Siderophore Conjugates of Daptomycin are Potent Inhibitors of

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Siderophore Conjugates of Daptomycin are Potent Inhibitors of Carbapenem Resistant Strains of Acinetobacter baumannii Manuka Ghosh, Yun-Ming Lin, Patricia A. Miller, Ute Möllmann, William Boggess, and Marvin J. Miller ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00150 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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ACS Infectious Diseases

Siderophore Conjugates of Daptomycin are Potent Inhibitors of Carbapenem Resistant Strains of Acinetobacter baumannii Manuka Ghosh,1 Yun-Ming Lin,1 Patricia A. Miller,1,2 Ute Möllmann,1 William C. Boggess2 and Marvin J. Miller1,2* 1

Hsiri Therapeutics, Rosetree Corporate Center, 1400 N. Providence Road, Building 1, Suite

115S, Media, PA 19063. 2

Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre

Dame, Notre Dame, IN 46556. *email: [email protected]

The development of resistance to antibiotics is a major medical problem. One approach to extending the utility of our limited antibiotic arsenal is to repurpose antibiotics by altering their bacterial selectivity. Many antibiotics that are used to treat infections caused by Gram-positive bacteria might be made effective against Gram-negative bacterial infections, if they could circumvent permeability barriers and antibiotic deactivation processes associated with Gramnegative bacteria. Herein we report that covalent attachment of the normally Gram-positive only

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antibiotic, daptomycin, with iron sequestering siderophore mimetics that are recognized by Gram-negative bacteria provides conjugates that are active against virulent strains of Acinetobacter baumannii, including carbapenemase and cephalosporinase producers. The result is the generation of a new set of antibiotics designed to target bacterial infections that have been designated as being of dire concern. KEYWORDS: Sideromycin, siderophore, daptomycin, conjugate, Gram-negative, antibiotic In February, 2017, the World Health Organization (WHO) published a list of antibiotic resistant bacteria that are responsible for deadly infections with prioritized emphasis on the need for discovery and development of new antibiotics.1

Of critical concern (Priority 1) are

carbapenem resistant strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae. Carbapenems are among the last resort antibiotics for treatment of infections due to these Gram-negative organisms and bacterial incorporation of β-lactamases, including carbapenemases, has dramatically enhanced antibiotic resistance. The lipopeptide antibiotic, daptomycin, itself is a Gram-positive only antibiotic.2 However, we recently reported that a conjugate (1)3 of daptomycin and a bis-catechol, monohydroxamate siderophore mimic (2) related to fimsbactin (3), an important iron sequestering siderophore used by pathogenic strains of Acinetobacter baumannii,4 had outstanding in vitro and in vivo antibiotic activity against A. baumannii. Since daptomycin is not a β-lactam and thus not susceptible to β-lactamases, we anticipated that, in contrast to our previously described carbacephalosporin conjugate 4,5,6 conjugate 1 and additional siderophore daptomycin conjugates might be active against β-lactamase producing bacteria, including carbapenemase and cephalsporinase containing Gram-negative bacteria. Herein we report that


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conjugate 1 and two new siderophore-daptomycin conjugates 5 and 6 have potent activity against strains of A. baumannii that produce carbapenemases and cephalsporinases.



Figure 1. Synthetic sideromycins 1, 2, 4-6, and the natural siderophore, fimsbactin A (3). RESULTS AND DISCUSSION: Siderophores are biosynthesized by bacteria to sequester iron, using a molecular recognition initiated, active transport process that is often essential for bacterial growth and virulence during bacterial infections.7

Natural sideromycins are covalent combinations of

siderophores and antibiotics that utilize the active recognition and transport processes of siderophores to deliver antibiotics to other bacteria and give the producing bacteria selective growth advantages.8 Synthetic sideromycins like 1 are designed to mimic this Trojan horse antibiotic strategy. In addition to determining whether mixed-ligand daptomycin conjugate 1 was active against β-lactamase producing strains of A. baumannii, we also synthesized and tested conjugates (5 and 6) with alternate siderophore mimetics that have been shown to be effective for delivery of β-lactam antibiotics to other Gram-negative bacteria. The choice of the new siderophore components was based on literature precedent that used iron chelators that closely mimic natural siderophores. Specifically, Möllmann and Heinisch, et al reported that the tetra acetylated form of synthetic bis-catechol conjugate 7 (HKI-9924154) was recognized by outer membrane siderophore receptors of targeted Gram-negative bacteria, and was extraordinarily active against tested strains of Pseudomonas aeruginosa, E. coli, S. maltophilia and Klebsiella pneumoniae both in vitro and in vivo.9 Cheng, et al described the design and synthesis of tricatechol-ampicillin conjugates that had excellent activity against select strains of P. aeruginosa10 and Nolan’s group reported that enterobactin (tri-catechol) β-lactam conjugates are active against pathogenic strains of E. coli.11 We anticipated that syntheses and studies of related daptomycin


