Special Issue: Emerging Peptide Science in Australia Guest Editors: Prof. David J. Craik (University of Queensland) and Prof. Richard J. Payne (University of Sydney) EDITORIAL Emerging peptide science in Australia David J. Craik and Richard J. Payne, Peptide Science 2018, doi: 10.1002/pep2.24080 REVIEWS Decarboxylative couplings as versatile tools for late-stage peptide modifications Lara R. Malins, Peptide Science 2018, doi: 10.1002/pep2.24049 Bridged bicyclic peptides: Structure and function Varsha J. Thombare and Craig A. Hutton, Peptide Science 2018, doi: 10.1002/pep2.24057 Molecular simulations of venom peptide-membrane interactions: Progress and challenges Evelyne Deplazes, Peptide Science 2018, doi: 10.1002/pep2.24060 Interaction of Cationic Antimicrobial Peptides from Australian Frogs with Lipid Membranes Shiying Zhu, Marc-Antoine Sani and Frances Separovic, Peptide Science 2018, doi: 10.1002/pep2.24061 Folding of granulin domains Mohadeseh Dastpeyman, Michael J. Smout, David Wilson, Alex Loukas and Norelle L. Daly, Peptide Science 2018, doi: 10.1002/pep2.24062 Disordered epitopes as peptide vaccines Christopher A. MacRaild, Jeffrey Seow, Sreedam C. Das and Raymond S. Norton, Peptide Science 2018, doi: 10.1002/pep2.24067 ARTICLES A comparison of pseudoproline substitution effects on cyclisation yield in the total syntheses of segetalins B and G Michelle S. Y. Wong and Katrina A. Jolliffe, Peptide Science 2018, doi: 10.1002/pep2.24042 A silicon-labelled amino acid suitable for late-stage fluorination and unexpected oxidative cleavage reactions in the preparation of a key intermediate in the Strecker synthesis Kymberley R. Scroggie, Lisa J. Alcock, Maria J. Matos, Gonçalo J. L. Bernardes, Michael V. Perkins and Justin M. Chalker, Peptide Science 2018, doi: 10.1002/pep2.24069 The binding of boronated peptides to low affinity mammalian saccharides Wioleta Kowalczyk, Julie Sanchez, Phillipe Kraaz, Oliver E. Hutt, David N. Haylock and Peter J. Duggan, Peptide Science 2018, doi: 10.1002/bip.23101 Covalent conjugation of cationic antimicrobial peptides with a β-lactam antibiotic core Wenyi Li, Neil M. O'Brien-Simpson, James A. Holden, Laszlo Otvos, Eric C. Reynolds, Frances Separovic, Mohammed Akhter Hossain and John D. Wade, Peptide Science 2018, doi: 10.1002/pep2.24059
Special Issue: Emerging Peptide Science in Australia Guest Editors: Prof. David J. Craik (University of Queensland) and Prof. Richard J. Payne (University of Sydney) Preparation and cellular uptake of bicyclic-peptide cargo clicked to cell penetrating peptides Ketav Kulkarni, Gabrielle M. Watson, Jianrong Sang and Jacqueline A. Wilce, Peptide Science 2018, doi: 10.1002/pep2.24037 Converting polar cyclic peptides into membrane permeable molecules using N-methylation Leo L. H. Lee, Laura K. Buckton and Shelli R. McAlpine, Peptide Science 2018, doi: 10.1002/pep2.24063 Amyloid aggregation and membrane activity of the antimicrobial peptide uperin 3.5 Lisandra L. Martin, Clemens Kubeil, Stefania Piantavigna, Tarun Tikkoo, Nicholas P. Gray, Torsten John, Antonio N. Calabrese, Yanqin Liu, Yuning Hong, Mohammed A. Hossain, Nitin Patil, Bernd Abel, Ralf Hoffmann, John H. Bowie and John A. Carver, Peptide Science 2018, doi: 10.1002/pep2.24052 Oat of this world: Defining peptide markers for detection of oats in processed food Charlotte Dawson, Omar Mendoza-Porras, Keren Byrne, Thomas Hooper, Crispin Howitt and Michelle Colgrave, Peptide Science 2018, doi: 10.1002/pep2.24045
Received: 21 January 2018
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Revised: 24 January 2018
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Accepted: 13 February 2018
DOI: 10.1002/pep2.24059
ORIGINAL ARTICLE
Covalent conjugation of cationic antimicrobial peptides with a b-lactam antibiotic core Wenyi Li1,2,3
|
Neil M. O’Brien-Simpson4,5 | James A. Holden4,5 |
Laszlo Otvos6 | Eric C. Reynolds4,5 | Frances Separovic1,4
|
Mohammed Akhter Hossain1,2 | John D. Wade1,2 1
School of Chemistry, University of Melbourne, VIC 3010, Australia
2
The Florey Institute of Neuroscience and Mental Health, University of Melbourne, VIC 3010, Australia
3
Leibniz-Forschungs Institut f€ ur Molekulare Pharmakologie, Berlin 13125, Germany
4
Bio21 Institute, University of Melbourne, VIC 3010, Australia
5
Oral Health CRC, Melbourne Dental School, University of Melbourne, VIC 3010, Australia
6
OLPE LLC., Audubon, Philadelphia, Pennsylvania 19403
Correspondence Wenyi Li, School of Chemistry, University of Melbourne, VIC 3010, Australia. Email:
[email protected] And Neil M. O’Brien-Simpson, Bio21 Institute, Melbourne Dental School, University of Melbourne, VIC 3010, Australia. Email:
[email protected] And John D. Wade, The Florey Institute of Neuroscience and Mental Health, University of Melbourne, VIC 3010, Australia. Email:
[email protected] Funding information ARC Discovery Project Grants, Grant Numbers: DP150103522, DP140102127; NHMRC Project Grants, Grant Numbers: APP1029878, APP1008106; NHMRC (Australia) Principal Research Fellow, Grant Number: APP1117483; Victorian Government’s Operational Infrastructure Support Program
