitro. In an implanted biofilms infection mice model, topical
subcutaneous injection of FOTyr-AMP allowed synergetic eradication of bacterial
biofilms and potent antibacterial activity, superior to antibiotic Cephalosporin C. Given
low hemolysis effect, little influence on the blood pressure and potent in vivo efficacy of
FOTyr-AMP, it is clear that subcutaneous administration of FOTyr-AMP could be a
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promising approach for the intervention of medical device-related biofilm infections with
desirable safety.
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INTRODUTION
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Implantable medical devices, such as intravascular catheters, heart valves, pace-
makers, prosthetic joints, dialysis catheters, dental implants, orthopaedic and trauma
devices, have benefited healthcare, treatment, and recovery of patients. However, high
risk of the medical device-related infections has remarkably threated the health of
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patients, causing undue distress . Especially, the biofilm formation on the surfaces of
the implants could protect bacterial species against antimicrobial agents and immune
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defenses, resulting in obstinate infection and increasing morbidity and mortality .
Biofilms are aggregates of microorganisms in which cells are frequently embedded in a
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self-produced matrix of extracellular polymeric substances . Once the biofilm is formed,
bacterial cells being protected inside the biofilm could perform high resistance to
antimicrobial agents over 10 -folds compared to their planktonic counterparts2, 5. In this
regard, most clinical drugs are assessed failed for treatment of such situations. Given
the key roles of biofilms in device-related infections and limited treatment options,
solutions towards opening the ‘black box’ of biofilm infections are urgently needed.
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As a well-known gas messenger molecule, nitric oxide (NO) is necessary in body’s
innate immune response to foreign pathogens, especially in macrophage defenses
against intracellular bacteria. NO as such, or together with reactive nitrogen species
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(RNS) which derived from the reaction of NO with reactive oxygen species (ROS), could
cause oxidative and nitrosative damage to microbial proteins, DNA, metabolic enzymes,
or exterior membrane structure. Furthermore, it was reported that NO could effectively
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induce biofilm dispersal in Pseudomonas aeruginosa , Staphylococcus aureus and
other bacterial species9-10. The possible mechanism is that NO could reduce the levels
of cyclic diguanylate (c-di-GMP), which promotes bacteria-surface attachment and then
formation of biofilms11-13. Therefore, NO-based antimicrobials, including enzyme-
triggered NO-donating agents, NO-releasing macromolecules and NO donor coated
nanoparticles, have attracted many attentions, representing a promising alternative for
the treatment of chronic biofilm infections14-16. However, in vivo therapeutic efficacy
against biofilms infection for these NO donating agents were not reported yet.
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Furoxans, a kind of NO donor, containing 1,2,5-oxadiazole 2-oxide heterocyclic
scaffold, can efficiently release NO under the attack of thiol-containing molecules
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(
Scheme S1), especially glutathione (GSH), at positions 3 and 417-18. Compared to other
NO donors, furoxans have a characteristic of prolonged duration of action19-21. Notably,
it was reported that GSH played an important role in P. aeruginosa biofilm formation
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and infection . Therefore, topical administration of furoxans could specifically release
NO under attack of GSH at the infection site to achieve biofilm dispersing effect.
Importantly, besides the biofilm eradication, bacteria killing is indispensable for biofilm
infections. Antimicrobial peptides (AMPs), possess unique membrane-permeabilizing
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activity, representing a promising therapeutic option against bacterial infections .
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Cationic peptides , a group of AMPs playing an important role in immune system,
possesses broad spectrum of antibacterial activity, low resistance and hemolysis. In
these regards, we hypothesized that whether hybridization of furoxan moiety with AMPs
could produce a hitherto-unknown NO-donating AMPs with dual biofilms eradication and
antimicrobial activities that could be more effective for the intervention of medical
device-related biofilm infections than monotherapy in vivo. To test this hypothesis, we
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selected and synthesized five cationic peptides 1-5 with straight chain which were
reported to have potentially broad antibacterial activity25-27 and tested the antibacterial
activity against Gram-positive S. aureus and Gram-negative E. coli. As shown in table
S1, peptide 5 exhibited the most potent antibacterial activity. Therefore, we conjugated
the N-terminal of peptide 5 with a furoxan moiety via a tyrosine-linkage and a click
reaction to produce a NO-donating AMP, FOTyr-AMP (Figure 1A). It was proposed that
FOTyr-AMP, containing a furoxan head and an AMP tail, could be endowed enhanced
antimicrobial efficacy to biofilm-exist infection owing to biofilms dispersing assisting with
antimicrobial activity (Figure 1B). We investigated the antimicrobial activities, biofilms
eradication effect of FOTyr-AMP against S. aureus and E. coli in vitro. Furthermore, we
evaluated the potential of FOTyr-AMP for the intervention of medical device-related
infections via topical administration strategy by using a biofilms infection model in mice.