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conjugates (5 and 6) would be especially interesting since O’Neill, et al have reported that some Gram-negative bacteria, including strains of E. coli, P. aeruginosa, E. cloacae, K. pneumoniae, M. catarrhalis, and S. typhirmuium do not have ample targets of daptomycin to allow it to be effective even if it could be internalized (permeate the outer membrane).12

Figure 2. Precedented β-lactam containing synthetic sideromycins, 7 and 8. Fortunately, since the daptomycin target tolerates acylation of the δ-amino group of the ornithine residue in daptomycin,13 release of the antibiotic14,15 from the siderophore component of 1 was not necessary after active transport into targeted A. baumanni. Thus, as shown below (Scheme 1), poly benzyl protected siderophore analog 9 was converted to the corresponding Nhydroxy succinimide (NHS) active ester and 10 was converted to a mixed anhydride with isobutyl chloroformate. The resulting carboxyl activated poly benzyl protected siderophores were then separately reacted with the free δ-amino group of daptomycin. The benzyl groups of the resulting protected conjugates were removed by standard hydrogenolysis to give the final conjugates 5 and 6, respectively. To enhance water solubility, the conjugates were treated with NaHCO3 to give the corresponding tetra sodium salts which were purified and characterized. Scheme 1. Syntheses of daptomycin conjugates 5 and 6.



With the new conjugates, 5 and 6, in hand, they, the original mixed ligand conjugate 1 and daptomycin were tested against the Gram-negative strains of A. baumannii, Pseudomonas aeruginosa, Shigella sonnei, Salmonella typhimurium, E. coli and Proteus mirabilis as shown in Table 1. Consistent with our earlier report,3 mixed ligand-daptomycin conjugate 1 displayed


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potent activity against all tested strains of A. baumannii, including outstanding MIC values of 0.2 µM against the carbapenemase and cephalosporinase producing strains (A. baumannii ATCC 17978 pNT165 and A. baumannii ATCC 17978 pNT320, respectively, Table 1, entries 7 and 10)! This mixed ligand conjugate was expected to be selectively recognized by A. baumannii since the siderophore component mimics fimsbactin, the natural siderophore biosynthesized for selective iron transport by strains of A. baumannii. Thus, lack of activity against other Gramnegative bacteria was not surprising. Interestingly, the new bis and tri-catechol conjugates (5 and 6, respectively) also had excellent activity against A. baumannii, but while bis-catechol conjugate 5 retained good activity against the carbapenemase and cephalosporinase producers (MIC = 1.6µM, entries 7 and 10, Table 1), tri-catechol conjugate 6 was less active (MI = 25µM) against those particular strains. Although the bis and tri-catechol conjugates were anticipated to be recognized by other Gram-negative bacteria,9,10 only the original mixed ligand conjugate (1) displayed any other activity (MIC = 0.8 µM against Salmonella typhimurium Enb-7,16 a strain that is unable to synthesize the natural tri-catechol siderophore, enterobactin, Table 1, entry 14). This general lack of activity is consistent with the report that the target for daptomycin is absent in E. coli and other Gram-negative bacteria.11 Lack of target is reasonable, but other points should be considered in future studies. Although acylation of ornithine in daptomycin is tolerated by Grampositive bacterial targets and by A. baumannii, might it be more sensitive in other Gram-negative bacteria? Could the daptomycin conjugates be substrates for efflux pumps in Gram-negative bacteria or is it possible that the warhead is not being delivered to the appropriate compartment since some siderophores deliver iron to the periplasm and others to cytoplasmic space?17,18



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Table 1. MIC values in µM of daptomycin conjugates and daptomycin against Gram-negative bacteria. Entry

