Polymyxin B3-Tobramycin Hybrids with Pseudomonas aeruginosa-Selective Antibacterial Activity and Strong Potentiation of Rifampicin, Minocycline, and Vancomycin

Article abstract of DOI:10.1021/acsinfecdis.7b00145

There is an urgent need to develop novel antibacterial agents able to eradicate drug-resistant Gram-negative pathogens such as Pseudomonas aeruginosa. Antimicrobial hybrids have emerged as a promising strategy to combat bacterial resistance, as a stand-al

Full text of DOI:10.1021/acsinfecdis.7b00145

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Article
Polymyxin B3 – tobramycin hybrids with Pseudomonas
aeruginosa-selective antibacterial activity and strong
potentiation of rifampicin, minocycline and vancomycin.
Ronald Domalaon, Xuan Yang, Yinfeng Lyu, George G Zhanel, and Frank M. Schweizer
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00145 • Publication Date (Web): 18 Oct 2017
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Polymyxin B3 – tobramycin hybrids with
Pseudomonas aeruginosaꢀselective antibacterial
activity and strong potentiation of rifampicin,
minocycline and vancomycin.
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Ronald Domalaon1, Xuan Yang1, Yinfeng Lyu1,3, George G. Zhanel2, Frank Schweizer*1,2
1Department of Chemistry, Faculty of Science, University of Manitoba, 144 Dysart Road, Winnipeg,
Manitoba, R3T 2N2, Canada.
2Department of Medical Microbiology and Infectious Diseases, Rady Faculty of Health Sciences,
University of Manitoba, 727 McDermot Avenue, Winnipeg, Manitoba, R3T 1R9, Canada
3Institute of Animal Nutrition, Northeast Agricultural University, Harbin, Heilongjiang, 150030, P.R.
China
* Corresponding author. Email address: schweize@cc.umanitoba.ca
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There is an urgent need to develop novel antibacterial agents able to eradicate drugꢀresistant
Gramꢀnegative pathogens such as Pseudomonas aeruginosa. Antimicrobial hybrids have
emerged as a promising strategy to combat bacterial resistance, as a standꢀalone drug but also as
an adjuvant in combination with existing antibiotics. Herein, we report for the first time the
synthesis and biological evaluation of polymyxinꢀaminoglycoside heterodimers composing of
polymyxin B3 covalently linked to tobramycin via an aliphatic hydrocarbon linker. The
polymyxin B3ꢀtobramycin hybrids demonstrate potent activity against carbapenemꢀresistant as
well as multidrugꢀ or extensively drugꢀresistant (MDR/XDR) P. aeruginosa clinical isolates.
Furthermore, the most potent hybrid was able to synergize with currentlyꢀused antibiotics against
wildꢀtype and MDR/XDR P. aeruginosa, but as well against Acinetobacter baumannii. The
promising biological activity described herein warrants additional studies into design and
development of new antimicrobial hybrids able to surmount the problem of antimicrobial
resistance.
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Keywords: antimicrobial hybrid, polymyxin, tobramycin, Pseudomonas aeruginosa, adjuvant,
rifampicin
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Antimicrobial resistance is a pressing concern worldwide.1–3 An increasing incidence rate
of drugꢀresistant bacterial infections has been observed in the past decade, to which effective
antibiotics are dwindling down to a few.4,5 Recently, the WHO released a list of priority
pathogens that necessitate new and effective antibiotic treatments.6 Carbapenemꢀresistant Gramꢀ
negative Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae lead the
list, with new treatments for these pathogens tagged as of critical importance. A major hurdle in
developing antimicrobials against Gramꢀnegative pathogens is attaining sufficient intracellular
accumulation. Intrinsic resistance, including a highly impermeable outer membrane (OM) and
overexpressed efflux pumps, of Gramꢀnegative bacteria prevent antimicrobials from achieving
intracellular concentrations required to induce the desired biological effect. Moreover, the
optimized physicochemical properties required for a molecule to traverse the OM are orthogonal
to that for the inner membrane.7 As a result of insufficient permeability, many agents with potent
(low nanomolar) activity against isolated enzymatic targets frequently exhibit poor whole cell
activity in Gramꢀnegative bacteria.
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Scientific ingenuity has led to various strategies in drug discovery. The covalent fusion of
two known drugs/pharmacophores or drug + delivery vehicle to form a unified heterodimer
construct known as an antimicrobial hybrid is a promising strategy that has yielded several drug
candidates.8–10 At least four antimicrobial hybrids are currently in clinical trials.11 The βꢀlactamꢀ
siderophore conjugate Cefiderocol12 is currently in phase III clinical trials for the treatment of
carbapenemꢀresistant Gramꢀnegative bacterial infections, as well as for complicated urinary tract
infections. Cadazolid13, consisting of a fused fluoroquinolone and oxazolidinone
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pharmacophores, is also currently in phase III clinical trials for the treatment Clostridium
difficileꢀassociated diarrhea. Two more hybrids (TDꢀ1607 and MCB3837) are in phase 1.11
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Our group recently reported tobramycinꢀfluoroquinolone hybrids that are able to
potentiate multiple classes of antibiotics against resistant Gramꢀnegative bacilli.14,15 For instance,
we described tobramycinꢀciprofloxacin14 and tobramycinꢀmoxifloxacin15 hybrids that are able to
sensitize fluoroquinoloneꢀresistant multidrugꢀ or extensively drugꢀresistant (MDR/XDR) P.
aeruginosa to ciprofloxacin, moxifloxacin and levofloxacin. We also developed tobramycinꢀ
efflux pump inhibitor hybrids16 and tobramycinꢀlysine peptoid hybrids17 that were found to
synergize with antibiotics subjected to intrinsic resistance in P. aeruginosa. Our results indicated
that tobramycin attached to a twelve carbonsꢀlong (C12) aliphatic hydrocarbon linker
(considered as an amphiphilic aminoglycoside) possesses intrinsic physicochemical properties
for potentiating multiple classes of antibiotics; while the second pharmacophore enhances the
antibacterial (biocidal) and/or adjuvant potency of the hybrid. Aminoglycosides are known to
possess concentrationꢀdependent bacterial killing. The aminoglycoside tobramycin eradicates
Gramꢀnegative bacteria by inhibition of protein translation at low concentrations while
membrane rupture occurs at higher concentrations.18 Mechanistic studies of the tobramycinꢀ
fluoroquinolone hybrids revealed that the protein translation inhibitory effects of tobramycin
were abolished while its membrane effects were significantly enhanced.14,15 In addition, we have
shown that tobramycinꢀbased hybrids disrupt the outer membrane and affect the proton motive
force of the cytoplasmic membrane in P. aeruginosa.15–17 Similar membrane actions were
reported for other amphiphilic aminoglycosides.19–29 Amphiphilic aminoglycosides were also
reported to possess beneficial immunomodulatory properties30, which may be retained in
tobramycinꢀbased hybrids.
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Polymyxin is a membraneꢀtargeting decalipopeptide antibiotic used to treat MDR/XDR
Gramꢀnegative bacterial infections. It is known to exhibit synergistic interactions with other
antibiotics by enhancing OM permeability31 and is inert to efflux32. Similar to the tobramycinꢀ
hybrid scaffold, polymyxin is also known to disrupt bacterial membranes and affect the proton
motive force.33,34 We therefore hypothesized that linking these two membranotropic
pharmacophores would result in a hybrid with augmented antibacterial and adjuvant properties
against MDR/XDR Gramꢀnegative pathogens. To the best of our knowledge, there has not been
any report of an antimicrobial hybrid containing an aminoglycoside fused to a polymyxin
pharmacophore. Herein, we describe for the first time the synthesis and biological evaluation of
polymyxin B3ꢀtobramycin hybrids.
