A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling

Inspired by natural sideromycins, the conjugation of antibiotics to siderophores is an attractive strategy to facilitate “Trojan horse” delivery of antibiotics into bacteria. Genome analysis of a soil bacterium, Dactylosporangium fulvum, found a “hybrid” biosynthetic gene cluster responsible for the production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containing siderophore named chlorodactyloferrin. While both of these natural products were synthesized independently, analysis of the culture supernatant also identified a conjugate of both molecules. We then found that the addition of ferric iron to purified chlorodactyloferrin and pyridomycin instigated their conjugation, leading to the formation of a covalent bond between the siderophore-catechol and the pyridomycin-pyridine groups. Using model reactants, this iron-based reaction was found to proceed through a Michael-type addition reaction, where ferric iron oxidizes the siderophore-catechol group into its quinone form, which is then attacked by the antibiotic pyridyl-nitrogen to form the catechol–pyridinium linkage. These findings prompted us to explore if other “cargo” molecules could be attached to chlorodactyloferrin in a similar manner, and this was indeed confirmed with a pyridine-substituted TAMRA fluorophore as well as with pyridine-substituted penicillin, rifampicin, and norfloxacin antibiotic analogues. The resultant biomimetic conjugates were demonstrated to effectively enter a number of bacteria, with TAMRA–chlorodactyloferrin conjugates causing fluorescent labeling of the bacteria, and with penicillin and rifampicin conjugates eliciting antibiotic activity. These findings open up new opportunities for the design and facile synthesis of a novel class of biomimetic siderophore conjugates with antibiotic activity.

The progress of reactions was routinely monitored by thin layer chromatography (TLC) using Merck commercial aluminum sheets coated with silica gel 60 F254.Visualization was achieved by monitoring fluorescence under UV light at 254 and 365 nm.Flash column chromatography was performed using prepacked silica gel cartridges (SiO2, 30-50 μm average diameter) and Interchim PuriFlash XS 420 system.UV detection at 250 nm and 280 nm was used to direct collections of the relevant fractions.
NMR spectra were recorded on Bruker Avance III300 spectrometer equipped with a 5 mm BBO (X-1 H) probe or a Bruker Avance IIIHD 600 equipped with a 5 mm cryogenic QCI ( 1 H/ 2 H/ 13 C/ 15 N/ 19 F) probe.The 1 H and 13 C spectra were referenced to the signals of residual organic solvents as internal references, to the DSS trimethylsilyl signal (0.00 ppm) for aqueous solutions and to the residual TFA signal for 19 F spectrum of 28.Indirect referencing was used for 15 N. Chemical shifts (δ) are in parts per million (ppm) downfield from tetramethylsilane (TMS).NMR coupling constants (J) are reported in Hertz (Hz), and splitting patterns are indicated as follows: s (singlet), br (broad singlet), d (doublet), dd (doublet of doublets), m (multiplet).Yields refer to chromatographically pure compounds as determined by TLC (single spot) or HPLC.Electrosynthesis was carried out on IKA ElectraSyn 2.0 potentiostat using the following conditions: 0.1 m NaOAc in DI water/acetonitrile (1:1, v/v), Ag/Ag+ as reference electrodes, C-glassy working electrode, and C-glassy counter electrode.Deionized water was prepared with a Milli-Q water purification system to a resistivity of 17 MΩ•cm.

Analytical mass spectrometry (UHPLC-MS)
Analytical (non-high resolution) spectrometry analysis (UHPLC-MS) were performed on an Ultimate 3000 UHPLC system, coupled with a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific).Chromatographic separation was achieved using an Acquity UPLC Peptide BEH C18-column (300Å, 1.7 µm, 2.1 mm x 100 mm).Mobile phase system was composed of solvent A (H2O, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), typically run through a linear gradient from 0% to 100% of solvent B in 10 min.Elution of compounds was monitored by UV absorbance at 215 nm and 254 nm, and by mass spectrometry electrospray ionization.