Abstract To combat the serious issue of increasing global antibiotic resistance, new antimicrobial therapies are urgently required. As one alternative, previously used antibiotics are being investigated for use in combination with more modern antibiotics including antimicrobial peptides. Towards this goal, 7aminocephalosporanic acid, the precursor to the conventional b-lactam antibiotic, cephalosporin, and the related compound, 7-aminodesacetoxycephalosporanic acid, were each chemically modified to enable their use in solid phase peptide synthesis for covalent conjugation via their 7-amino group and a glycolic linker to the N-terminus of a series of cationic antimicrobial peptides, MSI-78, CA(1-7)M(29)NH2 and des-Chex1-Arg20. Chemically functionalized Ca-Fm-protected 7-aminocephalosporanic acid and 7-aminodesacetoxycephalosporanic acid building blocks were separately prepared and attached by their 7-amino group to the N-terminus of each peptide on the solid phase via a glutaric anhydride linker. The resulting conjugated AMPs were assessed for antibacterial activity against a panel of six Gram-negative bacteria, including clinically isolated multi-drug resistant (MDR) pathogens. Only the conjugated MSI-78 analogues displayed significant activity against Acinetobacter baumannii and MDR A. baumannii 156 and enhanced activity against Klebsiella pneumoniae. Further work is required to optimize the conjugation of AMPs to 7-cephalosporanic acid and/or 7aminodesacetoxycephalosporanic acid to universally induce an enhanced effect on killing of drug sensitive and MDR bacterial strains that are common causes of hospital-acquired infections.
KEYWORDS
7-aminocephalosporanic acid, antimicrobial peptides, b-lactam antibiotic, cephalosporin, peptideantibiotic conjugates, solid phase peptide synthesis
1 | INTRODUCTION
Disease Control (CDC) reported that more than 2 million people become infected leading to 23 000 deaths due to these infections every year.
Antibiotic resistance in bacteria causing healthcare-associated infections
Moreover, the CDC has identified the six most deadly antibiotic-resistant
is increasing rapidly according to a recent global antimicrobial resistance
bacteria, including carbapenem-resistant Enterobacteriaceae, methicillin-
[1]
report from the World Health Organization (WHO).
Peptide Science. 2018;110:e24059. https://doi.org/10.1002/pep2.24059
The US Centre for
resistant Staphylococcus aureus, ESBL-producing Enterobacteriaceae,
wileyonlinelibrary.com/peptidesci
C 2018 Wiley Periodicals, Inc. V
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vancomycin-resistant Enterococci, multidrug resistant (MDR) Pseudomo-
C-terminal carboxylates to the 7-amino group of 7-ACA was sufficient to
nas, and MDR Acinetobacter. It is a matter of urgency that new classes of
result in significant antibacterial activity when the amino acids or dipepti-
antimicrobial agents to target these resistant bacteria are identified.
des alone had none.[28] This important observation, together with the
Antimicrobial peptides (AMPs), with their broad-spectrum activities and
fact that the authors used solution phase chemistry to attach the amino
distinct modes of action including host defense against pathogens, have
acids or dipeptides to the 7-amino group of 7-ACA, led us to undertake
been considered as promising potential alternatives to conventional
to examine the possibility of functionalizing 7-ACA and a related cephalo-
antibiotics.[2]
sporins precursor compound, 7-aminodesacetoxycephalosporanic acid (7-
Several earlier studies have suggested that combination of existing
ADCA) (Figure 1), to enable their use in solid phase peptide synthesis
conventional antibiotics or combining them with nonantibiotic drugs
(SPPS) for conjugating onto the N-terminus, rather than the C-terminus,
could enhance their antibacterial activity and/or modulate host defense
of selected cationic AMPs as a means of facilitating the production of
through increased effects on host innate immunity.[3–5] Recently,
novel potential antibacterial compounds. We report herein the develop-
hydrophobic cationic nanoparticles used to block the efflux pump were
ment and use of novel functionalized 7-ACA- and 7-ADCA-linked AMPs
shown to dramatically improve the activity of fluoroquinolone antibiot-
and the investigation of their activity on MDR bacteria.
ics for treatment of MDR bacterial strains.[6] Further, the organization of different antibacterial peptides or protein-peptides into functional
2 | MATERIALS AND METHODS
complexes have also been shown to expand their antimicrobial activity.[7] For example, defensins synergistically improved their binding
2.1 | Materials
affinity in the presence of granulocytic antibacterial proteins.[8] As well, the formation of a specific complex of the AMP magainin II and its syn-
The 9-fluorenylmethoxylcarbonyl (Fmoc)-L-amino acids, and Rink
thetic derivative PGLa showed synergism and together acted against
Amide resin were purchased from GL Biochem (Shanghai, China). N,N-
Several reviews
diisopropylethylamine (DIPEA), dimethylformamide (DMF), and tri-
have summarized the synergism between AMPs and small-molecule
fluoroacetic acid (TFA) were obtained from Auspep (Melbourne, Aus-
antibiotics or suggested the “revived” activity of conventional antibiot-
tralia).