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Figure 1. A) The synthetic route of FOTyr-AMP. N -AMP (green) was synthesized based
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on solid-phase peptide synthesis (SPPS) approach. Furoxan moiety FOTyr was
synthesized and characterized. Reagents and conditions: a) 5-hexynoic acid, HBTU, N-
methylmorpholine, DMF, overnight; b) CuSO , sodium ascorbate, THF, H O. B)
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Schematic illustration of biofilms eradi-cation and enhanced antibacterial activity of
FOTyr-AMP against biofilm protected bacterial infection.
RESULTS AND DISCUSSION
The five peptides (RLARIVVIRVAR-NH2 1; ILAWKWAWWAWRR-NH2 2;
KSRIVPAIPVSLL-NH 3; ILPWKWPWWPWRR 4; TRRKFWKKVLNGALKIAPFLLG 5)
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were synthesized by using a solid phase method and the procedure are described in
Scheme S2-6. The synthetic route of FOTyr-AMP is depicted in Figure 1A (detailed in
Scheme S7). The reaction of the N-terminus of peptide 5-resin with azide acetate, and
the subsequent deprotection by 5% triisopropylsilane (TIPS) in trifluoroacetic acid (TFA)
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solution produced azide derivative N -AMP. Meanwhile, tyrosine methyl ester
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substituted furoxan derivative FOTyr (11) was synthesized according to the previously
reported method28 (Scheme S7). The condensation reaction of FOTyr (11) with 5-
hexynoic acid furnished alkynyl-FOTyr (12). Finally, the click reaction of N -AMP and
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alkynyl-FOTyr (12) produced target molecule FOTyr-AMP.
The NO release from FOTyr and FOTyr-AMP in Gram-positive bacteria S. aureus and
Gram-negative E. coli cells were firstly investigated by measuring the concentrations of
nitrite (an oxidative metabolite of NO) in cell lysis after co-incubation for 4 h by Griess
assay. As shown in Table S2 and S3, FOTyr-AMP released significantly more amount
of NO than FOTyr in both S. aureus and E. coli strains, while the pretreatment of 1-
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chloro-2,4-nitrobenzene (CDNB, a well-known GSH-depleted agent) significantly decreased
the NO release from FOTyr-AMP in S. aureus strains, suggesting that FOTyr-AMP could
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effectively penetrate into bacteria cells and release NO under attack of thiol-containing
molecules. Additionally, the dose dependent hemolysis assay provided that FOTyr-AMP
possessed less hemolysis than AMP (Figure S1), and the half hemolytic concentration
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(
HC ) of FOTyr-AMP was greater than 380 μM.
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The antibacterial activity of FOTyr, peptide 5 (AMP) and FOTyr-AMP against S.
aureus and E. coli was evaluated by microdilution assay, and the corresponding
minimum inhibitory concentration (MIC) values were calculated and summarized in
Table S4. It was found that furoxan moiety FOTyr showed weak bacterial growth
inhibitory activity (MIC values were greater than 50 μM), and FOTyr-AMP exhibited
slightly enhanced bacterial growth inhibitory activity than AMP against S. aureus (2.2 vs
3.1 μM) and E coli (1.3 vs 2.0 μM), suggesting the introduction of furoxan moiety at the
N-terminus of AMP may not affect the membrane-permeabilizing activity of AMP, but
somehow improve the antibacterial activity due to the release of NO. Furthermore, by
using scanning electron microscopy (SEM), the morphologies of S. aureus and E. coli
cells treated by FOTyr-AMP (200 μM, 60 mins) confirmed that the membrane of bacteria
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was ruptured and the contents were partially spilled (Figure 2), indicating potent
bacteria killing effect of FOTyr-AMP.
