Solid-Phase Synthesis as a Platform for the Discovery of New Ruthenium Complexes for Efficient Release of Photocaged Ligands with Visible Light

Ruthenium-based photocaging groups have important applications as biological tools and show great potential as therapeutics. A method was developed to rapidly synthesize, screen, and identify ruthenium-based caging groups that release nitriles upon irradiation with visible light. A diverse library of tetra- and pentadentate ligands was synthesized on polystyrene resin. Ruthenium complexes of the general formula [Ru­(L)­(MeCN)_<i>n</i>]^<i>m</i>+ (<i>n</i> = 1–3, <i>m</i> = 1–2) were generated from these ligands on solid phase and then cleaved from resin for photochemical analysis. Data indicate a wide range of spectral tuning and reactivity with visible light. Three complexes that showed strong absorbance in the visible range were synthesized by solution phase for comparison. Photochemical behavior of solution- and solid-phase complexes was in good agreement, confirming that the library approach is useful in identifying candidates with desired photoreactivity in short order, avoiding time-consuming chromatography and compound purification

Ruthenium Tris(2-pyridylmethyl)amine as an Effective Photocaging Group for Nitriles

Ruthenium­(II) tris­(2-pyridylmethyl)­amine (TPA) is an effective caging group for nitriles that provides high levels of control over the enzyme activity with light. Two caged nitriles were prepared, [Ru­(TPA)­(MeCN)₂]­(PF₆)₂ (<b>1</b>) and [Ru­(TPA)­(<b>3</b>)₂]­(PF₆)₂ (<b>2</b>), where <b>3</b> is the cathepsin K inhibitor Cbz-Leu-NHCH₂CN, and characterized by various spectroscopic techniques and mass spectrometry. Both <b>1</b> and <b>2</b> show the release of a single nitrile within 20 min of irradiation with 365 nm light. Complex <b>2</b> acts as a potent, photoactivated inhibitor of human cathepsin K. IC₅₀ values were determined for <b>2</b> and <b>3</b>. Enzyme inhibition for <b>2</b> was enhanced by a factor of 89 upon exposure to light, with IC₅₀ values of 63 nM (light) and 5.6 μM (dark)

Solid-Phase Synthesis as a Platform for the Discovery of New Ruthenium Complexes for Efficient Release of Photocaged Ligands with Visible Light

Ruthenium-based photocaging groups have important applications as biological tools and show great potential as therapeutics. A method was developed to rapidly synthesize, screen, and identify ruthenium-based caging groups that release nitriles upon irradiation with visible light. A diverse library of tetra- and pentadentate ligands was synthesized on polystyrene resin. Ruthenium complexes of the general formula [Ru­(L)­(MeCN)_<i>n</i>]^<i>m</i>+ (<i>n</i> = 1–3, <i>m</i> = 1–2) were generated from these ligands on solid phase and then cleaved from resin for photochemical analysis. Data indicate a wide range of spectral tuning and reactivity with visible light. Three complexes that showed strong absorbance in the visible range were synthesized by solution phase for comparison. Photochemical behavior of solution- and solid-phase complexes was in good agreement, confirming that the library approach is useful in identifying candidates with desired photoreactivity in short order, avoiding time-consuming chromatography and compound purification

Effects of Methyl Substitution in Ruthenium Tris(2-pyridylmethyl)amine Photocaging Groups for Nitriles

Four complexes of the general formula [Ru­(L)­(CH₃CN)₂]­(PF₆)₂, [L = TPA (<b>5</b>), MeTPA (<b>6</b>), Me₂TPA (<b>7</b>), and Me₃TPA (<b>8</b>)] [TPA = tris­[(pyridin-2-yl)­methyl]­amine, where methyl groups were introduced consecutively onto the 6-position of py donors of TPA, were prepared and characterized by various spectroscopic techniques and mass spectrometry. While <b>5</b> and <b>8</b> were isolated as single stereoisomers, <b>6</b> and <b>7</b> were isolated as mixtures of stereoisomers in 2:1 and 1.5:1 ratios, respectively. Steric effects on ground state stability and thermal and photochemical reactivities were studied for all four complexes using ¹H NMR and electronic absorption spectroscopies and computational studies. These studies confirmed that the addition of steric bulk accelerates photochemical and thermal nitrile release

Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

<div><p>The cysteine protease cathepsin B has been causally linked to progression and metastasis of breast cancers. We demonstrate inhibition by a dipeptidyl nitrile inhibitor (compound <b>1)</b> of cathepsin B activity and also of pericellular degradation of dye-quenched collagen IV by living breast cancer cells. To image, localize and quantify collagen IV degradation in real-time we used 3D pathomimetic breast cancer models designed to mimic the <i>in vivo</i> microenvironment of breast cancers. We further report the synthesis and characterization of a caged version of compound <b>1</b>, [Ru(bpy)₂(<b>1</b>)₂](BF₄)₂ (compound <b>2</b>), which can be photoactivated with visible light. Upon light activation, compound <b>2</b>, like compound <b>1</b>, inhibited cathepsin B activity and pericellular collagen IV degradation by the 3D pathomimetic models of living breast cancer cells, without causing toxicity. We suggest that caged inhibitor <b>2</b> is a prototype for cathepsin B inhibitors that can control both the site and timing of inhibition in cancer.</p></div

Changes to the electronic absorption of compound 2 (25 μM) in a 2% acetone aqueous solution at irradiation times, t_irr, of 0, 3, 5, 7, 10 and 15 min (λ_irr ≥ 395 nm); the * denotes the mono-aqua intermediate.

<p>Inset: t_irr = 0.0, 0.5, 1.5, and 3.0 min.</p

Synthesis of the ruthenium-caged, nitrile-based inhibitor 2.

<p>Synthesis of the ruthenium-caged, nitrile-based inhibitor 2.</p

The ruthenium complex <i>cis</i>-[Ru(bpy)₂(MeCN)₂](PF₆)₂ (3) used for caging of inhibitor 2 does not affect degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>(A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control, dark-exposed ruthenium complex and light-exposed ruthenium complex. (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures incubated with DMSO (negative control), dark-exposed ruthenium complex or light-exposed ruthenium complex. Data shown are from 3 independent experiments (48 fields); mean ± SD.</p

Uncaged inhibitor 1 reduces total and pericellular degradation, but not intracellular degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>Quantification of degraded collagen IV in entire 3D volume of MDA-MB-231 and Hs578T structures at 4 days of culture: total degraded collagen IV, black bars; pericellular degraded collagen IV, open bars; and intracellular degraded collagen IV, gray bars. DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor 1. Data shown are from 3 independent experiments (48 fields); * p < 0.05; mean ± SD.</p

Uncaged inhibitor 1 reduces degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>(A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control and cysteine protease inhibitors (middle: 5 μM each of CA074 + CA074Me; right: uncaged inhibitor 1). (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures exposed to DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor <b>1</b>. Data shown are from 3 independent experiments (48 fields); * ≤ 0.05; mean ± SD.</p