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RESULTS AND DISCUSSION
The point of covalent attachment between the two chosen drugs is important to retain
meaningful antimicrobial activity. Literature data suggests that the Cꢀ5 position of the cyclitol
ring on tobramycin (TOB) is amenable to chemical modifications.19,35 Alanine scanning of
polymyxin B3 (PMB3) found the amine sideꢀchain of Lꢀ2,4ꢀdiaminobutyric acid (Dab) at amino
acid position 1 to be pliable as it does not contribute to polymyxin’s antibacterial activity.36
Therefore, we decided to fuse TOB and PMB3 at the aboveꢀmentioned position via copperꢀ
assisted azide alkyne cycloaddition (Figure 1). The spatial distance between the two fused
pharmacophores was also explored by incorporating hydrocarbon linkers of varying carbonꢀ
lengths, from four to twelve carbonsꢀlong.
The clinicallyꢀused polymyxin B is naturallyꢀisolated as a mixture of chemicallyꢀrelated
structure with minor differences on its fatty acyl, among which polymyxin B1 with 6ꢀ(S)ꢀ
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methyloctanoic acid and B2 with 6ꢀmethylheptanoic acid as the major component of the
mixture.37 Polymyxin B3 has an octanoic acid lipid portion that is more readily commercially
available relative to the other fatty acyls. Given that all components in the polymyxin B mixture
display similar antibacterial activity and do not interact synergistically with one another38, we
therefore decided to synthesize PMB3 (see Supporting information) as a reference. Following a
reported protocol39, standard solidꢀphase peptide synthesis on a Wang resin and subsequent
solutionꢀphase peptide intramolecular cyclization enable the preparation of PMB3. The
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polymyxin B3 to be fused with tobramycin (3) was synthesized similarly, with Lꢀ
propargylglycine (Pra) instead of Dab at amino acid position 1, to effectively install an alkyne
moiety (Scheme 1).
An azide moiety was installed on the tobramycin intermediates (7a-e) as outlined in
Scheme 2. All the reactive amine and alcohol groups, with the exception of the stericallyꢀ
hindered Cꢀ5 alcohol, were masked following a published protocol16. The aliphatic hydrocarbon,
in the form of a dibromoalkane, was appended to the free alcohol by phaseꢀtransfer catalysis
using tetrabutylammonium hydrogen sulfate (TBAHS) in anhydrous toluene. This resulted to a
tobramycinꢀintermediate that is already attached to the molecular linker (6a-e). The bromide was
then replaced by an azide to yield the desired intermediate.
The alkyneꢀ and azideꢀcontaining intermediates were then fused together via copperꢀ
assisted azide alkyne cycloaddition to form a 1,2,3ꢀtriazole ring linkage (Scheme 3). Copper
iodide triethylphosphite (CuI•P(OEt)3) was used as an efficient copper source in DMF, along
with the weak base diisopropylethylamine (DIPEA). The resulting heterodimer was then
deblocked using HCl in a methanol and tetrahydrofuran mixture to remove the Bocꢀ and
TBDMSꢀprotecting groups of the tobramycin portion. Subsequently, catalytic hydrogenolysis
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was used to deblock the remaining Cbz protecting groups of the polymyxin portion. Using this
strategy, we were able to prepare five polymyxin B3ꢀtobramycin hybrids (Figure 1) containing a
four (1a), six (1b), eight (1c), ten (1d) or twelve (1e) carbonsꢀlong aliphatic hydrocarbon linker.
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We assessed the antibacterial activity of the polymyxin B3ꢀtobramycin hybrids against a
panel of Gramꢀpositive and Gramꢀnegative pathogens (Table 1). The panel included clinical
isolates from Canadian hospitals obtained through the Canadian National Intensive Care Unit
(CANꢀICU)40 and Canadian Ward Surveillance (CANWARD)41 national surveillance studies.
Minimum inhibitory concentration (MIC) values are reported both in µg/mL and µM for
comparison. Antimicrobial hybrids have higher (2ꢀfold or more) molecular weight relative to
their respective parent antimicrobials and therefore would produce a higher MIC (less active) in
µg/mL as the value is dependent on the molecular weight. For instance, the molecular weight of
hybrid 1d (2859.46 g/mol) is double than the commerciallyꢀavailable polymyxin B (1301.56
g/mol) and 5ꢀfold higher than tobramycin (565.59 g/mol). An MIC value in µM would factor out
the molecular weight bias, to which should be used for comparison of the hybrids and parent
antibiotics. Previously reported amphiphilic tobramycin TOB-C1230, having a 12ꢀcarbons long
aliphatic chain attached to the Cꢀ5 position of tobramycin, was also included for comparison.
Similar to PMB3 and TOB-C12, the hybrids demonstrated moderate to poor activity against
Gramꢀpositive bacteria. Notably, 1d (8ꢀ32 µg/mL or 2.8ꢀ11.2 µM) and 1e (8ꢀ16 µg/mL or 2.8ꢀ
5.5 µM) displayed moderate activity across staphylococcal species that include methicillinꢀ
resistant strains. Against Gramꢀnegative bacteria, the hybrids exhibited potent antibacterial
activity against P. aeruginosa relative to other Gramꢀnegative bacilli. Compounds 1b and 1d
were the most potent hybrids and displayed equipotency compared to both PMB3 and TOB
against susceptible strains. For instance, PMB3, 1b and 1d demonstrated similar MICs (2 µg/mL
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or 0.7ꢀ1.1 µM) against a tobramycinꢀresistant P. aeruginosa (CANWARDꢀ2011 96846) clinical
isolate. All hybrids were notably potent relative to amphiphilic tobramycin reference TOB-C12
against P. aeruginosa isolates in the panel. Since TOB-C12 closely resembles the tobramycinꢀ
hydrocarbon linker portion of the hybrid, this data clearly showed the essentiality of polymyxin
B3 pharmacophore on the hybrid’s antibacterial activity. However, the activity of even the most
potent hybrid 1d was 8ꢀ or 16ꢀfold less relative to PMB3 and TOB against all susceptible
Escherichia coli isolates. Weak to poor activity was observed against Stenotrophomonas
maltophilia, A. baumannii and Klebsiella pneumoniae. Indeed, the polymyxinꢀtobramycin
hybrids displayed a relatively narrow spectrum Gramꢀnegative antibacterial activity limited to P.
aeruginosa. Conversely, PMB3 has broad spectrum activity against Gramꢀnegative bacilli but
poor activity against Gramꢀpositive organisms, while TOB demonstrated activity against both
Gramꢀpositive and Gramꢀnegative pathogens. While our current data cannot fully explain the
observed P. aeruginosaꢀspecific antibacterial activity of the hybrids, variations in the
exopolysaccharides and the lipopolysaccharides between Gramꢀnegative bacilli may be
considered. We postulate that these components in P. aeruginosa allows efficient interaction
(either through hydrogen bonding or electrostatic interaction) of the hybrid and the outer
membrane resulting to disruption and bacterial cell death.
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The possibility of nonꢀspecific membranolytic action had to be addressed42 as some of
the hybrids exhibited activity against both Gramꢀpositive (albeit moderate to weak) and Gramꢀ
negative bacteria. The ability of the hybrids to lyse eukaryotic membrane through hemolysis of
red blood cells was explored (Table 1). The concentration to induce 1% hemolysis (MHC) was
evaluated. No hemolysis was observed for any of the hybrids, nor for the membraneꢀactive
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PMB3, even at the highest concentration tested of 128 µg/mL. Therefore, the antibacterial
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activity observed was most likely specific to bacterial membranes.