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Figure 2. SEM images of FOTyr-AMP treated S. aureus and E. coli strains. Left:
Controls; Right: FOTyr-AMP (200 µM) treated bacteria (Up: S. aureus; Down: E. coli) for
60 mins. Red arrows: bacteria cells with broken cell wall/membrane and leakage of
contents. Bar: 5 µm.
Biofilm dispersal effect of FOTyr, AMP and FOTyr-AMP against S. aureus and E. coli
were quantitatively investigated by crystal violet staining assay. As shown in Figure 3,
the biofilm biomass of both S. aureus and E. coli in AMP treated groups (100 μM for 12
h) were similar to those in the vehicle control group, indicating little biofilm dispersing
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effect of AMP (Figure 3A). In sharp contrast, treatment of FOTyr and FOTyr-AMP (100
μM for 12 h) significantly reduced the biofilm biomass of S. aureus by 44.4% and
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64.1%, and reduced the biofilm biomass of E. coli by 81.2% and 80.9%, respectively
(Figure 3A). Interestingly, the addition of a NO scavenger PTIO (100 μM) totally
abrogated the biofilm eradication effect of FOTyr-AMP (Figure S2). Collectively, these
data suggest that the NO released from furoxan moiety under the attack of thiol-
containing molecules mainly contributed the biofilm dispersing effect of FOTyr-AMP. In
order to visualize the morphologies of the biofilms after treatment, the SEM images of
all groups were recorded. Briefly, S. aureus and E. coli were cultured on the silicon
wafers in Trypticase Soy Broth (TSB) medium for 24 h to allow the formation of biofilms,
followed by the addition of blank TBS, or AMP, FOTyr and FOTyr-AMP in TBS,
respectively, at a dose of 100 μM for further incubation of 12 h. As shown in Figure 3B,
in the blank TSB treated group (control), it can be seen a densified and thick biofilm,
while the treatment of AMP displayed weak biofilm dispersing effect. In sharp contrast,
the treatment of FOTyr-AMP dramatically dispersed the formed biofilms superior to
FOTyr.
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Figure 3. A) Quantitative calculation of biofilm biomass (S. aureus and E. coli) by crystal
violet staining assay. The statistical analysis of FOTyr, AMP and FOTyr-AMP (100 μM
for 12 h) were all compared with the control. Statistical significance; **/# #, p < 0.01;
two-sample t-Test analysis. Data: mean ± S.D. (n = 3). B) SEM images of biofilms after
treatment of blank TSB, 100 μM of FOTyr, AMP or FOTyr-AMP in TSB for 12 h. Bar: 20
µm.
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To fully understand the biofilm eradication dynamics and planktonic bacteria killing
effect of FOTyr-AMP, we designed a mixed assay (Figure 4A and 4C). Briefly, S. aureus
was added into a 16-cell plate and incubated for 24 h to allow biofilm grow on the
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bottom, which were timely recorded by using a real-time cell counter assay . After
discarding the supernatant and washing with saline, blank TSB, AMP, FOTyr-AMP and
a known antibiotic Cephalosporin C (CPC) in TSB were respectively added with a final
concentration of 100 μM and incubated for further 9 h. During this period, the biofilms on
the bottom of each groups were continually monitored every 15 mins to obtain a biofilm
quantity-time profile (Figure 4A). It was found that during the initial 24 h before the
administration of the agents, the biofilms of all groups in the 16-cell plate showed an
exponential growth between 0~7 h, then a stable maintenance lasted up to 24 h (Figure
S3). Compared with the naturally growth of the biofilm (TSB control), the group of AMP
and CPC had a significant inhibition of the continuous biofilm growth after treatment for
1-2 h, while the FOTyr-AMP treated group showed downward trend at the beginning of