[9–11]
Escherichia coli lipid extract membrane bilayers.
Piperidine,
triisopropylsilane
(TIPS),
anisole,
acetonitrile
For instance,
(CH3CN), ethyl (hydroxyimino)cyanoacetate (Oxyma), N,N’-diisopropyl-
MSI-78, a designed magainin family analogue, showed synergism in
carbodiimide, diethylether, ethyl acetate (EtOAc), dichloromethane
combination with b-lactam antibiotics against bacteria responsible for
(DCM),
bloodstream infections in vitro.[12] CA(1–7)M(2–9)NH2, a cecropin
(Fm-OH), triethylamine (Et3N), N,N0 -diisopropylcarbodiimide (DIC), and
A-melittin hybrid AMP, also improved its activity in combination with
1-hydroxybenzotriazole hydrate (HoBt hydrate) were all obtained from
[3,7]
ics co-administering with nonantibiotics drugs.
[13]
b-lactam antimicrobial agents.
Recently, the combination of M33,
a tetra-branched AMP, and levofloxacin, a fluoroquinolone antibiotic,
di-tert-butyl
dicarbonate
((Boc)2O),
9-Fluorenemethanol
Sigma (Sydney, Australia). The 7-ACA and 7-ADCA were both obtained from TCI Ltd (Australia).
displayed synergy-mediated activity against Gram-negative bacteria or restoration of levofloxacin action.[14] We have also previously shown the synergistic combination of proline-rich AMP (PrAMP) to recover the activity of conventional antibiotics.[15] Our laboratories have also recently further developed this PrAMP and found a class of multimer-
2.2 | The preparation of the cephalosporin derivatives 2.2.1 | 7-(tert-butoxycarbonyl)-aminocephalosporanic acid (2a) preparation
ized PrAMP with C-terminal functionalization that showed a broader
The 7-ACA (1a, 1.5 g, 5.5 mM) was dissolved in 20 mL dioxane/dH2O
spectrum or activity and increased potency as potential antibiotic
(1:1 v/v) with a round bottom flask. The solution became clear after Et3N (1.1 equiv, 841 mL) added. Then (Boc)2O (1.1 equiv, 1.3 g) was
[16–20]
alternatives.
The penicillin and related beta-lactams comprise one of the most
added to mixture and stirred overnight at room temperature. After-
In general, these
wards, the mixture was acidified with 1 N HCl to pH 2 and extracted
b-lactam antibiotics can inhibit bacterial cell wall synthesis by the inhi-
with EtOAc three times. The combined organic parts were washed
[21,22]
widely used and oldest antibiotic therapies.
bition of penicillin-binding proteins and derange the activity of polymer
with saturated NaHCO3 to obtain the aqueous layer. Such aqueous
peptidoglycan biogenesis machinery,[23] which has been deactivated by
parts were acidified with 1 N HCl to pH 2 and extracted with EtOAc
bacterial beta-lactamase to open the beta-lactam ring. Previously, one
for three times. The organic phase was dried with sodium sulfate and
class of the beta-lactam antibiotics, cephalosporins/cefazolin, which
concentrated. The mixture was purified by flash chromatography
possesses the core structure, 7-aminocephalosporanic acid (7-ACA),
(MeOH:DCM 10:90, with 1% acetic acid) to afford the product 7-(tert-
has been covalently conjugated with several small molecules to
butoxycarbonyl)aminocephalosporanic acid 2a (1.7 g, yield 84%). 1H
enhance and/or recover their biological activity, including oxazolidi-
and
[24]
13
C NMR spectra were obtained at room temperature using an
quinolones (at the C3’ position),
Agilent 400-MR (400 MHz) spectrometer with the deuterated solvent
metallopolymers (via ionic complexation),[25] siderophore (C7 position
as a reference and a sample concentration of about 10 mg mL21. 1H
substituent),[26] and host defense peptide (via a cleavable carbamate-
NMR(400 MHz, DMSO-d6) d 8.01 (d, J 5 8.8 Hz), 5.48 (dd, J 5 8.2, 4.7
none (at the C3’ position of 7-ACA),
[27]
1,4-triazole linker at the C3’ position).
Interestingly, a report
showed that a number of amino acids or dipeptides attached via their
Hz), 5.08-4.94 (m), 4.69 (d, J 5 12.8 Hz), 3.64-3.54 (m), 3.47 (d, J 5 18.1 Hz), 2.03 (d, J 5 1.5 Hz), 1.40 ppm.
[13]
C NMR (DMSO-d6): d 171.31,
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General conditions for the synthesis of Fm-protected cephalosporanic acid precursors. a 5 7-ACA; b 5 7-ADCA. (i) (Boc)2O (1.1 equiv), Et3N (1.1 equiv), DCM; (ii) Fm-OH (1.05 equiv), DCC (1 equiv), HOBt (1 equiv) (or EDC (1 equiv), DMAP (0.1 equiv)), and (iii) 20% TFA in DCM
FIGURE 1
165.91, 164.01, 156.17, 80.27, 67.45, 63.85, 61.80, 61.78, 60.86,
stirring for 2 h at room temperature. Then, the reaction solvents were
58.92, 29.13, 26.67, 21.68, 15.19 ppmIR 3345.7, 2979.5, 2936.2,
evaporated under nitrogen flow. Afterwards, the residual parts were dis-
2595.5, 1783.2, 1715.8, 1523.6, 1456.6, 1369.5, 1330.0, 1249.9,
solved in diethylether and coevaporated under vacuum to remove the
1161.1, 1117.3, 1053.0, 1028.1, 956.2, 605.9 cm21. HRMS (ESI):
rest TFA to afford the product (9H-fluoren-9-yl)methyl (6R,7R)-3-(ace-
[C15H20N2O7S 1NH4], Cal: 390.1329, Found: [C15H20N2O7S 1NH4]:
toxymethyl)-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
390.1328.