High-resolution Mass spectrometry
High resolution mass spectrometry of purified compounds was performed by the ARIADNE-ADME platform (Institut Pasteur de Lille, France), using a quadrupole time-of-flight (TOF) LCT Premier XE mass spectrometry machine (Waters).

HPLC purification
Compounds were purified with a Semi-preparative RP-HPLC systems PLC 2020 (Gilson) using either a semipreparative XBridge™ Peptide BEH C18-column (10 x 250 mm, 130 Å, 5 micron) or preparative XBridge™ Peptide BEH C18-OBDTM column (19 x 150 mm, 130 Å, 5 micron) from Waters.Compounds were eluted using the same mobile phase system as described above for UHPLC-MS.The corresponding fractions were assayed by analytical UHPLC-MS to determine the molecular weights and purities, and fractions with the desired characteristics were pooled together.Acetonitrile was evaporated and the aqueous solution was lyophilized to obtain the compound of interest.

Comp. 11 (4-chloro-2,3-dihydroxybenzoyl-glycinate methyl ester)
The procedure for preparing 4-chloro-2,3-dioxosulfinylbenzoyl chloride was identical to that of 2,3dioxosulfinylbenzoyl chloride 5 .Under nitrogen atmosphere, a solution of freshly prepared 4-chloro-2,3dioxosulfinylbenzoyl chloride (2 g, 7.94 mmol) in anhydrous DCM (20 mL) was cooled to 0 C.This solution was then added dropwise, through a cannula, to a stirred mixture of triethylamine (9 mL, 64.53 mmol), DMAP (200 mg, 1.64 mmol) and glycine methyl ester hydrochloride (2 g, 15.93 mmol) in anhydrous DCM (40 mL) at 0 C (icewater bath).The reaction mixture was allowed to gradually warm to room temperature while stirring for 9 h.The volatiles were removed by rotary evaporation, and the obtained residue was dissolved in ethyl acetate (100 mL).The organic layer was then washed with 5% citric acid (2 × 30 mL), brine (1 × 30 mL), dried over magnesium sulphate, filtered, and concentrated under reduced pressure.Subjection of the obtained residue to flash chromatography (silica gel, DCM/MeOH/AcOH in 96/3/1, v/v/v) and concentration of the relevant fractions under reduced pressure afforded the title compound as an off-white solid.Yield: 900 mg (44%); m.p. 156-158 C.Under nitrogen atmosphere, to an ice-cold solution of catechol 5 (20 mg, 0.1 mmol) and 3-methylpyridine (40 µL, 0.42 mmol) in acetonitrile (5 ml) under vigorous stirring iron trichloride hexahydrate (80 mg, 0.29 mmol) was added in one portion.A sudden change of the color to intense blue was observed.The reaction mixture was stirred at room temperature for 12 h.The reaction mixture was concentrated by rotary evaporation, and the residue was dissolved in a minimum amount of methanol/water mixture (1:1, v/v) and purified using Sephadex LH-20 with methanol/water (8:2, v/v).Yield as a red solid (iron salt): 32 mg (59%).The obtained product was further purified by depositing the product on a C18-column and eluting first with a 5% EDTA solution of followed by wateracetonitrile gradient.To replace traces of complexed iron, an analytical amount of the product was dissolved in 1M solution of GaCl3 and purified using preparative HPLC chromatography with water and acetonitrile as eluents.HRMS for 16a: formula: C14H13ClNO4+, calculated: 294.0533, found: 294.0539.