the treatment (Figure 4B). These results indicated that AMP and CPC could efficiently
kill the freed bacteria outside of biofilm leading to the inhibition of the biofilm growth,
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whereas FOTyr-AMP actively disperse the biofilm, freed the protected bacteria and kill
planktonic bacteria resulting a dispersing and eradication of the biofilm. The same
assay was conducted for FOTyr and FOTyr plus AMP. As shown in Figure S4-S5, FOTyr
plus AMP had a sustaining and stable inhibitory effect on the biofilm growth during the whole
period after treatment, while FOTyr alone was not able to suppress the biofilm growth
during the last 2h, indicating the addition of AMP indeed synergistically inhibited the
biofilm growth due its antibacterial activity. To further confirm this conclusion, we carried
out another assay to evaluate both biofilm eradication (biofilm on the bottom) and
antibacterial activity (in supernatant) of the test compounds (Figure 4C). S. aureus
strains was added into a confocal dish and incubated for 24 h. After discarding the
supernatant and washing with saline, blank TSB, FOTyr, AMP, FOTyr-AMP and CPC in
TBS were respectively added with a final concentration of 100 μM and incubated for
further 12 h. After incubation finished, the biofilms on the bottom of the confocal dish
and the supernatant were separated. The former ones were conducted 3D confocal
imaging to visualize the live bacteria cells by using live cells fluorescence staining assay
(Figure 4D), while the latter ones were measured the OD values to calculate the living
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planktonic bacterial cells in the supernatant (Figure 4E). 3D confocal imaging exhibited
that the treatments of CPC were not able to effectively decrease the living bacterial cells
in the biofilm on the bottom of the confocal dish compared with the solvent control
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(Figure 4D). Dramatically, the treatment of FOTyr-AMP almost completely killed the
bacterial cells in the biofilm on the bottom, superior to AMP and FOTyr (Figure 4D).
Accordingly, FOTyr-AMP also significantly killed the living planktonic bacterial cells in
the supernatant, superior to CPC (Figure 4E). Interestingly, although FOTyr significantly
decreased the living bacterial cells in the biofilm on the bottom of the confocal dish
(Figure 4D), it displayed weak bacterial cells killing activity in the supernatant (Figure
4E). Collectively, these data demonstrated that FOTyr-AMP, containing furoxan head
and AMP tail, not only effectively dispersed biofilm, but also exhibited potent
antibacterial activity superior to AMP and CPC, suggesting a promising agent for the
intervention of biofilm infection.
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Figure 4. A) Schematic illustration of dynamic detection of biofilm. B) Biofilm index of S.
aureus by the treatment of the test compounds. After one day of biofilm formation (for
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4 h, at 37 ℃ under 5% CO ), the medium was changed to TSB, AMP (100 μM in TSB),
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CPC (100 μM in TSB), FOTyr-AMP (100 μM in TSB), respectively, and the biofilm
indexes were continuously monitored every 15 mins for 9 h by xCELLigence RTCA DP
Instrument. C) Schematic illustration of the mixed assay to evaluate the biofilm
eradication (biofilm on the bottom) and antibacterial activity (in supernatant) of the test
compounds. D) 3D confocal images of the biofilm on the bottom of the confocal dishes
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for each group after 12 h of incubation. Bar 20 μm. E) Calculation of the living bacterial
cells in the supernatant separated from the confocal dishes.