carboxylate TFA salts 4a (1.4 g, yield 96%) without further purification. 1H NMR (400 MHz, DMSO-d6) d 8.91, 7.89 (dd, J 5 7.4, 2.8
2.2.2 | (9H-fluoren-9-yl)methyl (6R,7R)-3-(acetoxymethyl)7-((tert-butoxycarbonyl)amino)-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate (3a) preparation
Hz), 7.69-7.64 (m), 7.45-7.40 (m), 7.36-7.31 (m), 6.78 (dd, J 5 84.8, 1.1 Hz), 5.09 (dd, J 5 38.7, 1.4 Hz), 4.82-4.77 (m), 4.75 (d, J 5 3.9 Hz), 4.69-4.65 (m), 4.45 (d, J 5 12.8 Hz), 4.37-4.32 (m), 1.91 ppm. 13C NMR
The 7-(tert-butoxycarbonyl)Aminocephalosporanic acid (2a, 1.7 g, 4.5
(101 MHz) d 169.76, 166.42, 159.60, 158.67, 143.27, 140.84, 127.76,
mM) was dissolved in 20 mL DCM, followed by DCC (1 equiv, 0.93 g)/
127.12, 125.82, 124.86, 121.65, 120.13, 118.77, 116.68, 115.35,
HOBt (1 equiv, 0.61 g) and stirred for 5 min. Then FmOH (0.95 equiv,
68.99, 66.64, 64.83, 62.40, 58.78, 49.29, 46.27, 20.40 ppm. IR 3440.2,
0.84 g) was added to the mixture and reacted overnight. Afterwards,
3069.6, 2037.3, 1784.6, 1744.5, 1673.1, 1451.4, 1406.1, 1382.8,
the mixture was filtered and extracted by DCM for three times. The
1320.4, 1267.4, 1178.8, 1029.4, 742.9, 723.7 cm21. HRMS (ESI):
combined organic phase was washed with saturated NaHCO3, 1 N HCl
[C24H22N2O5S 1H]: Cal: 449.1249, Found: [C24H22N2O5S 1H]:
and water. Then it was dried with sodium sulfate, concentrated under
449.3874
vacuum and purified by flash chromatography (petroleum ether–ethyl acetate, 3:1) to afford the product (9H-fluoren-9-yl)methyl (6R,7R)3-(acetoxymethyl)-7-((tert-butoxycarbonyl)amino)-8-oxo-5-thia-1-aza1
2.2.4 | 7-(tert-butoxycarbonyl)aminodesacetoxycephalosporanic acid (2b) preparation
H
The 7-ADCA (1b, 1.5 g, 7 mM) was dissolved in 30 ml Dioxane/dH2O
NMR (400 MHz, DMSO-d6) d 7.97 (d, J 5 7.6 Hz), 7.92-7.86 (m),
(1:1 v/v) with a round bottom flask. The solution became clear after
bicyclo[4.2.0]oct-2-ene-2-carboxylate 3a (1.4 g, yield 56.7%).
7.70-7.63 (m), 7.47-7.38 (m), 7.38-7.29 (m), 6.67, 5.00, 4.88, 4.71-
Et3N (1.1 equiv, 1.1 mL) added. Then (Boc)2O (1.1 equiv, 1.68 g) was
4.61 (m), 4.55, 4.46 (d, J 5 12.5 Hz), 4.40-4.32 (m), 1.90 (d, J 5 2.2
added to mixture and stirred overnight at room temperature. After-
C NMR d 170.26, 143.85, 141.32, 141.26, 128.21,
wards, the mixture was acidified with 1 N HCl to pH 2 and extracted
127.57, 125.32, 120.58, 79.58, 66.88, 66.86, 65.50, 53.21, 50.03,
with EtOAc for three times. The combined organic parts were washed
Hz), 1.40 ppm.
13
46.76, 46.75, 28.48, 20.92 ppm.IR 3419.1, 2977.3, 1781.6, 1742.6,
with saturated NaHCO3 to obtain the aqueous layer. Such aqueous
1717.3, 1627.9, 1518.7, 1478.5, 1451.3, 1369.3, 1323.9, 1226.2,
parts were acidified with 1 N HCl to pH 2 and extracted with EtOAc
1162.1, 1036.3, 742.4 cm21. HRMS (ESI): [C29H30N2O7S 1NH4]: Cal:
for three times. The organic phase was dried with sodium sulfate and
568.2112, Found: [C29H30N2O7S 1NH4]: 568.2110.
concentrated. The mixture was purified by flash chromatography (MeOH:DCM 10:90 v/v, with 1% acetic acid) to afford the product 7-
2.2.3 | (9H-fluoren-9-yl)methyl (6R,7R)-3-(acetoxymethyl)7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate TFA salts (4a) preparation
yield 97.6%). 1H and
The (9H-fluoren-9-yl)methyl (6R,7R)-3-(acetoxymethyl)-7-((tert-butoxy-
1331.9, 1251.0, 1161.3, 1117.1, 1053.7, 711.7 cm21. HRMS (ESI):
carbonyl)amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
[C13H18N2O5S 1NH4], Cal: 332.1275, Found [C13H18N2O5S 1NH4]:
(3a, 1.4 g, 2.7 mM) was dissolved in 20 mL DCM, followed by 5 mL TFA
332.1273.