Comp. 16c (silver oxide oxidation)
The synthetic procedure was carried by following a similar literature protocol 7 .A solution of 5 (20 mg, 0.1 mmol) in dry DMF (5 mL) was stirred with Ag2O (91 mg, 0.39 mmol) for two hours at 0 C.Formation of the respective oquinone was confirmed by LC-MS.Using a syringe-fitted 200 nm Teflon filter, the dark-green solution was rapidly filtered into a mixture of 3-methylpyridine (40 µL, 0.41 mmol), MeOH (5 mL) and acetic acid (60 µL, 1 mmol).The reaction mixture was stirred at 0 C for 1 hr and then allowed to warm to room temperature for 1 hr.The organic solvents were removed by rotary evaporation.Diethyl ether (10 mL) was added to the obtained residue.The resulting precipitate was washed and centrifuged several times with fresh portions of diethyl ether.The residue was then dissolved with 5 molar equivalents of hydrochloric acid in DI water (20 mL) and purified by preparative HPLC with water and acetonitrile (with 0.1% TFA) as eluents to give a white solid after lyophilization.Yield: 30 mg (92%).

Comp. 17a (FeCl3 oxidation)
Formation of the title product was confirmed on the analytical scale and checked by LC-MS using catechol 6 (10 µL, 10 mM soln in MeOH), 3-methylpyridine (10 µL, 10 mM soln in MeOH) and iron trichloride (10 µL, 100 mM soln in DI water) as starting compounds and following the same procedure as in the case of 16a.Formation of the title product 17a was confirmed by LC-MS.

Comp. 17b (iodine oxidation)
Using catechol 6 (303 mg, 1.80 mmol), 3-methylpyridine (350 µL, 3.61 mmol) and iodine (732 mg, 2.88 mmol) as starting compounds, the title compound was prepared as a brownish solid according to the method used to A reduced version of 10% Pd/C catalyst was obtained by applying H2 pressure (ca. 2 atm) to the Pd catalyst (100 mg) in a mixture of methanol/ethanol (3:1, v/v) for 1 h in a 50 mL two-chamber hydrogen generation apparatus ("H-tube") 8 .The pressure was carefully released, and solid 18 b (70 mg, 0.18 mmol) was added to the suspension in one portion under nitrogen.The reaction mixture was cooled down with ice-water, and the hydrogen pressure was reapplied.The reaction mixture was stirred vigorously and gradually warmed to room temperature over a period of 16 h while under H2 pressure.The hydrogen pressure was released, and the reaction mixture was transferred to a 50 mL plastic tube using methanol.The obtained suspension was centrifuged, and the supernatant was collected by decantation.The washing procedure was repeated two more times using methanol to wash the catalyst.The combined organic solution was concentrated under vacuum.The residue was extracted into ethyl acetate (100 mL), washed with aqueous sodium bicarbonate (2 × 20 mL), dried (MgSO4), and concentrated by rotary evaporation.The obtained residue was resuspended in DMSO/water mixture (1:1, v/v) and applied to a C18-column.A water/acetonitrile (with 1% TFA) linear gradient system was employed to provide a cherry-like gum after lyophilization of the respective fractions; yield: 33 mg (57%).

Comp. 19a (FeCl3 oxidation)
Formation of the title product was confirmed on the analytical scale by LC-MS using catechol 5 (10 µL, 10 mM soln in MeOH), 4-methylpyridine (10 µL, 10 mM soln in MeOH) and iron trichloride (10 µL, 100 mM soln in DI water) as starting compounds and following the same procedure as in the case of 17a.

Comp. 19b (iodine oxidation) 18f 18b
Comp. 23-2 (723 mg) was hydrogenated over 10% palladium on carbon (ca.0.2 g) in a mixture of EtOH/EtOAc for 16 h using in situ generated hydrogen in a 20 mL Coware apparatus 5 .After completion, the catalyst was removed by filtration through Celite and the filtrate was evaporated under vacuum.The obtained residue was purified using a gradient elution from water to 100% acetonitrile, both with 0.1% TFA.Yield: 162 mg (21%) of the title comp.23 as a white solid.HRMS for 23: formula: C15H18N3O4S+, calculated: 336.09, found: 336.17.