Strain

1

5

6

Daptomycin

1 2 3 4 5 6 7

A. baumannii ATCC 17961 0.2 0.4 0.2 >50 A. baumannii ARC 3484 0.4 1.6 3 >50 A. baumannii ARC 3486 0.4 0.8 3 >50 A. baumannii ARC 5079 0.8 3 12.5 >50 A. baumannii ARC 5081 0.8 50 12.5 >50 A. baumannii ATCC 17978 0.4 1.6 0.8 >50 A. baumannii ATCC 17978 0.2 1.6 0.4 >50 pNT165 (a) 8 A. baumannii ATCC 17978 0.2 0.8 0.4 >50 pNT221 9 A. baumannii ATCC 17978 0.2 1.6 0.8 >50 pNT255 10 A. baumannii ATCC 17978 0.2 1.6 0.4 >50 pNT320 (b) 11 P. aeruginosa KW799/wt >50 >50 >50 >50 12 P. aeruginosa ARC3502 >50 >50 >50 >50 13 S. typhimurium ATCC 13311 >50 nt >50 >50 14 S. typhimurium enb - 7 (c) 1.6 50 nt >50 15 Shigella sonnei X813 >50 >50 >50 >50 16 E. coli DCO >50 >50 >50 >50 17 Proteus mirabilis X235 >50 >50 >50 >50 (a) carbapenamase producing (b) cephalosporinase producing (c) cannot synthesize enterobactin MH160 broth (MH media containing 160 µM bipyridyl + 110 µg/mL CaCl2) was used. MIC values are the average of ≥3 assays. nt = not tested. Although the activity of conjugate 1 was better than that of 5 and 6, occasionally, during the antibacterial assays of 1

in 96 well plates, growth in a single well would occur at

concentrations above the MIC but always < 12µM in 1. These colonies were selected, studied and again found to be resistant at concentrations 50 0.4 0.8 PU 1.6 0.8 BAA 1710 2 A. baumannii ATCC 0.4 0.8 3 >50 0.4 1.0~PU 0.6~ 0.4 BAA 1793 3 A. baumannii ATCC 0.4 1.6 1.6 >50 0.4 0.4 0.8 0.4 BAA 1797 4 A. baumannii ATCC 0.4 0.8 1.6 >50 0.4 0.6~ 0.8 0.4 BAA 1800 MH160 broth (MH media containing 160 µM bipyridyl + 110 µg/mL CaCl2) was used. Combinations kept the concentration of daptomycin at a constant level. 1 + 6 mixed in 1:1 molar ratio with daptomycin at 200µM before addition to assay. The same procedure was followed for 1 + 5 and 5 + 6. The combination of 1 + 5 + 6 was made 1:1:1 molar ratio with the daptomycin concentration at 200µM before addition to the assay. MIC values are the average of ≥3 assays. CONCLUSION: Overall, the activity of daptomycin-siderophore mimic conjugates, 1, 5, 6, and combinations thereof demonstrates that a Gram-positive antibiotic, daptomycin in this case, might be repurposed to develop much needed antibiotics that are effective against specific Gramnegative bacteria. The ability to specifically target and circumvent multidrug resistance in strains of A. baumannii, including β-lactamase producing strains, addresses an especially significant area of concern.19,20



EXPERIMENTAL SECTION

General Methods. All solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated. Isobutyl chloroformate (ClCO2i-Bu) was used from Acros Seal anhydrous bottles. Technical grade tetrahydrofuran (THF) was freshly distilled over sodium before use. OmniSolv® LC-MS Water from EMD Millipore was used for all reactions, purifications, and work-up purposes. Reactions were conducted under an atmosphere of dry argon unless otherwise stated. For column chromatography purifications, Sorbent Technologies silica gel 60 (32−63 µm) was used. Reverse phase chromatographic purifications were performed on Teledyne Instruments’ 30 gram RediSep Rf Gold® C18Aq reversed-phase columns (column volume: 26.4 mL, average particle size: 20 to 40 µm, average pore size: 100 Å) at a flow rate of 35 mL/min. Thin layer chromatography (TLC) was performed with Al-backed Merck 60-F254 or Al-backed Merck RP-C18 F256 silica gel plates using a 254 nm lamp and aqueous FeCl3 for visualization. HPLC-MS mass measurements were used to determine purity as well as structural consistency. All characterized compounds were determined to be ≥99% pure. The HPLC-MS studies were performed with a Bruker MicrOTOF-QII Quadrupole Time-of-Flight mass spectrometer operating in positive ion mode with acquisition mass range 50-3000 u. Electrospray ionization source parameters were capillary voltage = 2200 v, end slate offset = -500 v, nebulizer gas pressure = 5 bar, dry gas flow rate = 10 L/min, and dry gas temperature = 220 oC. Liquid separation was performed on a Dionex UltiMate 3000 RSLC with mobile phases consisting of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The mobile phase gradient at 0.4 mL/min was 10% of B for 2 min followed by linear ramp of 100% of B at 18 min and a return to initial conditions from 18.1 to 20 min. The mobile phase for the iron complex