(tert-butoxycarbonyl)-aminodesacetoxycephalosporanic acid 2b (2.1 g, 13
C NMR spectra are given in the SI. IR 3418.9,
2979.1, 2933.1, 1775.3, 1711.2, 1636.0, 1522.2, 1455.8, 1368.8,
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2.2.5 | (9H-fluoren-9-yl)methyl (6R,7R)-7-((tertbutoxycarbonyl)amino)-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate (3b) preparation
reversed-phase high performance liquid chromatography (RP-HPLC) in
The 7-(tert-butoxycarbonyl)-aminodesacetoxycephalosporanic acid (2b,
time-of-flight mass spectrometry (MALDI-TOF MS) (Supporting Infor-
2.1 g, 6.8 mM) was dissolved in 20 mL DCM, followed by EDC (1 equiv,
mation Figure 1).
water and acetonitrile with 0.1% TFA. The final products were characterized by RP-HPLC and matrix-assisted laser desorption/ionization
1.3 g)/DMAP (0.1 equiv, 83 mg) at 08C for 5 min. Fm-OH (1.05 equiv, 1.4 g) was then added to the mixture and reacted overnight. Afterwards, the mixture was extracted with DCM for 3 times and the com-
2.4 | Circular dichroism spectroscopy
bined organic phase were washed with 1N HCl and saturated NaCl.
CD spectra were acquired between 190 and 280 nm using a
Then it was dried with sodium sulfate, concentrated under vacuum and
Chirascan-plus instrument (Applied Photophysics, Leatherhead, UK).
purified by flash chromatography (petroleum ether–ethyl acetate, 4:1,
The cell path length was 0.1 cm and all measurements were recorded
v/v) to afford the product (9H-fluoren-9-yl)methyl (6R,7R)-7-((tert-
at 258C. Peptides were prepared in phosphate buffer (pH 7.4) and ana-
butoxycarbonyl)amino)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
lyzed as previously described.[19] The spectra in Figure 3 are the aver-
2-ene-2-carboxylate 3b (2.1 g, yield 61.4%). 1H and
C NMR spectra
age of 3 scans with the subtraction of the buffer solution spectrum.
are given in the SI. IR 3397.2, 2977.2, 1780.4, 1718.4, 1509.1, 1450.8,
Spectra were zeroed at 280 nm and normalized to give units of mean
1391.8, 1326.3, 1248.1, 1161.3, 1108.8, 1151.1, 759.3, 741.4 cm21.
residue ellipticity (MRE) according to Equation 1:
HRMS
(ESI):
[C27H28N2O5S
1NH4],
Cal:
13
510.2057,
Found ½u5
[C27H28N2O5S 1NH4]: 510.2026.
2.2.6 | (9H-fluoren-9-yl)methyl(6R,7R)-7-amino-3-methyl-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate TFA salts (4b) preparation
u ðc3l3NrÞ
(1)
where u is the recorded ellipticity in millidegrees, c is the peptide concentration in dmol/litre, l is the cell path-length in cm, and Nr is the number of residues per peptide.
The (9H-fluoren-9-yl)methyl (6R,7R)-7-((tert-butoxycarbonyl)amino)-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
(3b,
1.9g, 4 mM) was dissolved in 20 ml DCM, followed by 5 mL TFA stir-
2.5 | Antibacterial assay
ring for 2 h at room temperature. Then, the reaction solvents were
Antibacterial assays were undertaken to determine the minimal inhib-
evaporated under nitrogen flow. Afterwards, the residual parts were
itory concentrations (MIC) as described previously.[20] A panel of
dissolved in diethylether and co-evaporated under vacuum to remove
Gram-negative
nosocomial
bacteria,[30]
Klebsiella
pneumoniae
the rest TFA to afford the product (9H-fluoren-9-yl)methyl(6R,7R)-7-
ATCC13883, Acinetobacter baumannii ATCC 19606, Pseudomonas
amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxy-
aeruginosa ATCC 47085, as well as clinical isolates of strains MDR K.
late TFA salts 4a (2.0 g, yield 99%) was used without further purifica-
pneumoniae (FADDI-KP628), A. baumannii (FADDI-AB156), P. aerugi-
tion. 1H and
C NMR spectra are given in the SI. IR 3441.4, 2952.7,
nosa (FADDI-PA067) were chosen to test the antibacterial activities
1782.4, 1674.4, 1478.7, 1450.6, 1399.1, 1319.0, 1203.0, 1050.1,
of the cephalosporanic acid core and the non-conjugated and conju-
13
759.4, 741.9 cm21. HRMS (ESI): [C22H20N2O3S 1H], Cal: 393.1267, Found [C22H20N2O3S 1H]: 393.1319.
gated cationic AMPs with b-lactam cephalosporin cores, with 2.5 3 105 cells mL21 in MHB at 378C immediately prior to the determination of MIC.