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The antipseudomonal potency of 1b and 1d was further tested against clinical isolates, all
(except two) were carbapenemꢀresistant, MDR/XDR strains (Table 2). According to Clinical and
Laboratory Standards Institute (CLSI)43, the susceptibility breakpoint for both imipenem (IMI)
and meropenem (MER) in P. aeruginosa is ≤2 µg/mL (~ ≤6 µM). As expected, 1b and 1d
demonstrated relatively potent activity (MIC of 2ꢀ16 µg/ml or 0.7ꢀ5.7 µM) against MDR/XDR
P. aeruginosa strains, which was significantly more potent than the MICs obtained with IMI and
MER. It was apparent that among the two hybrids, 1d possessed slightly better activity relative
to 1b. Hybrid 1d displayed similar MIC to PMB3 in most P. aeruginosa clinical isolates (except
for 101885, 259ꢀ96918 and 264ꢀ104354 strains). Similarly, 1d displayed similar MICs to TOB
in tobramycinꢀsusceptible and significantly lower MICs in tobramycinꢀresistant strains.
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All the strains tested at this point were colistinꢀsusceptible and warranted assessment in
colistinꢀresistant strains. Colistin (COL), also known as polymyxin E, displays a resistance
breakpoint of ≥4 µg/mL (~ ≥3.2 µM) in P. aeruginosa according to CLSI43. The antibacterial
activity of polymyxin B3ꢀtobramycin hybrids was then evaluated in seven colistinꢀresistant P.
aeruginosa clinical isolates (Table 3). Compounds 1b (4ꢀ32 µg/mL or 1.4ꢀ11.4 µM) and 1d (8ꢀ
16 µg/mL or 2.8ꢀ5.6 µM) displayed slightly better or similar MICs to PMB3 (4ꢀ128 µg/mL or
2.3ꢀ72.7 µM) and COL (4ꢀ1024 µg/mL or 3.2ꢀ807.9 µM) against most isolates. It was not
surprising as the major polymyxin resistance mechanism is through membrane modification,
resulting in the loss of overall negative charge in lipopolysaccharides studded on the OM.44–46
This effectively confers resistance to most cationic membraneꢀtargeting antimicrobials such as
polymyxins and antimicrobial peptides. Since we fused two OMꢀacting cationic pharmacophores
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(amphiphilic tobramycin and polymyxin), we expected that resistance due to OM modification
would lead to reduced activity of the hybrids. However, both 1b and 1d displayed lower MICs
relative to PMB3 and COL against colistinꢀresistant P. aeruginosa 101243 clinical isolate.
Specifically, 1d displayed an MIC (16 µg/mL or 5.6 µM) of ~8ꢀfold better than PMB3 (128
µg/mL or 72.7 µM) and ~128ꢀfold better than COL (1024 µg/mL or 807.9 µM). Subtly, both
hybrids also displayed a ~4ꢀfold lower MIC than PMB3 against strain 100403. This observation
suggests that the polymyxin B3ꢀtobramycin hybrids possess enhanced membrane effects relative
to polymyxins alone.
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We recently described antimicrobial hybrids that are able to potentiate clinicallyꢀused
antibiotics against MDR/XDR P. aeruginosa clinical isolates.14–17 Encouraged by these results,
but also from reports of polymyxins interacting synergistically with select antibiotics31, we
evaluated the most potent polymyxin B3ꢀtobramycin hybrid 1d as a potential adjuvant in P.
aeruginosa. We assessed synergy of 1d with a panel of 26 clinicallyꢀused antibiotics in wildꢀtype
P. aeruginosa PAO1 (Figure 2). Synergistic combinations were scored by their fractional
inhibitory concentration (FIC) index. FIC value of ≤ 0.5, 0.5 < x < 4 or ≥ 4 denotes for
synergistic, additive or antagonistic interaction, respectively.47 Polymyxin B3ꢀtobramycin hybrid
1d was found to be synergistic with tetracycline antibiotics minocycline, doxycycline and
tigecycline. It also potentiated the outer membraneꢀimpermeable antibiotics rifampicin,
vancomycin and novobiocin, but also the effluxꢀsusceptible trimethoprim, chloramphenicol,
clindamycin and linezolid. PMB3 and TOB were assessed in combination with the ten aboveꢀ
mentioned antibiotics (Figure 3A and 3B) to investigate whether the adjuvant property of the
hybrid stem from either parent pharmacophores. Both PMB3 and TOB were not able to
potentiate all ten antibiotics, suggesting the fusion of both pharmacophores to be causal of the
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observed adjuvant property. To validate these observations, we performed the same
checkerboard study using the ten antibiotics in combination with either 1d, PMB3 and TOB
against MDR P. aeruginosa 259ꢀ96918 clinical isolate (Figure 3C, 3D and 3E). Similarly, hybrid
1d displayed strong synergy, yet neither PMB3 nor TOB were able to potentiate all of the ten
antibiotics. Therefore, our results indicate that the potent adjuvant property of polymyxin B3ꢀ
tobramycin heterodimer 1d arises from the hybridization of PMB3 and TOB.
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Out of the ten antibiotics, we decided to pursue further evaluation of hybrid 1d in
combination with either minocycline (Table 4), rifampicin (Table 5) or vancomycin (Table 6)
against MDR/XDR P. aeruginosa clinical isolates. Notably, we included colistinꢀ and
tobramycinꢀresistant MDR P. aeruginosa 101243 in the panel. The three antibiotics were chosen
due to their strong synergism; but also that the presence of ≤¼ × MIC (≤4 µg/mL) of hybrid 1d
brought down the antibiotic’s MIC in PAO1 and/or 259ꢀ96918 strains below their interpretative
susceptibility breakpoint. There are no established susceptibility breakpoints for the three chosen
antibiotics in P. aeruginosa (neither CLSI nor European Committee on Antimicrobial
Susceptibility Testing [EUCAST]). Therefore, we have used the established susceptibility
breakpoint for other organisms as representative of clinically achievable concentrations as
comparison for Pseudomonas spp. According to CLSI43, the minocycline breakpoint in
Acinetobacter spp. is ≤ 4 µg/mL, rifampicin breakpoint in Staphylococcus spp. is ≤ 1 µg/mL and
vancomycin breakpoint in Enterococcus spp. is ≤ 4 µg/mL. Hybrid 1d brought down the MIC of
minocycline in all tested P. aeruginosa strains below its susceptibility breakpoint. We previously
reported that the tobramycinꢀhybrid scaffold, indiscriminate of the other appended
pharmacophore, affected the electrical component (ꢁΨ) of the proton motive force resulting in
an increase in the ꢁpH component for compensation.16,17 This leads to an enhanced tetracycline
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(such as minocycline) uptake as it is known to be ꢁpHꢀdependent.48 Our results corroborate that
the intrinsic nature of the tobramycin scaffold to influence the proton motive force is retained in
polymyxin B3ꢀtobramycin hybrids. The outer membraneꢀimpermeable rifampicin was also found
to be synergistic with 1d across the panel of MDR/XDR P. aeruginosa clinical isolates.