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consisted of water (A) and acetonitrile (B). UV-VIS spectra were recorded on a Dionex UltiMate 3000 RS Diode Array Detector over the wavelength range of 190-400 nm. The LC column was a Thermo Scientiffic Acclaim RSLC 120 C18 with 2.2 µm particle size, 120 Å pore size and 2.1 X 100 mm dimensions heated at 40oC. 1D 1H and 13C{1H} spectra of the compounds were recorded on a Varian DirectDrive 600 spectrometer operating at a proton resonance frequency of 599.98 MHz. Sodium 2,2'-((3S,6S,9R,15S,18R,21S,24S,30S,31R)-30-((S)-2-((R)-4-amino-2-((S)-2decanamido-3-(1H-indol-3-yl)propanamido)-4-oxobutanamido)-3carboxylatopropanamido)-3-(2-(2-aminophenyl)-2-oxoethyl)-6-((S)-1-carboxylatopropan-2yl)-24-(3-(2-(N-(4-(2,3-dihydroxybenzamido)butyl)-2,3dihydroxybenzamido)acetamido)propyl)-9-(hydroxymethyl)-18,31-dimethyl2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28nonaazacyclohentriacontane-15,21-diyl)diacetate (5). Preparation of the NHS-ester of 9. To a solution of benzyl-protected siderophore analog 9 (246 mg, 0.315 mmol) in anhydrous DCM (15.0 mL) cooled to 0 oC was added NHS (128 mg, 1.12 mmol, 3.5 equiv), and EDC (210 mg, 1.10 mmol, 3.5 equiv). The reaction mixture was stirred under an atmosphere of argon keeping the temperature at 0 oC for 1 h and then at room temperature overnight. Analysis (TLC) of an aliquot showed nearly 30% of the unreacted starting materials. Additional amount of NHS (128 mg, 1.12 mmol, 3.5 equiv), and EDC (210 mg, 1.10 mmol, 3.5 equiv) was added to the reaction mixture and stirring was continued under an argon atmosphere overnight. Analysis of the product after usual work up showed complete consumption of starting siderophore analog 9. Coupling of the NHS-ester of 9 with Daptomycin.



In a separate HCl-washed round bottom flask, Daptomycin (407 mg, 0.252 mmol, 0.8 equiv. relative to 9), NaHCO3 (129 mg, 1.54 mmol, 5.0 equiv. relative to daptomycin) and DMAP (4.0 mg, 0.032 mmol) were dissolved in H2O (14 mL) and THF (20 mL), the resulting Na-salt of daptomycin with the free δ-amino group was purged with argon and cooled to 0 oC. The NHS ester of the acid 9 described above was filtered slowly into the Na-salt over 20 min and purged with argon. The nearly homogeneous reaction mixture was stirred under an atmosphere of argon overnight at room temperature. THF was then evaporated from the reaction mixture and the aqueous layer was acidified with 3N HCl to pH 3 and lyophilized to obtain a light yellowish solid. The reaction mixture was analyzed by LCMS. LCMS (m/z): [M+2H]2+calcd for C120H145N19O33, 1191.0199; found, 1191.0210; tR 26 min. The crude compound was dissolved in CH3CN/water (1:1, 20 mL) and was purified by reversed phase chromatography (RediSep Rf Gold reversed-phase C18 high performance columns, 30 g) using a gradient 10-100% CH3CN/H2O as the eluent to give fractions containing the benzyl-protected conjugate contaminated with ~30% of the siderophore analog 9. Hydrogenolytic Deprotection of Benzyl-Protected Daptomycin-Containing Sideromycin (5a). The above crude benzyl protected conjugate (400 mg) without further purification was diluted with a mixed solvent of H2O / THF (1:1, 44 mL) in a HCl-washed flask. The reaction mixture was purged with argon, and treated with 10% Pd-C (60 mg), and flushed thoroughly with hydrogen gas with intermediate vacuum evacuation. The resulting suspension was stirred under an atmosphere of hydrogen (1 atm) for 8h. The hydrogenolysis was monitored by reverse phase C18 TLC (40% CH3CN / H2O). The reaction mixture was purged with argon, filtered through a sintered funnel and washed with THF/ H2O (1:1, 10 mL), concentrated to remove THF, and lyophilized to get the fully deprotected bis-catechol-daptomycin conjugate 5 contaminated with