2.3 | Peptide preparation The peptides were synthesized by Fmoc/tBu solid-phase methods[29] on a CEM Liberty microwave-assisted synthesizer using RAM-resin. Standard Fmoc-chemistry was used throughout with a 4-fold molar
2.6 | Fractional inhibitory concentration index (FICI) calculation
excess of the Fmoc-protected amino acids in the presence of four-fold
The synergistic activity of conjugated cationic AMPs with b-lactam
HCTU and 8-fold DIPEA. The final amino acids were deprotected with
cephalosporin cores was then evaluated using FICI. This was calculated
20% piperidine followed an addition with glutamic anhydride in pres-
using the following formula, which was adapted from previous syner-
ence of DIPEA. Then, Fm-protected cephalosporin core was coupled
gistic studies[5,6,31,32]:
on the solid phase with peptide activated by DIC/Oxyma (2 equiv to resin) for 6 h at room temperature. The peptides were cleaved from the solid support resin with TFA in the presence of anisole, TIPS as the scavenger (ratio 95:2:3) for 2 h
FICI5FICAMPs 1FICb2lactam cephalosporin cores 5 MICconjugated AMPs =MICAMPs 1 MICconjugated AMPs =MICb2lactam cephalosporin cores
(2)
at room temperature. Followed by filtration to remove the resin, the fil-
Based on a previously reported investigation,[33] an FICI 0.5 rep-
trate was concentrated under a stream of nitrogen, and the peptide
resents synergistic activity; between 0.5 and 1 is partial synergistic
products were precipitated in ice-cold diethyl ether and washed for
activity; between 1 and 4 represents an additive effect or indifferent;
three times. The peptides were then purified with moderate yield by
and an FICI > 4 represents antagonistic activity.
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General conditions for the synthesis of cationic antimicrobial peptides containing the b-lactam antibiotic precursors, 7-ACA or 7-ADCA. a) glutaric anhydride (3 equiv), DIPEA(3 equiv), DMF, 1 h; b) DIC (2 equiv to resin), Oxyma (2 equiv to resin), DMF, 20 min; c) 4, DMF, 6 h; d) TFA, anisole, TIPS (ratio v/v 95: 3: 2), 2 h; and e) 20% piperidine in DMF, 25 min
FIGURE 2
2.7 | Cell proliferation/cytotoxicity test The cell proliferation of HEK-293 (ATCC® CRL-1573TM) and H-4-II-E (ATCC® CRL-1548TM) cells were tested with cephalosporanic acid core and the nonconjugated and conjugated cationic AMPs with b-lactam cephalosporin cores as described before.[20] Briefly, HEK-293 and H-4-II-E cells were grown to 80% confluency and serial dilutions of
resistant to the final acidic cleavage conditions of SPPS.[34] However, the total synthesis of cephalosporin showed the stability of its b-lactam ring in presence of TFA.[35] Therefore, the cephalosporin beta-lactam core, 7ACA, and also 7-ADCA were chosen for functionalization and conjugation with a series of cationic AMPs on the solid phase. To minimize the need for acid treatment, fluorenylmethyl (Fm)-protection was used[36] for the preparation of Ca-Fm-protected 7-ACA and
test compounds were added in 50 lL aliquots to a final incubation vol-
7-ADCA (Figure 1) which are suitable for use following Fmoc-SPPS.
ume of 200 lL. After incubation (18 h, humidified atmosphere, 5% CO2
Importantly, we have observed that these compounds very readily absorb
at 378C) the level of proliferation was determined using CellTiter 96
moisture leading to decomposition during the storage after few days in
AQueous Non-Radioactive Cell Proliferation Assay (Promega) as
the fridge or room temperature and therefore need to be used promptly.
described,[20] and measured at 490 nm. Camptophecin (10 nM) was
Each of the cationic AMPs were assembled on Rink amide resin
used as a positive control. Data is from three biological replicates and is
and was followed by an additional reaction with glutaric acid anhydride
expressed as the concentration in lM required to inhibit 100% of cell
(Figure 2). Initially, we tried to activate the Ca-Fm-protected cephalo-
viability.
sporin building block with HCTU/DIPEA, HBTU/DIPEA or HATU/ DIPEA for conjugation of this b-lactam to the cationic AMPs. However,
3 | RESULTS AND DISCUSSION 3.1 | Conjugated AMPs on solid phase
the desired products could not be observed or reacted only in trace amounts. For this reason, we undertook conjugation of Ca-Fm-functionalized 7-ACA and 7-ADCA via their 7-amino group and a glutaric anhydride linker to the solid phase-bound cationic AMPs, including
Like the studies that covalently combined conventional antibiotics and
MSI-78, CA(1–7)M(2–9)NH2 and Chex1-Arg20, following a pre-
small molecules, we undertook to prepare similar conjugates of 7-ACA
activation with DIC/Oxyma (2 equiv to resin) for 20 min and reacting
and 7-ADCA with AMPs via SPPS. In general, beta-lactam rings are not
on the solid phase for 6 h (Figure 2). These conjugated peptides were
6 of 9
|
T AB LE 1
LI
ET AL.
Conjugated AMPs produced and used in this study
No.
Peptide
Structure
Yield
1
CA(1–7)M(2–9)-ADCA
2.4%
2
CA(1–7)M(2–9)-ACA
1.9%
3
CA(1–7)M(2–9)NH2
4
MSI-78-ADCA
1.5%
5
MSI-78-ACA
2.6%
6
MSI-78
7
des-Chex1-Arg20-ADCA
2%
8
des-Chex1-Arg20-ACA
3%
9
Chex1-Arg20*
KWKLFKKIGAVLKVL-NH2
GIGKFLKKAKKFGKAFVKILKK-NH2
Chex-RPDKPRPYLPRPRPPRPVR-NH2
11.2%
10.1%
35%
obtained following TFA cleavage (TFA:TIPS:anisole, 95:3:2 v/v) and
activity against MDR A. baumannii. Interestingly, 7-ACA alone was
subsequent RP-HPLC purification (Supporting Information Figure S1) in
found to have very poor antibacterial activity (MIC) against all the bac-
overall good yields (Table 1).