Rifampicin’s MIC dropped below its susceptibility breakpoint in almost all (except one) clinical
P. aeruginosa strains in the presence of ≤¼ × MIC (≤4 µg/mL) 1d. In fact, we observed 8ꢀ to
≥2048ꢀfold potentiation of rifampicin against the MDR/XDR P. aeruginosa panel. The
combination of vancomycin and 1d was found to be synergistic in seven out of eight P.
aeruginosa strains. Compound 1d reduced the MIC of vancomycin below its susceptibility
breakpoint in half of the strains tested. The degree of vancomycin potentiation by 1d appeared to
be strainꢀspecific. For instance, vancomycin only demonstrated a 4ꢀfold potentiation in strain
101243 while it resulted in a 1024ꢀfold potentiation in strain 264ꢀ104354. Combination of PMB3
with the three antibiotics exhibited slightly weaker to similar effects relative to 1d across the
panel (see Supporting information). TOB combination with the three antibiotics mostly showed
additive interactions although synergy with rifampicin was observed in half of the strains and
weak synergy with vancomycin was found in two strains tested (see Supporting information).
Our data support the notion that the potent adjuvant properties of polymyxin B3ꢀtobramycin
hybrids in P. aeruginosa arise from the conjugation of both PMB3 and TOB components.
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Synergism of compound 1d with either minocycline, rifampicin or vancomycin against
other Gramꢀnegative bacilli was then explored (Table 7). Checkerboard assay was performed
against three A. baumannii strains, including ATCC reference strain and two clinical isolates.
Compound 1d displayed poor antibacterial activity by itself (MIC of >64 µg/mL). Interestingly,
minocycline was not potentiated against the three isolates (Table 7). We believe that this might
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be due to the fact that the three organisms were already susceptible (below MIC breakpoint) to
minocycline. However, 4 µg/mL of hybrid 1d reduced the MIC of rifampicin (3/3 strains) and
vancomycin (2/3 strains) below their susceptibility breakpoint. Similar to our previous
observations in P. aeruginosa, synergism with vancomycin against A. baumannii appeared to be
strainꢀspecific. The adjuvant potency of 1d was also evaluated against an MDR K. pneumoniae
strain, to which hybrid 1d exhibited poor activity by itself (MIC of >64 µg/mL). Minocycline,
rifampicin and vancomycin were all synergistic with 1d against the K. pneumoniae strain (Table
7).
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CONCLUSION
The synthesis of polymyxinꢀaminoglycoside hybrids possessing meaningful biological
activity was achieved. Specifically, polymyxin B3 was covalently linked to tobramycin using
copperꢀassisted azide alkyne cycloaddition at functional sites amenable to modification. The
polymyxin B3ꢀtobramycin hybrids displayed potent P. aeruginosaꢀselective activity. We
speculate that differences in the exopolysaccharides and lipopolysaccharides between Gramꢀ
negative bacilli are responsible for the observed differences. The most potent hybrid 1d also
synergized clinicallyꢀused antibiotics in wildꢀtype and MDR/XDR P. aeruginosa, with MIC
reductions (minocycline, rifampicin and vancomycin) below susceptibility breakpoints.
Furthermore, hybrid 1d reduced the MIC of rifampicin and vancomycin below their
susceptibility breakpoint in A. baumannii. We highlight the potential of this class of
antimicrobial hybrids not only as a standꢀalone antimicrobial but also in combination with other
existing antibiotic classes.
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EXPERIMENTAL SECTION
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General. Reagents and solvents were purchased commercially and used without purification
unless otherwise noted. Compounds were purified, as specified in their synthesis, by either
normalꢀphase flash chromatography, using Kiesel gel 40 (40ꢀ63 µm) purchased from Merck, or
reverseꢀphase flash chromatography, using C18 silica gel (40ꢀ63 µm) purchased from Silicycle
(USA). TLC was performed on silica gel 60 F254 (0.25 mm) acquired from Merck (USA) to
monitor reactions and was visualized by both ultraviolet light and ninhydrin staining solution.
All reported hybrids/final compounds were found to have purity of ≥ 95% as determined by
highꢀperformance liquid chromatography (HPLC) analysis via Breeze HPLC Waters with 2998
PDA detector (1.2 nm resolution) coupled to Phenomenex Synergi Polar (50 x 2.0 mm) 4 micron
reverseꢀphase column with phenyl etherꢀlinked stationary phase. All purified compounds were
characterized using 1ꢀdimensional and 2ꢀdimensional NMR experiments such as 1H, 13C, COSY,
HSQC and HMBC. All NMR measurements were recorded from either AMXꢀ500 (500 MHz) or
AMXꢀ300 (300 MHz) Bruker instrument (Germany). The molecular weight for all compounds
were recorded by either ESIꢀMS on a Varian 500ꢀMS Ion Trap Mass Spectrometer (USA) or
MALDIꢀTOFꢀMS on a Bruker Ultraflextreme (Germany), using 2,5ꢀdehydroxybenzoic acid as
the matrix.
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Chemistry. The synthesis of intermediate compounds 4, 5 and 6a-e follows our previously
reported protocol16 and is described in the Supporting information.
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Synthesis of uncyclized PMB3ꢀCbzꢀalkyne (2). Solidꢀphase peptide synthesis following a
standard fluorenylmethyloxycarbonyl (Fmoc) protection strategy on a Wang pꢀalkoxybenzyl
alcohol resin was utilized. A published protocol39 was followed with several modifications.
Amino acids with protected functional groups were purchased and used to yield the uncyclized
lipopeptide, having a free carboxylic acid Cꢀterminus of Thr10 and free amine sideꢀchain of Dab4.
Therefore, all the Dab amino sideꢀchain, with the exception of Dab4 which was protected with
Boc, were protected with Cbz protecting group. The two Thr hydroxyl sideꢀchains were
protected with tꢀBu. Pra was used instead of Dab for amino acid position 1 to effectively install
an alkyne functional group. Briefly, the protocol is as follows. Fmoc removal was done by
subjecting the resin to 4:1 DMF:piperidine solution for 15 minutes, and repeated twice to ensure
complete Fmoc removal. Coupling was achieved by the addition of a preꢀactivated coupling
solution [Fmocꢀamino acid or lipid tail (3 mol. eq.) was preꢀmixed with the coupling reagent
N,N,N′,N′ꢀtetramethylꢀOꢀ(benzotriazolꢀ1ꢀyl)uronium tetrafluoroborate (TBTU) (3 mol. eq.) and
Nꢀmethylmorpholine (8 mol. eq.) in DMF 5 minutes prior] to the immobilized peptide on solid
support followed by gentle agitation using N2 gas for 45 minutes. Both Fmoc removal and
coupling steps were followed with thorough washing of the beads using DMF (3x), DCM (3x)
and DMF (3x). A small amount of resin was subjected to Kaiser test (5% chloranil in DMF) after
each coupling and Fmoc removal step to qualitatively ensure that the reaction went to
completion. Consecutive Fmoc removal and coupling steps were performed to yield the resinꢀ
immobilized desired lipopeptide. Cleavage from the solid support was performed by the addition
of 95:5 TFA:water solution followed by gentle stirring for 30 minutes. The solvent was removed
in vacuo to obtain a dry crude. MALDIꢀTOFꢀMS was performed to check for the presence of 2.
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Synthesis of PMB3ꢀCbzꢀalkyne (3). The crude containing compound 2 was mixed with
benzotriazoleꢀ1ꢀylꢀoxyꢀtrisꢀpyrrolidinoꢀphosphonium hexafluorophosphate (PyBOP) (4 mol.
eq.), hydroxybenzotriazole (HOBt) (4 mol. eq.) and Nꢀmethymorpholine (10 mol. eq.) in DMF
under very dilute and anhydrous conditions, followed by vigorous stirring for 2 hours, to afford
the intramolecular cyclization via amide bond formation between the Thr10 carboxyl end and
Dab4 amine sideꢀchain. The solvent was removed in vacuo. Precipitation of the product from the
crude was induced by addition of cold water. The precipitate was filtered and obtained as a pale
brown solid. DCM was added to partially dissolve the product and was evaporated in vacuo to
dryness. MALDIꢀTOFꢀMS was performed to confirm product 3.
