Re(I) carbonyl complexes: Multimodal platforms for inorganic chemical biology

TL;DR: Metal complexes are reviewed and potential as probes or drugs relying on their photophysical properties are reviewed, before focusing on their use as multimodal probes for the labelling and imaging of peptides and proteins.

About: This article is published in Coordination Chemistry Reviews.The article was published on 2017-11-15 and is currently open access. It has received 88 citations till now.

Summary

Introduction

• Fluorescence microscopy is one of the most widely used techniques for the visualization and study of biological processes.
• Fluorescence techniques also suffer from some drawbacks, such as photobleaching and photodamage of cells.
• IR spectroscopy and IR-imaging involve excitation in the vibrational levels.
• Nevertheless, IR imaging of live cells is possible and has been successfully performed [11] .
• Indeed, their use for correlative fluorescence and infrared imaging was recently exemplified which demonstrates their validity as Single Core Multimodal Probes for Imaging [10, [12] [13] [14] .

1.1. Spectroscopic properties of luminescent Re(I) tricarbonyl complexes

• Re(I) fac-tricarbonyl complexes bearing low energy π * orbitals (e.g. α-diimine or dipicolylaminederived ligands) display attractive luminescence properties and biocompatibility features .
• Modifications of the X ligand may also alter the luminescence properties of the complex.
• Excitation wavelength, on the other hand, is generally in the 340-360 nm range, which is not favorable for excitation of biological tissues.
• Interestingly, though, Re(I) tricarbonyl complexes bearing a bis-quinoline ligand could be imaged in cells by fluorescence microscopy with excitation at 488 nm [50, 51] .
• All other measurements were made in aerated solution or it was not specified if the measurement was in degassed acetonitrile. [a].

1.2. Re(I) tricarbonyl complexes with specific cell localization

• Owing to their luminescence properties, Re(I) tricarbonyl complexes have been imaged in biological contexts and in particular in cells.
• A thiolreactive chloromethyl group was, for instance, appended to a Re(I) tricarbonyl complex in order to immobilized the complex into the mitochondria through its reaction with reduced thiols [39] .
• A few years ago, Policar et al. developed Re(CO) 3 complexes for correlative imaging.
• Colocalisation studies with known markers of the different cell organelles showed that the complexes were found in the endoplasmic reticulum, but not in the Golgi nor in the mitochondria.
• Of note, they observed that the organic organelle stains could show dramatic photobleaching upon prolonged irradiation, whereas the complexes showed stable luminescence intensity throughout these photobleaching experiments.

1.3.1. CO releasing Re(CO)

• X complexes CO is endogenously produced through heme degradation by the heme oxygenase (HO) enzyme [56] .
• At low doses, CO was recently shown to play beneficial roles in various physiopathological situations such as inflammation and oxidative stress or during ischemia-reperfusion [57] [58] [59] .
• Both the frontier orbitals (HOMO 3σ and LUMO 2π) can interact with a d-orbital from a metal center to create a M-CO bond.
• Efforts have also been devoted to the development of photo-activable CO-RMS.
• The main limitation of this complex relies on the fact that high-energy light is required to promote CO release.

1.3.2. Rhenium based complexes for Photodynamic therapy (PDT)

• Photodynamic therapy relies on the use of a non toxic photosensitizer which, upon irradiation and in presence of dioxygen, will generate toxic species.
• This results in oxidative damage of cellular components and then induces cell death.
• There are only scarce examples of use of Re(I) complexes for this application.
• In both cases, the complexes were incubated on HeLa cells and were found cytotoxic after irradiation only, with IC 50 in the nanomolar range.
• As discussed later in this review (Section 2.2), Gasser et al also designed Re(CO) 3 complexes and peptide conjugates for PDT [73] .

1.4 Re(CO)x complexes for vibrational imaging

• Beside their interesting properties in the UV-visible region, as mentioned earlier, metal-carbonyl complexes present attractive infrared properties, with intense absorption bands in the 1800-2200 cm −1 range.
• The first use of these unique properties in a biological context was a Carbonyl Metallo-ImmunoAssay (CMIA) developed by Jaouen's group.
• More recently, Policar et al. have tagged a mestranol derivative 20 and showed that the labelled molecule could be imaged in MDA-MB-231 and MCF-7 breast cancer cell lines using synchrotron based FTIR microspectroscopy (SR-FTIR-MS), AFMIR and confocal Raman microscopy [10] .
• Interestingly, correlative imaging could also be performed using synchrotron-based UV microspectroscopy (SR-UV-MS), wide field and confocal fluorescence microscopies.
• The deeper penetration of IR radiation makes IR imaging more suitable for tissues imaging.