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To verify the potential of biofilms eradication and antibacterial activity of FOTyr-AMP
in vivo, we modified a previously reported biofilms infection model to mice31-32. As
shown in Figure 5A, we cultured biofilms of S. aureus on the surface of
polydimethylsiloxane (PDMS) slices (0.3 × 0.3 cm) overnight, and then the PDMS slices
were subcutaneously implanted into the back of the mice. Twelve hours after
implantation, blank solvent, FOTyr, AMP, FOTyr-AMP, FOTyr-AMP plus NO scavenger
PTIO, and CPC with the same dose of 0.25 μmol/kg in 5% DMSO in saline, were
respectively subcutaneously administrated to mice every 12 h for 3 days. Then, the
PDMS slices were removed from mice for ultrasound, and the bacterial cells dropped
from the PDMS slices were quantitatively counted. It was found that in the control group
the skin near the implanted site had obvious ulceration, indicating a serious bacterial
infection, while in the FOTyr-AMP treated group no visible ulceration was observed
(
Figure 5B). Plate colony counting assay33 demonstrated that FOTyr-AMP (Log CFU
10
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value 4.54 ± 0.30) significantly decreased colony counts than solvent control (Log CFU
10
value 6.40 ± 0.28), superior to AMP (5.40 ± 0.14), CPC (4.97 ± 0.21) and FOTyr (4.91 ±
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0.49). Notably, the co-treatment of NO scavenger PTIO (0.25 μmol/kg) significantly
abrogated the activity of FOTyr-AMP, decreasing Log CFU value to 5.92 ± 0.13 (Figure
10
5C), indicating the contribution of NO. In addition, the vicinity tissues of the implanted
site were taken to perform H&E staining to evaluate bacterial infection. We found that
FOTyr-AMP significantly decreased the bacterial inflammation in the vicinity tissues of
the implanted site, superior to the antibiotic CPC and AMP groups (Figure 5D). Notably,
we found that the subcutaneous administration of FOTyr-AMP (0.172 μmol/kg) in rats
had little influence on the blood pressure within 6 h after injection (Table S5). Given the
low hemolysis effect, little influence on the blood pressure and potent in vivo efficacy of
FOTyr-AMP, it is clear that subcutaneous administration of FOTyr-AMP could be a
promising approach for the intervention of medical device-related infections with
desirable safety.
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Figure 5. A) Schematic diagram of biofilm infection mice model. B) State diagram of the
implanted site every 12 h after PDMS slices were surgically implanted. Black arrows:
obvious ulceration sites. C) Bacterial culture counting on the implanted PDMS after
ultra-sound. The data were expressed as mean ± SD (n = 3), one-way ANOVA with a
Turkey comparisons post hoc test. ** p<0.05. D) H&E staining of the vicinity tissues of
the implanted site (Bar 50 μm).
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CONCLUSION
Herein, we have synthesized five antibacterial peptides and selected the most potent
one to conjugate its N-terminal with a furoxan moiety to offer a hitherto-unknown NO-
donating AMP FOTyr-AMP. Notably, the conjugation of AMP with the NO donor
provided dual biofunctions of biofilm eradiation and antibacterial activity. Dynamics
studies of biofilm dispersing showed that FOTyr-AMP released NO from the furoxan
head to sustainably eradicate biofilm, subsequently free the bacterial cells, which could
be more sensitive to the AMP tail. Furthermore, we established an implanted biofilms
infection mice model, to the best of our knowledge for the first time, to evaluate the in
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vivo therapeutic efficacy of the NO donating AMP for implantable medical device-related
biofilms infections. It was found that FOTyr-AMP significantly decreased bacterial cells
on the PDMS slices, relieved the skin ulceration and decreased the bacterial
inflammation in the vicinity tissues of the implanted site, superior to AMP and antibiotic
CPC. Importantly, subcutaneous administration, GSH specific NO-release and bacterial
targeting of AMP all contributed to minimize NO-mediated off-target effects. In
conclusion, FOTyr-AMP exhibited dual biofilms eradication and antibacterial activities,
warranting further investigation for the intervention of implantable medical device-
related biofilm infections. Our strategy may also expand the NO-based therapeutics for
clearing biofilm-based infections.
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EXPERIMENTAL SECTION
Chemistry. General information. All commercially available compounds were used without
further purification, unless otherwise noted. Analytical and preparative TLC was performed on
silica gel (200 - 300 mesh) GF/UV 254 plates, and the chromatograms were visualized under UV
1
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light at 254 and 365 nm. H-NMR and C-NMR spectra were recorded on a Bruker AVANCE
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III HD 400 ( H, 400 MHz; C, 100 MHz) spectrometer at 303 K, using TMS as an internal
standard. MS spectra were recorded with a Mariner mass spectrometer (ESI) and high-resolution
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mass spectrometry (HRMS) spectra on an Agilent Technologies LC/MSD TOF instrument.
Solutions after reactions and extractions were concentrated using a rotary evaporator operating at
a reduced pressure of ~20 Torr. HPLC was performed on a Shimadzu Series (LC-20AT) using an
InertSustain 5 µm C18 column (4.6 × 250 mm) to determine the purity of the test compounds.