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General Procedure for the conversion of bromoꢀ to azidoꢀtobramycin intermediate (7a-e). The
bromoalkylated tobramycin intermediate (6a-e) and sodium azide (3 mol. eq.) were dissolved in
DMF. The resulting solution was stirred at 75 ˚C for 3 hours and transferred to a separatory
funnel. Cold water was added followed by an extraction with ethyl acetate (3x). The combined
organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvent was
removed in vacuo and the resulting solid was dried under high vacuum for more than 24 hours to
afford 7a-e as an opaque solid (91ꢀ97% yield). Rf = 0.52 (hexanes/ethyl acetate = 4:1). Detailed
characterization of compounds 7a-e are described in the Supporting Information.
Synthesis of polymyxin B3ꢀtobramycin hybrids (1a-e). The Cu+ source iodo(triethyl
phosphite)copper (I) was utilized for the formation of a 1,2,3ꢀtriazole ring linkage via copperꢀ
assisted azide alkyne cycloaddition between the intermediate compounds 7a-e and 3.
Unfortunately, the resulting compounds 8a-e proved to have very limited solubility that it did not
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permit a proper purification using neither normalꢀ nor reverseꢀphase flash chromatography.
Therefore, the crude was coꢀdistilled with toluene (3x) and left drying under highꢀvacuum for at
least 24 hours. No further steps were taken and the compounds were used as is in the consecutive
steps.
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Deprotection were done systematically. First, removal of Bocꢀ and TBDMSꢀ protecting
groups were performed. Compounds 8a-e were dissolved in methanol (3 volume ratio eq.)
followed by the addition of HCl (4 volume ratio eq.) and tetrahydrofuran (3 volume ratio eq.).
The acidic solution was stirred for 2.5 hours. The solvent was then removed in vacuo followed
by coꢀdistillation with toluene (3x). The resulting dry compound were crudely purified as the
Bocꢀ and TBDMSꢀdeprotection procedure also resulted to the partial deprotection of Cbzꢀ
protecting groups. The partial deprotection of Cbz groups, which occurred at a slow rate, was
deemed negligible as the subsequent step entitled their global deprotection. Therefore, reverseꢀ
phase flash chromatography was performed using an eluent mixture of water and methanol (both
spiked with 0.1% TFA), from 25% to 100% methanol in water ratio (12.5% stepꢀwise), to afford
compound 9a-e that also contained a minor amount of partially Cbzꢀdeprotected product.
Finally, Cbzꢀdeprotection of the hybrid was achieved by catalytic hydrogenolysis. The
resulting material 9a-e (from the prior step) was dissolved in a mixture of 4:5:1 methanol:acetic
acid:water. Palladium on carbon (Pd/C) was added and the compound was subjected to H2 gas
(balloon) with constant stirring until all the Cbz groups were removed. It was then filtered via
Nylon filter followed by washing of ample amounts of methanol. The filtrate was evaporated to
dryness in vacuo. The crude was then purified using reverseꢀphase flash chromatography having
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an eluent mixture of water and methanol (both spiked with 0.1% TFA), from 0% to 50%
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methanol in water ratio (12.5% stepꢀwise), to afford compounds 1a-e as an opaque solid.
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PMB3ꢀtriazoleꢀC4ꢀTobramycin (1a). 1H NMR (500 MHz, Methanolꢀd4) δ 7.91 – 7.83 (m, 1H,
triazole), 7.32 – 7.20 (m, 5H, DꢀPhe6 aromatic), 5.48 – 5.38 (m, 1H, Hꢀ1’), 5.09 (d, J = 3.2 Hz,
1H, Hꢀ1’’), 4.74 – 4.66 (m, 1H, Pra1α), 4.53 (dd, J = 8.8, 4.4 Hz, 1H, Dab5α), 4.50 – 4.42 (m, 2H,
DꢀPhe6α + Dab8α), 4.42 – 4.08 (m, 11H, Thr2α + Thr2,10β + Leu7α + Dab3,4,9α + C4’s ꢀCH2ꢀN=Nꢀ +
Hꢀ4 + Hꢀ5’), 4.05 (d, J = 4.6 Hz, 1H, Thr10α), 3.95 – 3.62 (m, 11H, C4’s ꢀCH2ꢀOꢀ + Hꢀ5 + Hꢀ6 +
Hꢀ2’ + Hꢀ4’ + Hꢀ2’’ + Hꢀ4’’ + Hꢀ5’’ + Hꢀ6’’), 3.56 – 3.40 (m, 4H, Dab4γ1 + Hꢀ1 + Hꢀ3 + Hꢀ3’’),
3.28 – 3.19 (m, 3H, Pra1β1 + Hꢀ6’), 3.17 – 2.91 (m, 12H, Pra1β2 + Dab4γ2 + DꢀPhe6β + Dab3,5,8,9γ),
2.52 – 2.42 (m, 2H, Dab3β1 + Hꢀ2eq), 2.29 – 1.90 (m, 16H, Dab3β2 + Dab4,5,8,9β + PMB3 C8’s ꢀ
CH2COꢀ + C4’s ꢀCH2CH2ꢀN=Nꢀ + Hꢀ2ax + Hꢀ3’), 1.69 – 1.47 (m, 5H, Leu7β1 + C4’s ꢀCH2CH2ꢀOꢀ
+ PMB3 C8’s ꢀCH2CH2COꢀ), 1.38 – 1.26 (m, 9H, Leu7β2 + PMB3 C8’s ꢀCH2ꢀ), 1.25 – 1.02 (m,
6H, Thr2,10γ), 0.89 (t, J = 7.2 Hz, 3H, PMB3 C8’s ꢀCH3), 0.87 – 0.58 (m, 7H, Leu7γ + Leu7δ). 13C
NMR (126 MHz, Methanolꢀd4) δ 175.48 (PMB3 C8’s carbonyl), 173.91, 172.88, 172.58, 172.50,
172.29, 171.98, 171.87, 171.54, 171.32, 170.98, 143.19 (triazole C without H), 135.82 (DꢀPhe6 C
without H), 128.86, 128.34, 126.72, 123.42 (triazole C with H), 101.07 (Cꢀ1’’), 92.78 (Cꢀ1’),
82.50, 81.72 (Cꢀ6), 76.81, 76.17, 74.08, 72.02, 69.05, 66.41, 65.83, 65.29, 63.50, 59.86, 59.71,
56.46, 55.03, 53.51 (Pra1α), 51.79, 51.67, 51.49, 49.97 (Dab5α), 49.67, 49.62, 48.46, 48.42, 39.27
(Leu7β), 38.45, 38.39, 38.35, 36.65, 36.60, 36.44, 36.36, 36.17, 36.00, 35.39, 31.43, 30.25, 28.88,
28.67, 28.34, 27.69, 26.96, 26.48, 26.38, 25.91, 25.84, 25.51, 23.51, 22.24, 22.17, 20.02, 19.81,
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19.76, 19.18, 18.69, 12.98 (PMB3 C8’s ꢀCH3). MALDIꢀTOFꢀMS m/z calcd for C72H138N23O22
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(M+H)+ monoisotopic peak: 1749.039, found: 1749.012.