1.5. Re(CO) 3 as surrogates for 99m Tc(CO) 3

• Radiotracers and radiopharmaceuticals are widely used in nuclear medicine for diagnosis and treatment.
• Finally, non-radioactive isostructural Re(I) tricarbonyl complexes can be prepared and characterized without handling radioactive materials.
• Various proteins have been targeted, including Translocator Protein TSPO [82] , metallothioneins [83] , G Protein-Coupled Estrogen Receptor GPER/GPR30 [84] or Carbonic Anhydrase IX [85] .
• Peptides were also labelled with Re/Tc tricarbonyl complexes, generally in order to target specific cell lines or events [86] [87] [88] [89] .

2.1. Conjugation to single amino-acids and Single Amino-Acid Chelate (SAAC) strategy

• Single amino-acids have been derivatized with luminescent Re(CO) 3 complexes in order to incorporate them into peptides, or to study their photophysical properties as such in cuvette or in a cellular context [22, 43, 90, [95] [96] [97] .
• The study particularly focused on the use of the rhenium complex as photo-oxidant to generate tyrosyl radicals by exciting the MLCT.
• The amino acids were linked to the complex through the pyridine ligand, at either the meta (23) or para (24) position on the pyridine.
• Both SAAC and SAAC-Re could be incorporated by automated peptide synthesis into the formylated MLF peptide fMLF peptide (fMLF(SAAC-Re)G, 27, Figure 12 ), a peptide sequence targeting the formyl peptide receptor (FPR).

2.2. N-terminal labelling of peptide

• This was the case for the labeled peptide 29 that exhibited more pronounced differences in its cellular uptake between the cell lines than the parent complex, suggesting some selectivity in its uptake mechanism.
• Neither the parent complex 30 nor the Bombesin conjugate 32 displayed cytotoxicity in the dark, whereas conjugation to NLS sequence increased the cytotoxicity of the compound 31.
• The goal of the study was to design Re/Tc tricarbonyl complexes that could be addressed selectively to the nucleus of cancer cells.
• More generally, quantification using fluorescence can be difficult due to the dependence of quantum yield with the environment.

3. Labelling of proteins with Re(CO) 3 complexes

• As emphasized in this review, Re(CO) 3 complexes are interesting IR and luminescent probes for bioimaging.
• This has motivated the development of Re(CO) 3 complexes suitable for the labelling of more complex biomolecules such as proteins.
• These bio-reactive Re complexes are the first step towards the multimodal imaging of proteins in cells.

3.1. In vitro covalent labelling of proteins

• Luminescent Re(I) tricarbonyl complexes have been functionalized with thiol-or amine-reactive groups in order to label peptides or proteins in vitro.
• Between four and five cystein residues were labelled, over the 35 cystein residues BSA and HSA contain.
• The position, width, lifetimes of signal of IR probes may for instance give information on the local conformation and dynamics of a protein or on the local electric field near a catalytic site.
• An interesting alternative takes advantage of the reactivity of aqua Re(I) tricarbonyl complexes with amino acids side chains such as histidine imidazole [86, [122] [123] [124] [125] .

3.2. Non covalent labelling of proteins

• The protein labelling methods described above, although useful for proof of concept and for in vitro studies of purified proteins, are difficult to apply for the selective labelling of proteins in a complex cell environment.
• To the best of their knowledge, fluorescence cell imaging of proteins with Re(I) tricarbonyl has only been performed using non-covalent methods, i.e. by labelling a specific ligand of the protein of interest with a luminescent Re(I) tricarbonyl complex.
• Biotinylated complexes 4 and 5 were designed as cross-linkers for avidin.
• Similarly, Re(I) tricarbonyl complexes were appended to estrogen derivatives to target estrogen receptors [9, 20] or to indoles to target indole-binding proteins [20] .
• Doyle et al. conjugated luminescent Re(I) bisquinoline complexes to folic acid and to vitamin B12 to target folate receptor and cubilin receptor, respectively [50, 97] .

Conclusion

• Owing to their unique photophysical properties, Re(I) metal carbonyl complexes have been extensively used in biological contexts as probes, 99m.
• Tc surrogates, drugs and as photosensitizers for photodynamic therapies.
• And in particular for the imaging of peptides and proteins.the authors.
• Several strategies aiming at labelling peptides and proteins with Re complexes have been highlighted.
• No rational have clearly emerged so far.

Figures

Table 1. Photophysical properties of selected Re(I) tricarbonyl complexes in acetonitrile at 298K.

Frequently asked questions

Q1. What is the effect of a nucleophilic substitution on the label?