Individual compounds with a purity of > 95% were used for biological experiments.
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General synthesis protocol for peptides. The resin was soaked in DMF for 2 h and
then soaked in DMF containing 20% piperidine for 15 min at the room temperature to
remove the Fmoc group. Then the resin was washed for 3 times with dichloromethane
and DMF alternately, after that 3-5 particles were taken out into 1.5 mL centrifuge tube
containing Kaiser test solution, heated to boiling about 1min (if particles turned black,
the protective group has been removed). The obtained resin was filtered and reacted
with the Fmoc-AA-OH (10 equiv.) in the presence of hexafluorophosphate benzotriazole
tetramethyl uronium (HBTU, 10 equiv.) and N-methylmorpholine (5%) in DMF. One hour
later, the resin was washed for 3 times with dichloromethane and DMF alternately, 3-5
particles were taken out into 1.5 mL centrifuge tube containing Kaiser test solution,
heated to boiling about 1min (if particles did not turn black, the coupling has been
completed). After that, the resin was filtered and then soaked in DMF containing 20%
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piperidine to remove the Fmoc group. The similar condensation and deprotection
reactions were conducted in the order of the AA sequence to eventually furnish peptide
resin. Finally, 5% triisopropylsilane (TIPS) in trifluoroacetic acid (TFA) solution was used
to slice peptide from resin, as well as the Boc groups from Lysine and Tryptophan, Pbf group
form Arginine, tBu group from Threonine. The Kaiser test solution was composed of one
drop Ninhydrin solution (0.5 g Ninhydrin in 10 mL Ethanol), one drop phenol solution (20
g phenol in 5 mL Ethanol) and one drop ascorbic acid solution (0.1 g ascorbic acid in 5
mL Ethanol).
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Peptide 1, RLARIVVIRVAR-NH , a total yield of 17%, R: Arginine; V: Valine; L: Leucine;
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+
A: Alanine; I: Isoleucine. ESI MS (m/z): [M + 2H] calcd for C H N O , 711.0; found,
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123 25 12
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11.7. Peptide 1 combines two protons, therefore, the molecular weight of the peptide 1 is
11.7*2-2=1421.4, M calcd for C H N O , 1420.8.
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123 25 12
Peptide 2, ILAWKWAWWAWRR-NH , a total yield of 15%, R: Arginine; K: Lysine; W:
2
Tryptophan; L: Leucine; A: Alanine; I: Isoleucine. ESI MS (m/z): [M + 2H]2+ calcd for
C H N O , 914.5; found, 914.7. Peptide 2 combines two protons, therefore, the molecular
94 128 26 13
weight of the peptide 2 is 914.7*2-2=1827.4, M calcd for C H N O , 1827.0.
9
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128 26 13
Peptide 3, KSRIVPAIPVSLL-NH , a total yield of 16%, K: Lysine; S: serine; R: Arginine; I:
2
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+
Isoleucine; V: Valine; P: Proline; A: Alanine; L: Leucine. ESI MS (m/z): [M + 3H] calcd for
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C H N O , 464.9; found, 464.4. Peptide 3 combines three protons, therefore, the molecular
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121 18 15
weight of the peptide 3 is 464.4*3-3=1390.2, M calcd for C H N O , 1391.0.
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121 18 15
Peptide 4, ILPWKWPWWPWRR, a total yield of 30%, I: Isoleucine; L: Leucine; P: Proline;
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2
3
4
5
6
7
8
9
0
W: Tryptophan; R: Arginine; K: Lysine; V: Valine. ESI MS (m/z): [M + 3H]3+ calcd for
C H N O , 636.8; found, 636.4. Peptide 4 combines three protons, therefore, the molecular
1
00 134 25 14
weight of the peptide 4 is 636.4*3-3=1906.2, M calcd for C H N O , 1906.0.