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PMB3ꢀtriazoleꢀC6ꢀTobramycin (1b). 1H NMR (500 MHz, Methanolꢀd4) δ 7.91 – 7.84 (m, 1H,
triazole), 7.34 – 7.19 (m, 5H, DꢀPhe6 aromatic), 5.43 (d, J = 2.7 Hz, 1H, Hꢀ1’), 5.11 (d, J = 3.3
Hz, 1H, Hꢀ1’’), 4.69 – 4.62 (m, 1H, Pra1α), 4.52 (dd, J = 8.7, 4.3 Hz, 1H, Dab5α), 4.46 (dd, J =
8.7, 4.7 Hz, 1H, Dab8α), 4.42 (dd, J = 9.4, 6.7 Hz, 1H, DꢀPhe6α), 4.40 – 4.14 (m, 10H, Thr2α +
Thr2,10β + Dab3,4,9α + C6’s ꢀCH2ꢀN=Nꢀ + Hꢀ4 + Hꢀ5’), 4.14 – 4.09 (m, 1H, Leu7α), 4.07 (d, J = 4.6
Hz, 1H, Thr10α), 3.94 – 3.88 (m, 1H, Hꢀ6), 3.85 – 3.64 (m, 8H, C6’s ꢀCH2ꢀOꢀ + Hꢀ5 + Hꢀ4’ + Hꢀ
2’’ + Hꢀ4’’ + Hꢀ6’’), 3.61 – 3.55 (m, 2H, Hꢀ2’ + Hꢀ5’’), 3.54 – 3.43 (m, 4H, Dab4γ1 + Hꢀ1 + Hꢀ3
+ Hꢀ3’’), 3.34 – 3.31 (m, 1H, Hꢀ6’eq), 3.25 – 3.19 (m, 2H, Pra1β1 + Hꢀ6’ax), 3.19 – 2.88 (m, 12H,
Pra1β2 + Dab4γ2 + DꢀPhe6β + Dab3,5,8,9γ), 2.53 – 2.43 (m, 2H, Dab3β1 + Hꢀ2eq), 2.29 – 1.85 (m,
16H, Dab3β2 + Dab4,5,8,9β + PMB3 C8’s ꢀCH2COꢀ + C6’s ꢀCH2CH2ꢀN=Nꢀ + Hꢀ2ax + Hꢀ3’), 1.66 –
1.60 (m, 2H, C6’s ꢀCH2CH2ꢀOꢀ), 1.59 – 1.47 (m, 3H, Leu7β1 + PMB3 C8’s ꢀCH2CH2COꢀ), 1.44 –
1.26 (m, 13H, Leu7β2 + PMB3 C8’s ꢀCH2ꢀ + C6’s ꢀCH2ꢀ), 1.24 – 1.03 (m, 6H, Thr2,10γ), 0.89 (t, J
= 7.2 Hz, 3H, PMB3 C8’s ꢀCH3), 0.87 – 0.59 (m, 7H, Leu7γ + Leu7δ). 13C NMR (126 MHz,
Methanolꢀd4) δ 175.64 (PMB3 C8’s carbonyl), 175.38, 174.53, 172.74, 172.64, 172.62, 172.48,
172.17, 171.91, 171.74, 171.47, 142.12 (triazole C without H), 135.51 (DꢀPhe6 C without H),
128.85, 128.34, 126.72, 123.53 (triazole C with H), 101.07 (Cꢀ1’’), 92.70 (Cꢀ1’), 82.24, 81.56
(Cꢀ6), 76.37, 75.62, 73.95, 72.59, 69.07, 66.33, 66.04, 65.06, 63.64, 59.92, 59.82, 56.41, 55.00,
53.43 (Pra1α), 52.18, 51.94, 51.67, 50.03 (Dab5α), 49.73, 48.41, 47.50, 39.30 (Leu7β), 38.54,
36.61, 36.45, 36.28, 36.01, 35.40, 31.43, 29.71, 29.47, 28.87, 28.83, 28.69, 28.60, 28.54, 27.69,
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calcd for C80H142N23O22 (M+H)+ monoisotopic peak: 1777.070, found: 1777.073.
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PMB3ꢀtriazoleꢀC8ꢀTobramycin (1c). 1H NMR (500 MHz, Methanolꢀd4) δ 7.88 – 7.81 (m, 1H,
triazole), 7.35 – 7.18 (m, 5H, DꢀPhe6 aromatic), 5.43 (d, J = 2.7 Hz, 1H, Hꢀ1’), 5.12 (d, J = 3.3
Hz, 1H, Hꢀ1’’), 4.70 – 4.62 (m, 1H, Pra1α), 4.52 (dd, J = 8.8, 4.4 Hz, 1H, Dab5α), 4.49 – 4.44 (m,
1H, Dab8α), 4.42 (dd, J = 9.7, 6.8 Hz, 1H, DꢀPhe6α), 4.39 – 4.14 (m, 10H, Thr2α + Thr2,10β
+
Dab3,4,9α + C8’s ꢀCH2ꢀN=Nꢀ + Hꢀ4 + Hꢀ5’), 4.13 – 4.08 (m, 1H, Leu7α), 4.06 (d, J = 4.6 Hz, 1H,
Thr10α), 3.91 (t, J = 9.3 Hz, 1H, Hꢀ6), 3.83 – 3.64 (m, 8H, C8’s ꢀCH2ꢀOꢀ + Hꢀ5 + Hꢀ4’ + Hꢀ2’’ +
Hꢀ4’’ + Hꢀ6’’), 3.61 – 3.56 (m, 2H, Hꢀ2’ + Hꢀ5’’), 3.56 – 3.43 (m, 4H, Dab4γ1 + Hꢀ1 + Hꢀ3 + Hꢀ
3’’), 3.29 – 3.25 (m, 1H, Hꢀ6’eq), 3.25 – 3.19 (m, 2H, Pra1β1 + Hꢀ6’ax), 3.19 – 2.85 (m, 12H,
Pra1β2 + Dab4γ2 + DꢀPhe6β + Dab3,5,8,9γ), 2.54 – 2.43 (m, 2H, Dab3β1 + Hꢀ2eq), 2.31 – 1.85 (m,
16H, Dab3β2 + Dab4,5,8,9β + PMB3 C8’s ꢀCH2COꢀ + C8’s ꢀCH2CH2ꢀN=Nꢀ + Hꢀ2ax + Hꢀ3’), 1.67 –
1.60 (m, 2H, C8’s ꢀCH2CH2ꢀOꢀ), 1.59 – 1.47 (m, 3H, Leu7β1 + PMB3 C8’s ꢀCH2CH2COꢀ), 1.41 –
1.25 (m, 17H, Leu7β2 + PMB3 C8’s ꢀCH2ꢀ + C8’s ꢀCH2ꢀ), 1.24 – 1.02 (m, 6H, Thr2,10γ), 0.90 (t, J
= 7.3 Hz, 3H, PMB3 C8’s ꢀCH3), 0.88 – 0.58 (m, 7H, Leu7γ + Leu7δ). 13C NMR (126 MHz,
Methanolꢀd4) δ 175.34 (PMB3 C8’s carbonyl), 173.91, 173.16, 172.64, 172.50, 172.20, 171.97,
171.81, 171.48, 171.28, 170.95, 142.97 (triazole C without H), 135.85 (DꢀPhe6 C without H),
128.86, 128.34, 126.71, 123.28 (triazole C with H), 101.10 (Cꢀ1’’), 92.67 (Cꢀ1’), 82.22, 81.57
(Cꢀ6), 76.41, 75.56, 73.93, 72.58, 69.07, 66.28, 65.26, 63.66, 59.94, 59.82, 56.41, 54.99, 53.53
(Pra1α), 52.01, 51.73, 51.45, 50.00 (Dab5α), 49.95, 49.76, 48.48, 47.68, 39.28 (Leu7β), 38.58,
36.67, 36.60, 36.52, 36.45, 36.40, 36.00, 35.38, 31.43, 29.88, 29.84, 29.70, 29.62, 29.39, 29.31,
29.26, 28.86, 28.83, 28.70, 28.60, 28.33, 27.70, 26.13, 26.06, 26.01, 25.54, 25.51, 25.48, 25.44,
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23.54, 22.25, 22.16, 20.04, 19.18, 18.74, 12.99 (PMB3 C8’s ꢀCH3). MALDIꢀTOFꢀMS m/z calcd
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PMB3ꢀtriazoleꢀC10ꢀTobramycin (1d). 1H NMR (500 MHz, Methanolꢀd4) δ 7.87 – 7.82 (m, 1H,
