Bioorthogonal Labeling, Bioimaging, and Photocytotoxicity Studies of Phosphorescent Ruthenium(II) Polypyridine Dibenzocyclooctyne Complexes

Abstract

The synthesis, characterization, photophysics, lipophilicity, and cellular properties of new phosphorescent ruthenium(II) polypyridine complexes functionalized with a dibenzocyclooctyne (DIBO) or amine moiety [Ru(N^N)2 (L)](PF6 )2 are reported (L=4-(13-N-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonyl) amino-4,7,10-trioxa-tridecanyl-aminocarbonyl-oxy-methyl)-4'-methyl-2,2'-bipyridine bpy-DIBO, N^N=2,2'-bipyridine bpy (1 a), 1,10-phenanthroline phen (2 a); L=4-(13-amino-4,7,10-trioxa-tridecanylaminocarbonyl-oxy-methyl)-4'-methyl-2,2'-bipyridine bpy-NH2 , N^N=bpy (1 b), phen (2 b)). The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction of the DIBO complexes 1 a and 2 a with benzyl azide were studied. Also, the DIBO complexes 1 a and 2 a can selectively label N-azidoglycans located on the surface of CHO-K1 and A549 cells that were pretreated with 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine (Ac4 ManNAz). Additionally, the intracellular trafficking and localization of these biomolecules were monitored using laser-scanning confocal microscopy. Interestingly, the biolabeling and cellular uptake efficiency of the DIBO complexes 1 a and 2 a were cell-line dependent, as revealed by flow cytometry and ICP-MS. Furthermore, the complexes showed good biocompatibility toward the Ac4 ManNAz-pretreated cells in the dark, but exhibited photoinduced cytotoxicity due to the generation of singlet oxygen.

Structural Manipulation of Ruthenium(II) Polypyridine Nitrone Complexes to Generate Phosphorogenic Bioorthogonal Reagents for Selective Cellular Labeling

“…The electronic absorption spectra and data of complexes 1-5 in MeOH are presented in Figure 1a and Ta ble S1 (in the Supporting Information), respectively.A ll the complexes exhibited intense absorption bands at % 250-339nma nd weakerb ands at % 392-469nm, which are attributable to spin-allowed intraligand ( 1 IL) (p!p*) (diimine) and metal-to-ligand charge-transfer ( 1 MLCT) (dp(Ru)!p*(diimine)) transitions, respectively. [13,15] Upon irradiation, all the complexes displayed very weak orange-red emission.T he photophysical data of the complexes are summarized in Ta ble 1a nd the emission spectra in de-gassedM eOH at 298 Ka re shown in Figure 1b.T he complexes show ab road and featureless emission band at % 599-660 nm in fluid solutionsa t2 98 Ka nd their emission maxima were significantlyb lueshifted upon cooling to 77 K, suggestive of spinforbidden 3 MLCT (dp(Ru)!p*(diimine)) character in their emissive states. [13,15,16] Importantly,t he complexes showedm uch lower emissionq uantum yields than common ruthenium(II) polypyridine complexes, [13, 15f,g] which is ac onsequence of efficient quenching associatedw ith the rapid C=Ni somerization of the nitrone moiety.…”

“…[13,15] Upon irradiation, all the complexes displayed very weak orange-red emission.T he photophysical data of the complexes are summarized in Ta ble 1a nd the emission spectra in de-gassedM eOH at 298 Ka re shown in Figure 1b.T he complexes show ab road and featureless emission band at % 599-660 nm in fluid solutionsa t2 98 Ka nd their emission maxima were significantlyb lueshifted upon cooling to 77 K, suggestive of spinforbidden 3 MLCT (dp(Ru)!p*(diimine)) character in their emissive states. [13,15,16] Importantly,t he complexes showedm uch lower emissionq uantum yields than common ruthenium(II) polypyridine complexes, [13, 15f,g] which is ac onsequence of efficient quenching associatedw ith the rapid C=Ni somerization of the nitrone moiety. [10] Interestingly,t he bpy-nitrone-Me complex 1 was less emissive than its bpy-nitrone-Ph counterpart complex 5,i ndicating that N-methyl nitrone quenches the emission of ruthenium(II) complexesm ore significantly than Nphenylnitrone.…”

“…Since bioorthogonal cell surfacel abeling can be viewed as ap romising strategy to facilitate the cellular uptake of membrane-impermeable fluorophores [25] and transition-metal complexeso fh igh formal cationic chargea nd polarity, [13] we herein propose an explanation in Scheme3 for the aforementioned results. Considering the very low uptake of complexes 1-3 and 5 by BCN-C10-untreated cells, it is reasonable that these complexesa re hardly permeable acrossc ell membranes, whichi s actuallya dvantageous for labeling BCN-C10 at the surface of pretreated cells( Scheme3,b ottom left).…”

Luminescent Ruthenium(II) Polypyridine Complexes for a Wide Variety of Biomolecular and Cellular Applications

“…Our group has developed complexes 19 and 20 (Chart 5), luminescent ruthenium(II) polypyridine complexes functionalized with a dibenzocyclooctyne unit capable of strainpromoted alkyne−azide cycloaddition. 28 (CHO)-K1 and human lung adenocarcinoma (A549) cells were grown with or without pretreatment of 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine. The complexes are able to selectively label the N-azidoglycans located on the cell surface of pretreated CHO-K1 and A549 cells.…”

Synthesis of a Novel Fluorescent Ruthenium Complex with an Appended Ac4GlcNAc Moiety by Click Reaction

“…Microscopic inspection is a powerful and widely used tool for high-resolution and non-invasive imaging of target molecules in their native environments [ 12 ]. With spectroscopic probes such as alkyne-functionalized rhodamine or 1,8-naphthalimide derivatives, imaging of O -GlcNAc proteins in their native environment is feasible [ 13 , 14 , 15 , 16 ].…”