100 134 25 14
Peptide 5, TRRKFWKKVLNGALKIAPFLLG, a total yield of 27%, T: Threonine; R:
Arginine; K: Lysine; F: Phenylalanine; W: Tryptophan; V: Valine; L: Leucine; N: Asparagine;
-
G: Glycine; A: Alanine; I: Isoleucine; P: Proline. MALDI-TOF MS (m/z): [M - H] calcd for
C H N O , 2554.5; found, 2554.4.
1
23 201 34 25
Synthesis of N -AMP (N -TRRKFWKKVLNGALKIAPFLLG): Fmoc-Gly-OH Wang-
3
3
resin was soaked in DMF for 2 h and then soaked in DMF containing 20% piperidine for
15 min at the room temperature to remove the Fmoc group. The obtained Gly-OH
Wang-resin was filtered and reacted with the second Fmoc-AA-OH (10 equiv.) in the
presence of HBTU (10 equiv.) and N-methylmorpholine (5%) in DMF. After the
condensation reaction was completed, the resin was filtered and then soaked in DMF
containing 20% piperidine to remove the Fmoc group. The similar condensation and
deprotection reactions were conducted in the order of the AA sequence to eventually
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furnish peptide 5-resin. Subsequently, N CH COOH (10 equiv.) was reacted with the
3
2
free amino group of peptide 5-resin in the presence of HBTU (10 equiv.) and DIPEA (10
0
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4
5
6
7
8
9
0
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0
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0
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9
0
equiv.) in DMF. Finally, 5% TIPS in TFA solution was used to slice N -AMP from resin,
3
as well as the Boc groups from lysine and tryptophan, Pbf group from arginine, tBu
group from threonine. N -AMP, N -TRRKFWKKVLNGALKIAPFLLG, a total yield of
3
3
39%, T: Threonine; R: Arginine; K: Lysine; F: Phenylalanine; W: Tryptophan; V: Valine;
L: Leucine; N: Asparagine; G: Glycine; A: Alanine; I: Isoleucine; P: Proline. MALDI-TOF
-
MS (m/z): [M - H] calcd for C H N O , 2637.6; found, 2636.7.
1
25 206 37 26
Synthesis of 4-(4-((N-Tert-butoxycarbonyl) L-alanine methyl ester-3-yl) phenoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (compound 10). Compound 9 was prepared
starting from thiophenol (6), via an addition reaction with chloroacetic acid, an oxidation
reaction with hydrogen peroxide, and the subsequent reaction with fuming nitric acid in
28 1
the presence of acetic acid, by using a previously reported method . H NMR (300 MHz,
CDCl ) for compound 9: δ 8.24 – 8.11 (m, 2H), 7.81 (dd, J = 12.5, 7.3 Hz, 1H), 7.66 (q, J = 7.8
3
Hz, 2H). Compound 9 (0.36 g, 1.00 mmol) was reacted with Boc-Tyr methyl ester (0.44
g, 1.50 mmol) in CH Cl (15 mL) in the presence of DBU (0.46 g, 3.00 mmol) for 2 h
2
2
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under vigorous stirring. After the reaction was completed monitored by TLC, the mixture
was washed with water (50 mL), HCl aqueous solution (2 M, 50 mL) and brine (50 mL),
dried over Na SO and then evaporated under vacuum. The crude product was purified
1
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5
5
5
5
5
5
5
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0
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0
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0
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by flash chromatography on silica gel using petroleum ether and ethyl acetate (100:1 to
1:10) for eluting to give target compound 10 (0.33 g, 62.8%) as a white solid. MS (m/z):
+
1
[
M + Na] calcd for C H N NaO S, 542.1; found, 542.3. H NMR (300 MHz, CDCl ) δ
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3
9
3
8
.13 – 8.05 (m, 2H), 7.79 (t, J = 7.5 Hz, 1H), 7.65 (t, J = 7.8 Hz, 2H), 7.26 – 7.20 (m, 4H), 5.04
(d, J = 6.9 Hz, 1H), 4.59 (d, J = 6.2 Hz, 1H), 3.72 (s, 3H), 3.11 (ddd, J = 32.8, 13.9, 6.1 Hz, 2H),
1
.42 (s, 9H) .