teria tested (Table 2, Supporting Information Figure S2) which supports a very early previous observation.[38,39] Yet 7-ACA is a critical interme-
3.2 | Antibacterial activity of the conjugated AMPs With the 7-ACA- and 7-ADCA-conjugated cationic AMPs in hand, we performed the antibacterial activity screening on a panel of Gramnegative bacteria, including K. pneumoniae, A. baumannii and P. aeruginosa. While the activity of the monomer PrAMP, Chex1-Arg20, was comparable to our previous study,[20] the conjugation of 7-ACA or ADCA to the des-Chex analogue did not result in significant improvement against these bacteria (Table 2). However, both b-lactam coreconjugated MSI-78 and CA(1–7)M(2–9)NH2 displayed a significant improvement in their antibacterial activity (Table 2) by 5–14 fold against the most important cause of hospital-acquired infection[37] A.
diate for the production of numerous members of the cephalosporin family of compounds bearing potent activities. For 7-ADCA, a report shows that the antibacterial activity ratio is 7-ACA:7-ADCA 5 15:1,[40] which is consistent with our observation at the highest tested concentration of its antibacterial activity (data not shown). The molecular mechanism whereby those conjugated AMPs containing b-lactam antibiotic that possess increased or possibly synergistic activity to attack their target pathogens will be the subject of further investigation using these new lead conjugates.
3.3 | Synergistic calculation
baumanni. Similar observations were made against a panel of clinical
Using Equation 2, the FICI[5,6,31,32] was calculated for the cationic
isolates of strains MDR K. pneumoniae (FADDI-KP628), A. baumannii
AMPs and their conjugated analogues against the MDR clinical patho-
(FADDI-AB156), P. aeruginosa (FADDI-PA067). However, most of these
gens using MIC values in Table 2. Because none of these analogues
AMPs and their conjugated analogues showed no activity against the
showed any activity against MDR K. pneumoniae (FADDI-KP628) and
MDR nosocomial pathogens. The exceptions, MSI-78-conjugated ß-lac-
P. aeruginosa (FADDI-PA067) (Table 2), their FICI were not calculated.
tam peptides, displayed 3- to 6-fold improvement of their antibacterial
In contrast, the FICI of CA(1–7)M(2–9)-ACA and CA(1–7)M(2–9)-
LI
T AB LE 2
No.
a
|
ET AL.
7 of 9
Antibacterial activity (MIC in mM) of the conjugated AMPs against a panel of Gram-negative bacteria K. pneumoniae
A. baumannii
P. aeruginosa
K. pneumoniae 028 a
A. baumannii 156
P. aeruginosa 067
17.3 6 3.6
n/a
1
6.8 6 2.7
10.5 6 1.46
16.2 6 1.7
n/a
2
5.6 6 2.7
6.0 6 3.7
14.9 6 4.6
n/a
20.7 6 1.1
n/a
3
3.9 6 0.15
>50
3.6 6 0.1
n/a
18.4 6 3.8
n/a
4
4.6 6 0.3
3.6 6 1.7
2.9 6 0.25
n/a
4.2 6 0.8
n/a
5
3.4 6 1.2
3.7 6 1.6
3.1 6 1.0
n/a
9.6 6 0.5
n/a
6
8.1 6 0.2
>50
3.6 6 0.2
n/a
32.1 6 6.6
n/a
7
15.9 6 5.7
>50
>50
n/a
n/a
n/a
8
23.9 6 7.0
>50
>50
n/a
n/a
n/a
9
0.80 6 0.02
>100
>50
n/a
n/a
n/a
7-ACA
940 6 200
470 6 260
>2 000
940 6 600
1 900 6 600
>2 000
Indicated no activity against this pathogen.
ADCA were 1.1 and 0.94, respectively, which indicated partial synergistic activity[33] between CA(1–7)M(2–9)NH2 and cephalosporanic acid. Moreover, the calculated FICI of MSI-78-ACA (0.3) and MSI-78-ADCA (0.13) showed a strong synergistic activity.
3.4 | Secondary structure study The conformational properties of the conjugated MSI-78, CA(1–7)M (2–9)NH2, des-Chex1-Arg20 peptides were assessed by CD spectroscopy. Even though there were some structural changes adopted after
CD spectra of 7-ACA- and 7-ADCA-conjugated cationic AMPs in 20 mM phosphate buffer (pH 7.4). All measurements were recorded at 258C
FIGURE 3
8 of 9
|
LI
the conjugation, the conjugated cationic AMPs still showed mainly ran-
ET AL.
RE FE RE NC ES
dom coil structures in phosphate buffer (20 mM) (Figure 3). The overall
[1] World Health Organization, Geneva, Switzerland 2014.
unchanged structure implied that the synergistic activity between cati-
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onic AMPs and cephalosporanic acid was not associated with changes
[3] D. Brown, Nat. Rev. Drug Discov. 2015, 14, 821.
in secondary structure.
[4] X.-L. Hu, D. Li, L. Shao, X. Dong, X.-P. He, G.-R. Chen, D. Chen, ACS Med. Chem. Lett. 2015, 6, 793.
3.5 | Cytotoxicity
[5] F.-Y. Chan, N. Sun, Y.-C. Leung, K.-Y. Wong, J. Antibiot. 2015, 68, 253.