triazole), 7.32 – 7.19 (m, 5H, DꢀPhe6 aromatic), 5.41 (d, J = 2.6 Hz, 1H, Hꢀ1’), 5.12 (d, J = 3.4
Hz, 1H, Hꢀ1’’), 4.68 – 4.62 (m, 1H, Pra1α), 4.52 (dd, J = 8.8, 4.5 Hz, 1H, Dab5α), 4.48 – 4.39 (m,
2H, DꢀPhe6α + Dab8α), 4.39 – 4.13 (m, 10H, Thr2α + Thr2,10β + Dab3,4,9α + C10’s ꢀCH2ꢀN=Nꢀ + Hꢀ
4 + Hꢀ5’), 4.13 – 4.08 (m, 1H, Leu7α), 4.06 (d, J = 4.7 Hz, 1H, Thr10α), 3.91 (t, J = 9.2 Hz, 1H,
Hꢀ6), 3.84 – 3.63 (m, 8H, C10’s ꢀCH2ꢀOꢀ + Hꢀ5 + Hꢀ4’ + Hꢀ2’’ + Hꢀ4’’ + Hꢀ6’’), 3.61 – 3.56 (m,
2H, Hꢀ2’ + Hꢀ5’’), 3.55 – 3.43 (m, 4H, Dab4γ1 + Hꢀ1 + Hꢀ3 + Hꢀ3’’), 3.28 – 3.25 (m, 1H, Hꢀ6’eq),
3.25 – 3.18 (m, 2H, Pra1β1 + Hꢀ6’ax), 3.17 – 2.87 (m, 12H, Pra1β2 + Dab4γ2 + DꢀPhe6β +
Dab3,5,8,9γ), 2.54 – 2.43 (m, 2H, Dab3β1 + Hꢀ2eq), 2.29 – 1.86 (m, 16H, Dab3β2 + Dab4,5,8,9β
+
PMB3 C8’s ꢀCH2COꢀ + C10’s ꢀCH2CH2ꢀN=Nꢀ + Hꢀ2ax + Hꢀ3’), 1.67 – 1.59 (m, 2H, C10’s ꢀ
CH2CH2ꢀOꢀ), 1.59 – 1.48 (m, 3H, Leu7β1 + PMB3 C8’s ꢀCH2CH2COꢀ), 1.39 – 1.25 (m, 21H,
Leu7β2 + PMB3 C8’s ꢀCH2ꢀ + C10’s ꢀCH2ꢀ), 1.23 – 1.02 (m, 6H, Thr2,10γ), 0.89 (t, J = 7.2 Hz, 3H,
PMB3 C8’s ꢀCH3), 0.87 – 0.58 (m, 7H, Leu7γ + Leu7δ). 13C NMR (126 MHz, Methanolꢀd4) δ
175.31 (PMB3 C8’s carbonyl), 173.91, 173.20, 172.64, 172.49, 172.17, 171.98, 171.70, 171.45,
171.27, 170.93, 142.96 (triazole C without H), 135.86 (DꢀPhe6 C without H), 128.86, 128.33,
126.71, 123.27 (triazole C with H), 101.10 (Cꢀ1’’), 92.68 (Cꢀ1’), 82.19, 81.57 (Cꢀ6), 76.43,
75.62, 73.92, 72.65, 69.07, 66.26, 65.27, 63.64, 59.97, 59.81, 56.36, 54.98, 53.48 (Pra1α), 51.93,
51.75, 51.45, 50.02 (Dab5α), 49.91, 49.76, 48.48, 47.56, 39.29 (Leu7β), 38.55, 36.68, 36.59,
36.53, 36.45, 36.00, 35.38, 31.43, 30.31, 29.95, 29.78, 29.56, 29.38, 29.25, 28.86, 28.79, 28.70,
28.58, 28.48, 28.32, 27.70, 26.90, 26.21, 26.16, 25.66, 25.54, 23.56, 22.25, 22.15, 20.05, 19.17,
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18.74, 12.99 (PMB3 C8’s ꢀCH3). MALDIꢀTOFꢀMS m/z calcd for C84H150N23O22 (M+H)+
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monoisotopic peak: 1833.133, found: 1833.192.
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PMB3ꢀtriazoleꢀC12ꢀTobramycin (1e). 1H NMR (500 MHz, Methanolꢀd4) δ 7.87 – 7.83 (m, 1H,
triazole), 7.34 – 7.18 (m, 5H, DꢀPhe6 aromatic), 5.42 (d, J = 2.7 Hz, 1H, Hꢀ1’), 5.12 (d, J = 3.2
Hz, 1H, Hꢀ1’’), 4.70 – 4.62 (m, 1H, Pra1α), 4.52 (dd, J = 8.8, 4.3 Hz, 1H, Dab5α), 4.48 – 4.40 (m,
2H, DꢀPhe6α + Dab8α), 4.39 – 4.14 (m, 10H, Thr2α + Thr2,10β + Dab3,4,9α + C12’s ꢀCH2ꢀN=Nꢀ + Hꢀ
4 + Hꢀ5’) 4.14 – 4.08 (m, 1H, Leu7α), 4.06 (d, J = 4.6 Hz, 1H, Thr10α), 3.91 (t, J = 9.4 Hz, 1H, Hꢀ
6), 3.84 – 3.65 (m, 8H, C12’s ꢀCH2ꢀOꢀ + Hꢀ5 + Hꢀ4’ + Hꢀ2’’ + Hꢀ4’’ + Hꢀ6’’), 3.61 – 3.55 (m,
2H, Hꢀ2’ + Hꢀ5’’), 3.54 – 3.43 (m, 4H, Dab4γ1 + Hꢀ1 + Hꢀ3 + Hꢀ3’’), 3.29 – 3.26 (m, 1H, Hꢀ6’eq),
3.24 – 3.18 (m, 2H, Pra1β1 + Hꢀ6’ax), 3.17 – 2.87 (m, 12H, Pra1β2 + Dab4γ2 + DꢀPhe6β +
Dab3,5,8,9γ), 2.53 – 2.44 (m, 2H, Dab3β1 + Hꢀ2eq), 2.29 – 1.86 (m, 16H, Dab3β2 + Dab4,5,8,9β
+
PMB3 C8’s ꢀCH2COꢀ + C12’s ꢀCH2CH2ꢀN=Nꢀ + Hꢀ2ax + Hꢀ3’), 1.68 – 1.60 (m, 2H, C12’s ꢀ
CH2CH2ꢀOꢀ), 1.59 – 1.48 (m, 3H, Leu7β1 + PMB3 C8’s ꢀCH2CH2COꢀ), 1.38 – 1.24 (m, 25H,
Leu7β2 + PMB3 C8’s ꢀCH2ꢀ + C12’s ꢀCH2ꢀ), 1.24 – 1.04 (m, 6H, Thr2,10γ), 0.89 (t, J = 7.3 Hz, 3H,
PMB3 C8’s ꢀCH3), 0.88 – 0.59 (m, 7H, Leu7γ + Leu7δ). 13C NMR (126 MHz, Methanolꢀd4) δ
175.30 (PMB3 C8’s carbonyl), 174.84, 174.39, 173.92, 173.21, 172.64, 172.51, 172.16, 172.00,
171.46, 170.94, 143.08 (triazole C without H), 135.86 (DꢀPhe6 C without H), 128.86, 128.33,
126.71, 120.82 (triazole C with H), 101.11 (Cꢀ1’’), 92.66 (Cꢀ1’), 82.33, 82.19, 81.58 (Cꢀ6),
80.61, 76.40, 75.59, 73.92, 72.66, 69.07, 66.27, 65.80, 65.26, 63.65, 63.12, 59.97, 59.81, 56.00,
54.98, 53.46 (Pra1α), 52.66, 52.01, 51.74, 51.47, 50.03 (Dab5α), 49.77, 48.48, 47.51, 39.31
(Leu7β), 38.55, 36.68, 36.66, 36.62, 36.53, 36.44, 36.00, 35.39, 31.44, 29.95, 29.79, 29.62, 29.49,
29.43, 29.35, 29.26, 29.16, 28.91, 28.87, 28.81, 28.77, 28.71, 28.58, 28.32, 27.69, 26.21, 26.16,
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25.70, 25.55, 23.56, 22.26, 22.16, 20.05, 19.17, 18.75, 13.00 (PMB3 C8’s ꢀCH3). MALDIꢀTOFꢀ
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MS m/z calcd for C86H154N23O22 (M+H)+ monoisotopic peak: 1861.164, found: 1861.144.
