Different Quenching Effect of Intramolecular Rotation on the Singlet and Triplet Excited States of Bodipy

It is well-known that the fluorescence of a chromophore can be efficiently quenched by the free rotor effect, sometimes called intramolecular rotation (IMR), i.e. by a large-amplitude torsional motion. Using this effect, aggregation induced enhanced emission (AIE) and fluorescent molecular probes for viscosity measurements have been devised. However, the rotor effect on triplet excited states was rarely studied. Herein, with molecular rotors of Bodipy and diiodoBodipy, and by using steady state and time-resolved transient absorption/emission spectroscopies, we confirmed that the triplet excited state of the Bodipy chromophore is not quenched by IMR. This is in stark contrast to the fluorescence (singlet excited state), which is significantly quenched by IMR. This result is rather interesting since a long-lived excited state (triplet, 276 μs) is not quenched by the IMR, but the short-lived excited state (singlet, 3.8 ns) is quenched by the same IMR. The unquenched triplet excited state of the Bodipy was used for triplet–triplet annihilation upconversion, and the upconversion quantum yield is 6.3%

Interconversion of 7α-hydroxy-, 7β-hydroxy- and 7-keto-DHEA by 11β-HSD1 in intact cells.

<p>HEK-293 cells transfected with a plasmid for 11β-HSD1 and either a control vector or a plasmid for H6PDH were incubated with 1 µM of 7α-hydroxy-DHEA (7α-OH-DHEA) (<i>A</i>), 7β-hydroxy-DHEA (7β-OH-DHEA) (<i>B</i>) or 7-keto-DHEA (<i>C</i>) in absence or presence of 11β-HSD1 inhibitor T0504, followed by determination of C7-oxygenated DHEA metabolites after 3 h (<i>A–C</i>). Alternatively, cells were incubated with either 7α-hydroxy-, 7β-hydroxy- or 7-keto-DHEA (7-oxo-DHEA) for 24 h, followed by determination of C7-oxygenated DHEA metabolites as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000561#s4" target="_blank">Materials and Methods</a> (<i>D</i>). Data are given as percentage of initially supplied substrate. A representative experiment from three independent transfections is shown. Hatched bars, 7α-hydroxy-DHEA; filled bars, 7β-hydroxy-DHEA, open bars, 7-keto-DHEA.</p

Minimized structures of DHEA analogs in 11β-HSD1.

<p><i>A</i>, the D ring is oriented towards the interior of 11β-HSD1. <i>B</i>, the A ring is oriented towards the interior of 11β-HSD1.</p

7-oxygenated neurosteroids compete with 11β-HSD1-dependent cortisone reduction

<p>11β-HSD activities were determined in lysates of HEK-293 cells expressing recombinant enzyme as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000561#s4" target="_blank">Materials and Methods</a>. IC₅₀ values are in µM. Data represent mean±S.D. from four independent experiments.</p

Control of the steady state ratio of cortisone to cortisol by 11β-HSD1 and H6PDH

<p>The ratio of cortisone to cortisol was measured after incubating HEK-293 cells expressing 11β-HSD1 or coexpressing 11β-HSD1 and H6PDH for 16 h in the presence of 200 nM of radiolabeled substrate. The effect of inhibitors was determined by coincubating cells with 20 µM of the corresponding compound. Data are given as percentage of total glucocorticoid and represent mean±SD, n = 4.</p

Superposition of 7β-hydroxy-DHEA on corticosterone in 11β-HSD1.

<p>In this model, the A ring of 7β-hydroxy-DHEA (7β-OH-DHEA) is oriented towards the interior of 11β-HSD1. The C7β-hydroxyl in 7β-hydroxy-DHEA superimposes nicely on the C11β-hydroxyl in corticosterone with equal predicted distances to C4 of the nicotinamide ring and the hydroxyl of the catalytic tyrosine.</p

Genetic analysis of the StAR gene.

<p>Upper panel, scheme of the identified mutations at the nucleotide (c.DNA) and protein (p.) level. Lower panel, family tree with individual electropherograms showing the novel StAR mutations.</p

Missense mutations identified in patients manifesting with non-classic StAR deficiency.

<p>*Mutation on partner allele is given in brackets (p.).</p><p>**Loss of function mutation manifesting clinically at birth with signs of classic StAR deficiency. Data given for comparison.</p><p>***<i>In vitro</i> activity (% of WT) is assessed by pregnenolone production (immunoassay) in COS cells transfected with expression vectors for wild-type or mutant StAR and F2 (the fusion protein P450 side-chain cleavage/adrenodoxin/adrenodoxin reductase). Note that data derive from different laboratories employing similar assay systems.</p

Exit of cholesterol from StAR as studied by steered MD simulations.

<p>StAR protein is shown as a ribbons model colored in a rainbow gradient from the amino terminus in blue to carboxy terminus in red. The A174–V179 loop that was observed to move and make way for the exit of cholesterol is shown in grey. Major amino acids involved in interaction of cholesterol with StAR and formation of cholesterol binding pocket are shown as stick models. The exit route of cholesterol observed during simulation is shown as a solid model in red. In case of S221-StAR a delay in exit of cholesterol was observed, potentially due to altered binding pattern caused by shift in H220 side chain and additional interactions with R188.</p

A closeup of the cholesterol binding pocket of StAR.

<p>After docking of cholesterol to both WT (A) and S221-StAR (B), we calculated potential residues interacting with cholesterol molecule during docking as well as exit of cholesterol from the binding cavity. In case of S221-StAR a loss of interaction with H220 was observed due to a shift in the H220 side chain. Cholesterol is shown as a ball and stick model in magenta. Amino acids interacting with cholesterol are shown as stick models.</p