Synthesis, Characterization, and in vitro Testing of a Bacteria-Targeted MR Contrast Agent

Abstract

A bacteria-targeted MR contrast agent, Zn-1, consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a Gd^III chelate has been synthesized and characterized. In vitro studies with S. aureus and E. coli show that Zn-1 exhibits a significant improvement in bacteria labeling efficiency vs. control. Studies with a structural analogue, Zn-2, indicate that removal of one Zn-dpa moiety dramatically reduces the agent's affinity for bacteria. The ability of Zn-1 to significantly reduce the T₁ of labeled vs. unlabeled bacteria, resulting in enhanced MR image contrast, demonstrates its potential for visualizing bacterial infections in vivo.

Keywords: Bacteria labeling; Imaging agents; Magnetic resonance imaging; Medicinal chemistry; Zinc.

Figure 1

Two bacteria-targeted Zn-dpa domains were conjugated to a macrocyclic Gd^III chelate to develop a bacteria-targeted MR probe. The probe's affinity for bacteria is due to the electrostatic attraction between the anionic bacteria membrane and the positive charge of the Zn-dpa moieties.

Figure 2

Structures of complexes used for bacteria labeling studies. The Zn^II-binding domains of the complexes are shown in blue, the Gd^III chelate is shown in red. After the addition of Zn^II, Zn-1 and Zn-2 have charges of 4+ and 2+, respectively, while Zn-3 is charge neutral.^[16] The positive charge of Zn-1 and Zn-2 is expected to enhance the complex's affinity for anionic bacterial cell membranes. Zn-3 was used as a control for low-affinity binding.

Figure 3

Aromatic region of 1-Eu COSY spectrum (60 °C, DMSO) used to assign peaks of the Zn^II binding domains of 1. The addition of Zn^II produces significant changes in the chemical shift of the protons on the Zn^II binding domain until a Zn^II:1-Eu ratio of 2 is reached.

Figure 4

Top: aromatic region of ¹H NMR spectrum of Eu-1 with increasing amounts of Zn^II. Bottom: Chemical shift of peaks c, e, f, and d (see Figure 3) as a function of Zn^II/Eu-1 ratio. It is clear that the peaks of the Zn^II binding domain shift only until a Zn^II/Eu-1 ratio of 2 is obtained, confirming that each Eu-1 binds two Zn^II ions.

Figure 5

In vitro cellular labeling of S. aureus and E. coli with Zn-1, Zn-2, and Zn-3. S. aureus (top) or E. coli (bottom) were incubated with increasing concentrations of Zn-1 (black bars), Zn-2 (gray bars), or Zn-3 (white bars) at room temperature in LB broth. Gd^III content was analyzed by ICP-MS and is represented as the total amount of Gd^III per sample. Data are represented as the means ±SEM.

Figure 6

S. aureus cultures were incubated with 300 μm Zn-1, Zn-2 or Zn-3. Cells were washed 3 times with LB-broth and resuspended in 1% agarose. Scale bar represents 300 μm. Top: T₁-weighted map was acquired at 7 T at 25 °C (T_R/T_E = 500/11 ms). Cells labeled with Zn-1 are clearly brighter than both unlabeled bacteria and bacteria treated with Zn-2 or Zn-3. Bottom: T₁ relaxation times of agarose suspensions of S. aureus were measured using a saturation recovery pulse sequence with an echo time (T_E) of 11 ms and repetition times (T_R) as indicated in the Experimental Procedures at 7 T and 1.41 T. Data are presented as the percent reduction in T₁ vs. bacteria incubated with LB media, washed, and suspended in agarose.

Scheme 1

Synthesis of the bacteria-targeted MR contrast agent 1 that contains two dipicolyl moieties, each of which is capable of binding one Zn^II ion to give the final Zn^II-bound complex an overall charge of 4+. The europium and terbium analogues were synthesized in a similar manner, using EuCl₃ and Tb(OAc)₃ in place of Gd(OAc)₃.