Upon reaction with a cystein side chain, a nucleophilic substitution occurs leading to the removal of the methanethiosulfonate group and the formation of a disulfide bond between the label and the protein. 

Q2. What are the contributions mentioned in the paper "Re(i) carbonyl complexes: multimodal platforms for inorganic chemical biology" ?

Bio-imaging, by enabling the visualization of biomolecules of interest, has proved to be highly informative in the study of biological processes. Here the authors review their applications and potential as probes or drugs relying on their photophysical properties, before focusing on their use as multimodal probes for the labelling and imaging of peptides and proteins. 

Q3. What was the effect of the IR modality on the peptide conjugate?

In skin biopsies, the IR modality also enabled them to quantify the penetration of the peptide conjugate into skin, while the fluorescence modality could be used to compare its localization with those of nuclei, using common fluorescence stain like DAPI. 

Q4. Why have they been imaged in biological contexts?

Owing to their luminescence properties, Re(I) tricarbonyl complexes have been imaged in biological contexts and in particular in cells. 

Q5. What are the main uses of Re(I) tricarbonyl complexes?

Beside their use as inert luminescent probes, Re(I) tricarbonyl complexes are attracting an increasing interest as potential photoresponsive drugs. 

Q6. What is the effect of the asymmetric complexes on the luminescence properties?

The resulting asymmetric complexes had enhanced luminescence properties, in particular longer emission wavelength (>600 nm in water) and good quantum yields in water for such complexes (around 0.15-0.2%). 

Q7. What biomolecules could be labelled and imaged?

Other biomolecules could also be labelled and imaged, e.g. biotin [100,101], folic acid [97] or thymidine derivatives [22,51,102]. 

Q8. Why is IR imaging more difficult than fluorescence?

Due to the low resolution of optical IR microscopy (see above), IR imaging of cells can be more challenging than fluorescence microscopy. 

Q9. How was the internalization of the complex observed?

After incubation in human prostatic carcinoma cells (PPC-1), the internalization of the complex into the cytoplasm was observed by fluorescence microscopy. 

Q10. Why do they have an increasing interest for fluorescence bioimaging?

Due to their photophysical features, Re(I) tricarbonyl have raised an increasing interest for fluorescence bioimaging applications. 

Q11. What is the peptide that was observed by fluorescence microscopy?

The Re(CO)3-labelled peptide could be observed by fluorescence microscopy in human leukocytes, and its localization was consistent with previous reports on chemotactic peptides (i.e. the peptide localized first at the membrane at 4°C and then into the cytoplasm when the temperature was increased to room temperature). 

Q12. How do the authors label proteins with Re(I) tricarbonyl?

To the best of their knowledge, fluorescence cell imaging of proteins with Re(I) tricarbonyl has only been performed using non-covalent methods, i.e. by labelling a specific ligand of the protein of interest with a luminescent Re(I) tricarbonyl complex. 

Q13. What is the common use of Re(I) complexes?

Although Ru(II) polypyridyl species have been shown to be efficient photosensitizers for 1 O2, there are only scarce examples of use of Re(I) complexes for this application. 

Q14. What was used to immobilize the complex into the mitochondria?

A thiolreactive chloromethyl group was, for instance, appended to a Re(I) tricarbonyl complex (1, R=Cl, Figure 3) in order to immobilized the complex into the mitochondria through its reaction with reduced thiols [39]. 

Q15. What is the possibility of using these complexes as biological surfaces?

Some of these complexes could even be embedded in a flexible polymeric silesquioxane (SSO) film while retaining their photophysical features, which opens up the possibility to use such films as biological or microbial-photoinactivating surfaces [70]. 

Q16. What are the common uses of Re(I) metal carbonyl complexes?

ConclusionOwing to their unique photophysical properties, Re(I) metal carbonyl complexes have been extensively used in biological contexts as probes, 99m 

Q17. What is the IR-visible region of metal-carbonyl complexes?

Beside their interesting properties in the UV-visible region, as mentioned earlier, metal-carbonyl complexes present attractive infrared properties, with intense absorption bands in the 1800–2200 cm −1 range. 

Q18. What are the properties of the re(i) fac-tricarbonyl complexe?

Re(I) fac-tricarbonyl complexes bearing low energy π* orbitals (e.g. α-diimine or dipicolylaminederived ligands) display attractive luminescence properties and biocompatibility features (Figure 1). 

Q19. What was the effect of phosphorylation of the internalized complex on mitochondria?

To explain this, it was postulated that phosphorylation of the internalized complex could lead to a thiol-reactive moiety, although no experimental evidence for this hypothesis was obtained.