In vitro cytotoxicity of the 7-ACA- and 7-ADCA- conjugated AMPs
[6] G. Akash, M. S. Neveen, D. Riddha, F. L. Ryan, B. Arafeh, M. Khatereh, C. Alexandre Rosa, P. Kenneth, M. Morteza, M. R. Vincent, Nano Futures 2017, 1, 015004.
were performed with HEK-293 and H-4-II-E cells by CellTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay. None of these peptides
showed any significant toxicity on both mammalian cell lines at the tested concentration range (Supporting Information Table S1).
4 | CONCLUSIONS In summary, a series of cationic AMPs, MSI-78, CA(1–7)M(2–9)NH2 and des-Chex1-Arg20, were conjugated with a prepared Ca-Fm-protected b-lactam-containing cephalosporin building blocks, 7-ACA and 7-ADCA, via the solid phase. The resulting conjugated AMPs were screened against a panel of nosocomial pathogens, including clinical isolated MDR pathogens. The conjugated MSI-78 analogues displayed significant synergistic antibacterial activity against the common cause of hospital-acquired infectious strain, A. baumannii and MDR A. baumannii 156. Furthermore, the secondary structure study did not indicate a relationship between synergistic activity and their structure. However, further optimization of the conjugation of such b-lactam cephalosporin precursors with cationic AMPs to induce increased synergistic antibacterial activity are under further investigation by our group. This will include the use of our functionalized cephalosporin precursors for direct on-resin acylation onto selectively Cb-deprotected side chain of Asp/Glu residues.
AC KNOW LE DGME NT S The authors kindly thank Dr Julien Tailhades for discussions and suggestions for the synthesis of b-lactam antibiotic building blocks. They thank Assoc. Prof. Craig Hutton (University of Melbourne) for access to facilities. A portion of this work was funded by ARC Dis-
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covery Project grants (DP150103522) to JDW and MAH, and
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(DP140102127) to FS, and NHMRC Project grants (APP1029878) to
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NMOBS and (APP1008106) to ECR and NMOBS. JDW is an
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NHMRC (Australia) Principal Research Fellow (APP1117483). WL
€ ollmann, Med. Chem. [24] S. Yan, M. J. Miller, T. A. Wencewicz, U. Mo Commun. 2010, 1, 145.
was the recipient of an MIRS PhD award and Dr Albert Shimmins postgraduate writing-up award. Research at the FINMH was also supported by the Victorian Government’s Operational Infrastructure
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Wenyi Li
http://orcid.org/0000-0003-3584-0301
Frances Separovic John D. Wade
http://orcid.org/0000-0002-6484-2763 http://orcid.org/0000-0002-1352-6568
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NEIL M. O'BRIEN‐SIMPSON graduated from Edin-
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burgh Napier University in 1992 with a BSc with honors in Science and Management Stud-
[31] S. Zhang, R. Li, J. Yu, Antimicrob. Agents Chemother. 2013, 57, 3395.
ies. He moved to Australia and completed a
[32] I. S. Hwang, J. S. Hwang, J. H. Hwang, H. Choi, E. Lee, Y. Kim, D. G. Lee, Curr. Microbiol. 2013, 66, 56.
PhD in peptide-polymer vaccines in 1998 at The University of Melbourne. He is currently a Professor and his research interests include
[33] F. C. J. Odds, Antimicrob. Chemother. 2003, 52, 1. [34] M. Tarbe, I. Azcune, E. Balentova, J. J. Miles, E. E. Edwards, K. M. Miles, P. Do, B. M. Baker, A. K. Sewell, J. M. Aizpurua, C. DouatCasassus, S. Quideau, Org. Biomol. Chem. 2010, 8, 5345.
antimicrobial peptides/materials, nanoparticles for peptide and vaccine
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Users Group.
delivery and bacterial outer membrane vesicles-host interactions. He has several editorial duties and is the current president of the Peptide
JOHN D. WADE obtained his PhD in 1979 at
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Monash University, Australia, and was then a
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ratory of Molecular Biology, Cambridge, UK. In
[38] H. Sumano, L. Ocampo, J. Azuara, J. Appl. Anim. Res. 1998, 13, 169.
roscience and Mental Health, University of
[39] B. Loder, G. G. F. Newton, E. P. Abraham, Biochem. J. 1961, 79, 408. [40] W. C. Topp, B. G. Christensen, J. Med. Chem. 1974, 17, 342.
Nuffield Foundation Fellow at the MRC Labo1983, he moved to the Florey Institute of NeuMelbourne, where he heads the Laboratory of Peptide Chemistry. He is an NHMRC Principal Research Fellow and a Fellow of the Royal Australian Chemical Institute and of the Royal Society of Chemistry.
AUT HOR B IOGR AP HIE S WENYI LI received his doctoral degree in September 2016 at the University of Melbourne under the supervision of Prof John D Wade
SUP POR TI NG INFOR MATION Additional Supporting Information may be found online in the supporting information tab for this article.
and Prof Frances Separovic. His doctoral thesis was awarded the Graham Johnston Best Thesis Award of the RACI. In October 2016, he joined Prof Christian Hackenberger’s group in LeibnizInstitute for Molecular Pharmacology, Germany. Currently, he is supported as Leibniz-DAAD postdoctoral fellow investigating the semisynthesis and physiological behavior of the Alzheimer’s disease-related tau protein.
How to cite this article: Li W, O’Brien-Simpson NM, Holden JA, et al. Covalent conjugation of cationic antimicrobial peptides with a b-lactam antibiotic core. Peptide Science. 2018;110: e24059. https://doi.org/10.1002/pep2.24059