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Microbiology. Bacterial isolates were either obtained from the American Type Culture
Collection (ATCC), Canadian National Intensive Care Unit (CANꢀICU) surveillance study40 or
the Canadian Ward Surveillance (CANWARD) study41. Strains from CANꢀICU and
CANWARD are clinical isolates recovered from patients suffering from a presumed infectious
disease admitted in a participating medical center across Canada.
Antimicrobial susceptibility assay. The in vitro antibacterial activity was assessed by microbroth
dilution susceptibility testing following the CLSI guidelines43. Overnight grown bacterial
cultures were diluted in saline to achieve a 0.5 McFarland turbidity, followed by 1:50 dilution in
MuellerꢀHinton broth (MHB) for inoculation to a final concentration of approximately 5 × 105
colony forming units/mL. The assay was performed on a 96ꢀwell plates to which the tested
agents were 2ꢀfold serially diluted in MHB and incubated with equal volumes of inoculum at 37
°C for 18 hours. The MIC was determined as the lowest concentration to inhibit visible bacterial
growth in form of turbidity, which was confirmed using EMax Plus microplate reader (Molecular
Devices, USA) at a wavelength of 590 nm. The broth with or without bacterial cells was used as
positive or negative control, respectively.
Checkerboard assay. The assay was performed in a 96ꢀwell plate as previously described16,17
.
Briefly, the antibiotic of interest was 2ꢀfold serially diluted along the xꢀaxis, while the adjuvant
was 2ꢀfold serially diluted along the yꢀaxis to create a matrix in which each well consist of a
combination of both at different concentrations. Bacterial cultures grown overnight were diluted
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in saline to 0.5 McFarland turbidity, followed by 1:50 dilution in MHB and inoculation on each
well to a final concentration of approximately 5 × 105 colony forming units/mL. Wells
containing only MHB with or without bacterial cells were used as positive or negative control,
respectively. The plate were incubated at 37 °C for 18 hours and examined for visible turbidity,
to which was confirmed using EMax Plus microplate reader (Molecular Devices, USA) at a
wavelength of 590 nm. The fractional inhibitory concentration (FIC) of the antibiotic was
calculated via dividing the MIC of antibiotic in the presence of adjuvant by the MIC of antibiotic
alone. Similarly, the FIC of adjuvant was calculated via dividing the MIC of adjuvant in the
presence of antibiotic by the MIC of adjuvant alone. FIC index was obtained by the summation
of both FIC values. FIC index was interpreted as synergistic, additive or antagonistic for values
of ≤ 0.5, 0.5 < x <4 or ≥4, respectively.47
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Hemolytic assay. The ability of the agents to lyse eukaryotic red blood cells were quantified by
the amount of hemoglobin released upon incubation with pig erythrocytes, following published
protocol17. Briefly, fresh pig blood (generously provided by Dr. Charles M. Nyachoti from
University of Manitoba) drawn from pig antecubital vein was centrifuged at 1000 × g for 5
minutes at 4 °C, washed with PBS three times and reꢀsuspended in the same buffer,
consecutively. Agents were 2ꢀfold serially diluted in PBS on 96ꢀwell plate and mixed with equal
volumes of erythrocyte solution. Post 1ꢀhour incubation at 37 °C, intact cells were pelleted by
centrifugation at 1000 × g for 5 minutes at 4 °C. The resulting supernatant was then transferred
to a new 96ꢀwell plate. The hemoglobin released was measured via EMax Plus microplate reader
(Molecular Devices, USA) at 570 nm wavelength. Erythrocytes in PBS with or without 0.1%
Triton Xꢀ100 was used as negative or positive control, respectively.
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ASSOCIATED CONTENT
Supporting information.
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Additional synthesis, characterization, NMR & HPLC spectra and microbiological studies are
provided in the supporting information. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
*Corresponding Author
Eꢀmail, Frank.Schweizer@umanitoba.ca. Phone: +1 (204) 474ꢀ7012. Fax: +1 (204) 474ꢀ7608.
Author contributions
F.S and G.G.Z supervised the studies. R.D developed the synthetic route. R.D conducted the
synthetic work. R.D and X.Y characterized the hybrids. R.D performed the microbiological
assays. R.D and Y.L performed the hemolytic assay. R.D and F.S wrote the manuscript and
supporting information.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The work was supported by NSERCꢀDG (261311ꢀ2013) and MHRC
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ABBREVIATIONS USED
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OM, outer membrane; MDR, multidrugꢀresistant; XDR, extensively drugꢀresistant; C4, aliphatic
hydrocarbon with four carbons; C6, aliphatic hydrocarbon with six carbons; C8, aliphatic
hydrocarbon with eight carbons; C10, aliphatic hydrocarbon with ten carbons; C12, aliphatic
hydrocarbon with twelve carbons; TOB, tobramycin; PMB3, polymyxin B3; Dab, Lꢀ2,4ꢀ
diaminobutyric acid; Pra, Lꢀpropargylglycine; TBAHS, tetrabutylammonium hydrogen sulfate;
CuI•P(OEt)3, copper iodide triethylphosphite; DIPEA, diisopropylethylamine; TBDMS, tertꢀ
butyldimethylsilyl; CANꢀICU, Canadian National Intensive Care Unit surveillance study;
CANWARD, Canadian Ward surveillance study; MIC, Minimum Inhibitory Concentration;
MHC, concentration to induce 1% hemolysis; CLSI, Clinical and Laboratory Standards Institute;
EUCAST, European Committee on Antimicrobial Susceptibility Testing; IMI, imipenem; MER,
meropenem; COL, colistin; TBTU, N,N,N′,N′ꢀtetramethylꢀOꢀ(benzotriazolꢀ1ꢀyl)uronium
tetrafluoroborate; PyBOP, benzotriazoleꢀ1ꢀylꢀoxyꢀtrisꢀpyrrolidinoꢀphosphonium
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hexafluorophosphate; HOBt, hydroxybenzotriazole
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