Zinc(II) coordination complexes as membrane-active fluorescent probes and antibiotics

Abstract

Molecular probes with zinc(II)-(2,2'-dipicolylamine) coordination complexes associate with oxyanions in aqueous solution and target biomembranes that contain anionic phospholipids. This study examines a new series of coordination complexes with 2,6-bis(zinc(II)-dipicolylamine)phenoxide as the molecular recognition unit. Two lipophilic analogues are observed to partition into the membranes of zwitterionic and anionic vesicles and induce the transport of phospholipids and hydrophilic anions (carboxyfluorescein). These lipophilic zinc complexes are moderately toxic to mammalian cells. A more hydrophilic analogue does not exhibit mammalian cell toxicity (LD(50) >50 microg mL(-1)), but it is highly active against the Gram-positive bacteria Staphylococcus aureus (MIC of 1 microg mL(-1)). Furthermore, it is active against clinically important S. aureus strains that are resistant to various antibiotics, including vancomycin and oxacillin. The antibiotic action is attributed to its ability to depolarize the bacterial cell membrane. The intense bacterial staining that was exhibited by a fluorescent conjugate suggests that this family of zinc coordination complexes can be used as molecular probes for the detection and imaging of bacteria.

Figure 1

CF transport from vesicles induced by addition of zinc complex 6 (1 µM) at 50 s to vesicles in 5 mM TES (pH 7.4) buffer with either 100 mM NaCl (◆) or 75 mM Na₂SO₄ (■), followed by vesicle lysis with Triton X-100 at 300 s. (left) Zwitterionic vesicles composed of POPC; (right) Anionic vesicles composed of POPG:POPC (1:9).

Figure 2

Dependence of CF release on zinc complexation to form transport active 6. Apo-6 (1 µM) was added at 50 s to POPC vesicles (25 µM). Inside the vesicles is 5 mM TES, 50 mM CF, 100 mM NaCl (pH 7.4) buffer; outside the vesicles is 5 mM TES, 100 mM NaCl (pH 7.4) buffer. Zn(NO₃)₂ (0 µM (■), 0.25 µM (×), 0.5 µM (●), 1 µM (◆) and 2 µM (◇)) was added at 100 s and the vesicles lysed with Triton X-100 at 750 s.

Figure 3

Initial CF flux at different concentrations of 6. Vesicles [25 µM lipid, POPC: cholesterol 7:3, encapsulating 5 mM TES, 100 mM NaCl, 50 mM CF (pH 7.4) buffer] were dispersed in 5 mM TES, 100 mM NaCl (pH 7.4) buffer and treated with varying concentrations of 6. The initial flux is the % CF transport at 20 s after addition of 6. The error bars reflect the averages of three independent experiments.

Figure 4

Fluorescence micrographs of E. coli (top row) and S. aureus (bottom row) cells, after treatment with zinc complex 7. The right panels are magnified to show single cells undergoing binary fission. Scale bar in each image represents 2 µm.

Figure 5

Membrane depolarization of S. aureus cells induced by 3 at 10 µg/mL (■), 1 µg/mL (▲), and buffer control (●).

Scheme 1

Association of zinc complex 2 with phosphate anion

Scheme 2

Phospholipid translocation

Scheme 3