Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy

We propose to monitor molecular vibrations to identify metal ion–ligand complexation by means of Raman spectroscopy, which has been applied to track vibrational modes of molecules and to obtain a structural fingerprint. We prepared ligand molecules for Zn2+ ion complexation with a dipycolylaminoethyl aniline (DPEA) skeleton and phenylacetylene unit as the Raman tag which showed a typical band around 2200 cm−1. Among the labeled ligands synthesized in this study, A-DPEA showed a strong band attributed to the acetylene unit at 2212 cm−1, while the addition of Zn2+ ion resulted in a band shift to 2220 cm−1 due to complex formation. The addition of other metal ions and titration experiments showed that A-DPEA bound with Zn2+ selectively with a dissociation constant (Kd) that was estimated to be 0.22 μM. We also conducted cellular experiments and found that complexation between A-DPEA and Zn2+ also occurred in cells, with a shift in the Raman signal of the ligand from 2212 to 2215 cm−1. Thus, complex formation of the metal ion was identified by monitoring the Raman band shift.


Measurement of Raman spectra of A-DPEA and B-DPEA.
A-DPEA or B-DPEA (1 mM) and ZnCl 2 (0-10 mM) in 2 ml of aqueous methanol solutions (methanol : water = 2 : 1, v/v, 100 mM HEPES, pH 7.0) were prepared. The solution was dropped on a glass slide, then a cover glass was placed on the sample. Raman spectra were measured using 532 nm excitation.

Dissociation of zinc ion from the complex by addition of EDTA.
To the complex consisted of A-DPEA-Zn 2+ (1 mM) in aqueous methanol solution (1:1), EDTA (2 mM) was added and then Raman spectra was measured using 532 nm excitation.
Cell culture. Human fibroblasts fibrosarcoma cell, HT1080, was cultured in Roswell Park Memorial Institute media (RPMI1640) supplemented with 10 % FBS, 1 % penicillin-streptomycin, and sodium pyruvate. The cells were maintained at 37 ˚C in 5% CO 2 / 95% air and were kept in a logarithmic growth phase by routine passages every 2−3 days. Prior to the use of cells, the densities of cells were determined using a hemocytometer.
Experiments using cell lysate. A549 cells and HT1080 cells were cultured in 10 dishes (90 % confluent in 100 mm dishes) and washed twice with ice-cold PBS(-). The cell lysate was then harvested by the freeze-thaw process. A-DPEA was dissolved in the cell lysate containing 1% DMSO and then Raman spectra were measured using 532 nm excitation.
Measurement of Raman spectra in living cells. HT1080 cells (5.0 × 10 3 cells) in the medium of RPMI1640 were seeded onto grass dish. Glass dish was maintained at 37 ˚C in 5% CO 2 / 95% air incubator for 24 h. HT1080 cells were incubated with 0.1 mM A-DPEA in RPMI1640 (DMSO 1%) for 30 min. After incubation, the cells were washed with PBS, and then incubated with 5 mM ZnCl 2 in RPMI1640 for 30 min. The cells were washed with PBS, added fresh RPMI1640 (without phenol red) and then Raman spectra were measured by Renishaw inVia Raman Microscope (Renishaw plc, UK). Raman spectra were measured using 532 nm excitation.