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the siderophore analog 9 as a purplish-white solid (300 mg), which was subjected to reverse phase chromatography (RediSep Rf Gold reversed-phase C18 high performance columns, 30 g), using a gradient 10-100% CH3CN / H2O as the eluent. The pure fractions were combined and lyophilized to obtain the fully deprotected conjugate 5 as an off white fluffy solid (50 mg, yield 10% overall 2-steps, purity >99 area %, UV). Na-salt Formation. The isolated salt of the conjugate (50 mg, 0.025mmol) was dissolved in CH3CN/ LCMS grade H2O (1:1, 10 mL) and treated with with NaHCO3 (8.1 mg, 0.099 mmol, 4 equiv). The aqueous phase after evaporation of the CH3CN layer was lyophilized to provide the desired Na salt of 5 in near quantitative yield. LCMS as neutral form (m/z): [M+2H]2+ calcd for C92H121N19O33, 1010.9260; found, 1010.9337; tR 11.2 min.

4-((1,7-Bis(2,3-bis(Benzyloxy)benzamido)-4-(3-(2,3bis(benzyloxy)benzamido)propyl)heptan-4-yl)amino)-4-oxobutanoic acid (10). To a solution of the methyl ester 10a (2.0 g, 1.6 mmol) in THF/water (1:1. 40 mL) was added NaOH (76 mg, 1.9 mmol, 1.2 equiv). The reaction mixture was left stirring overnight at room temperature. Usual work up after adjustment of pH to ~3 provided the benzyl-protected siderophore analog 10 (1.8 g, yield 90%); 1H NMR (600 MHz, CDCl3) δ 8.07 (t, J = 5.4 Hz, 3H), 7.67 (m, 3H), 7.35-7.26 (m, 30H), 7.08-7.09 (m, 6H), 5.7 (s, 1H), 5.09 (s, 6H), 5.04 (s, 6H), 3.10-3.13 (m, 6H), 2.57-2.59 (t, J = 6.6 Hz, 2H), 2.33-2.35 (t, J = 6.0 Hz, 2H), 1.42-1.45 (m, 6H), 1.16-1.20 (m, 6H); 13C NMR (150 MHz, CDCl3) δ 174.2, 165.5, 151.7, 146.7, 136.4, 128.7, 128.6, 128.3, 127.6, 127.0, 124.4, 123.2, 117.0, 76.34, 63.14, 58.55, 50.9, 39.9, 32.2, 31.7, 30.60, 23.24; LCMS (m/z): [M+H]+ calcd for C77H78N4O12, 1251.5689; found, 1251.5671; tR 1.5 min.



Sodium 2,2'-((3S,6S,9R,15S,18R,21S,24S,30S,31R)-30-((S)-2-((R)-4-amino-2-((S)-2decanamido-3-(1H-indol-3-yl)propanamido)-4-oxobutanamido)-3carboxylatopropanamido)-3-(2-(2-aminophenyl)-2-oxoethyl)-24-(3-(4-((1,7-bis(2,3dihydroxybenzamido)-4-(3-(2,3-dihydroxybenzamido)propyl)heptan-4-yl)amino)-4oxobutanamido)propyl)-6-((S)-1-carboxylatopropan-2-yl)-9-(hydroxymethyl)-18,31dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28nonaazacyclohentriacontane-15,21-diyl)diacetate (6). Formation of the Mixed Anhydride of 10. To a solution of benzyl-protected acid 10 (385 mg, 0.308 mmol) and N-methyl morpholine (42 µL, 0.385 mmol) in anhydrous THF (8.0 mL) cooled to 0 oC was added isobutyl chloroformate (50 µL, 0.385 mmol). The reaction mixture was stirred for 1h under an atmosphere of argon maintaining the temperature at 0 oC. The mixed anhydride formation was deemed complete when reversed-phase C18 TLC (65% CH3CN / 10 mM NH4OAc; FeCl3 stain) showed complete consumption of the starting siderophore 10. Coupling of the Mixed Anhydride of 10 with Daptomycin. In a separate HCl-washed round bottom flask, Daptomycin (500 mg, 0.308 mmol) and NaHCO3 (129 mg, 1.54 mmol, 5.0 equiv) were dissolved in H2O (20 mL) and the resulting Na-salt of daptomycin with the free δ-amino group was purged with argon and cooled to 0 oC. The mixed anhydride of 10 described above was filtered slowly into the Na-salt over 20 min, washing with additional THF (10 mL). The reaction mixture was stirred under an atmosphere of argon for 2h at 0 oC, followed by 4h at room temperature to give the benzyl-protected conjugate 6a. The reaction was monitored by reverse phase C18 TLC (~65% CH3CN / 10 mM NH4OAc; FeCl3 stain) for any remaining mixed anhydride or its precursor siderophore, and analyzed by LCMS.