Synthesis of 4-(4-(L-Alanine methyl ester-3-yl) phenoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide (compound 11, FOTyr). Compound 10 (0.20 g, 0.39 mmol) was
dissolved in 20% trifluoroacetic acid in CH Cl (5 mL), and the obtained mixture was
2
2
stirred at the room temperature for 3 h until compound 10 was totally deprotected. The
solvent was removed under vacuum to give the crude product, which was purified by
flash chromatography on silica gel using CH Cl and methanol (100:1 to 1:5) for eluting
2
2
+
to give target compound 11 (0.14 g, 87.0%) as a white solid. MS (m/z): [M + H] calcd
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for C H N O S, 420.1; found, 420.2. H NMR (300 MHz, CDCl ) δ 8.09 (d, J = 7.6 Hz,
18 18
3
7
3
2
H), 7.80 (dd, J = 15.1, 7.5 Hz, 1H), 7.65 (t, J = 7.8 Hz, 2H), 7.28 (s, 4H), 4.29 (s, 1H),
0
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.85 – 3.72 (m, 3H), 3.31 (s, 2H).
Synthesis of 4-(4-(N-5’-Hexynoyl-L-alanine methyl ester-3-yl) phenoxy)-3-
(
phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (compound 12, alkynyl-FOTyr). 5-Hexynoic
acid (0.08 g, 0.79 mmol) and HBTU (0.26 g, 0.68 mmol) were dissolved in DMF (3 mL),
to which was added FOTyr (11) (0.10 g, 0.24 mmol) and N-methylmorpholine (0.070 g,
0.70 mmol). The obtained mixture was stirred at the room temperature overnight. The
mixture was poured into cold water (30 mL), and was extracted with CH Cl (5 mL × 5).
2
2
The combined organic layers were washed with brine (5 mL), dried over Na SO and
2
4
then evaporated under vacuum. The crude product was purified by flash
chromatography on silica gel using petroleum ether and ethyl acetate (40:1 to 1:1) for
eluting to give target compound 12 (0.12 g, 97%) as a white solid. MS (m/z): [M + Na]+
+
calcd for C H N NaO S, 536.1; found, 536.0. HRMS (m/z): [M + H] calcd for
24 23
3
8
1
C H N O S, 514.1284; found, 514.1278. Error[ppm]=0.2. H NMR (400 MHz, CDCl ) δ
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24
3
8
3
8
.13 (d, J = 8.0 Hz, 2H), 7.82 (t, J = 8.0 Hz, 1H), 7.68 (t, J = 8.0 Hz, 2H), 7.22 – 7.20 (m,
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H), 5.98 (d, J = 8.0 Hz, 1H), 4.96-4.92 (m, 1H), 3.77 (s, 3H), 3.24-3.20 (m, 1H), 3.16-3.14
(m, 1H), 2.37 (t, J = 8.0 Hz, 2H), 2.25 (d, J = 8.0 Hz, 2H), 2.00 (s, 1H), 1.89-1.82 (m,
1
1
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H). 13C NMR (100 MHz, CDCl ) δ 170.92, 170.88, 157.41, 150.74, 137.01, 134.92,
3
33.80, 129.89, 128.88, 127.75, 119.08, 109.81, 82.40, 68.48, 52.01, 51.60, 36.50,
3.82, 22.98, 16.80.
Synthesis of FOTyr-AMP: Compound 12 (0.01 g, 0.019 mmol) and N -AMP (0.05 g,
3
0.019 mmol) were dissolved in a solution of water (2 mL) and tetrahydrofuran (2 mL)
containing copper sulfate (1.28 mg, 0.008 mmol) and vitamin C sodium salt (3.17 mg,
0.016mmol). The obtained mixture was stirred at the room temperature overnight. The
reaction solution was filtered and the filtrate was collected for dialysis (the molecular
weight cut off is 2000 D) to remove small molecules. Further lyophilization offered
FOTyr-AMP as a brown solid with a purity of 98.4% determined by HPLC. MALDI-TOF
-
MS (m/z): [M - H] calcd for C H N O S, 3152.7; found, 3153.6.
149 227 40 34
Biology. General information. Bacterial strains of Staphylococcus aureus (ATCC
6538) and Escherichia coli (ATCC 8099) were obtained from China General
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