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Hydrogenolytic Deprotection of Benzyl-Protected Daptomycin-Containing Sideromycin (6a). The reaction mixture containing the protected conjugate was diluted with LCMS grade water (30 mL) in a HCl-washed flask, resulting in a mixed solvent system of THF/water (2.8:1, 68 mL). The reaction mixture was purged with argon, treated with 10% Pd-C (100 mg), and flushed thoroughly with hydrogen gas with intermediate vacuum evacuation. The resulting suspension was stirred under an atmosphere of hydrogen (1 atm) for 8h. The hydrogenolysis was monitored by reverse phase C18 TLC (30% CH3CN in 10 mM NH4Ac, FeCl3 stain) until no remaining starting protected conjugate was apparent. The reaction mixture was purged with argon, filtered through a sintered funnel washing with water, and lyophilized to get the fully deprotected tricatechol-daptomycin conjugate 6 as an off-white solid (700 mg, >99% mass recovery). The crude conjugate 6 (700 mg) was purified by reverse phase chromatography (RediSep Rf Gold reversed-phase C18 high performance columns, 30 g) using a gradient 10-30% CH3CN/NH4OAc (10 mM) as the eluent. The desired fractions were collected and lyophilized to obtain slightly yellowish fluffy solid (100 mg) as the polyammonium salt of the conjugate 6. Na-salt Formation. The isolated polyammonium salt of the conjugate (100 mg, 0.042 mmol) was dissolved in CH3CN/ LCMS grade H2O (1:1, 10 mL), neutralized with 3 N HCl (pH~3) and then passed through a reverse phase column (30 g) eluting with water (100 mL, LCMS grade) followed by 50% CH3CN/water to remove inorganic salts. Lyophilization of the fraction containing the product provided the neutral conjugate 6 (70 mg, 0.030 mmol), which was dissolved in CH3CN / H2O (1:1, 13 mL) and treated with a solution of NaHCO3 (12.7 mg, 0.151 mmol, 5 equiv) in H2O (2 mL).



The aqueous phase after evaporation of the CH3CN layer was lyophilized to provide the desired Na salt of 5 in quantitative yield. LCMS as neutral form (m/z): [M+2H]2+ calcd for C107H141N21O37, 1156.9971; found, 1157.0073; Rt 11.4 min. Antibacterial Assays. MIC analyses were performed using iron depleted media as described previously.3

ASSOCIATED CONTENT Supporting Information The following file is available free of charge on the ACS Publications website at DOI: ….. Supporting information (PDF) includes copies of spectral/analytical data (LCMS and NMR) on key compounds. AUTHOR INFORMATION Corresponding Author *(M.J.M.) E-mail: [email protected] ORCID Marvin J. Miller: 0000-0002-3704-8214 Author Contributions The manuscript was written through contributions of all authors. M. G. and Y.-M. L. performed the syntheses and compound characterization, P. A. M. performed the bioassays. U. M.


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contributed to the development and implementation of the research plan. M. J. M. conceived, designed and directed the research plan. All authors have given approval to the final version of the manuscript. Notes M. G., Y.-M. L. and P. A. M. were employed by Hsiri Therapeutics. U. M. was a consultant for Hsiri Therapeutics. ACKNOWLEDGMENTS This work was supported by the Department of Defense (W81XWH-12-2-0015). REFERENCES

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and In Vitro Antibacterial Studies of Tris-catecholate Siderophore-Aminopenicillin Conjugates Reveals Selectively Potent Anti-Pseudomonal Activity. J. Am. Chem. Soc. 2012, 134, 98989901. 11

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Caselli, E.; Romagnoli, C.; Powers, R. A.; Taracila, M. A.; Bouza, A. A.; Swanson, H. C.;

Smolen, K. A.; Fini, F.; Wallar, B. J.; Bonomo, R. A.; Prati, F. Inhibition of AcinetobacterDerived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel β‐ Lactamase Inhibitors. ACS Infect. Dis., 2018, 4, 337-348.

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Siderophore Conjugates of Daptomycin are Potent Inhibitors of

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