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. 2008;47(5):898-901.
doi: 10.1002/anie.200703743.

Probing inducible nitric oxide synthase with a pterin-ruthenium(II) sensitizer wire

Edith C Glazer  1 , Yen Hoang Le Nguyen, Harry B Gray, David B Goodin
Affiliations

Probing inducible nitric oxide synthase with a pterin-ruthenium(II) sensitizer wire

Edith C Glazer et al. Angew Chem Int Ed Engl. 2008.
No abstract available

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Figures

Figure 1
Figure 1
Reconstitution of substrate- and tetrahydrobiopterin-free iNOSheme with wire 6. iNOSheme (~10 μM) was incubated at 25 °C with the pterin analogue at the specified concentration in the presence of 1 mM DTT, and the absorbance spectrum was taken after 2 hours. The spectrum of the DTT-equilibrated sample (without wire) shows the formation of a bis-thiolate heme. Inset shows the binding curve obtained from titration data, plotting the fractional difference in absorption (ΔA/ΔAtotal) as a function of concentration.
Figure 2
Figure 2
The steady-state Ru(II) luminescence spectrum of 6 (10 μM, red) is quenched in the presence of equimolar iNOSheme (black); it recovers upon addition of 10 μM (blue) and 100 μM (green) 4AH4B.
Figure 3
Figure 3
The relative fraction of the fast phase of luminescence decay for compound 6 (taken as fraction bound) as a function of [iNOSheme]free. An estimate for the dissociation constant of 6 to iNOSheme was obtained by fitting the curve to a single-site binding model (Kd=3.8±1.4 μM), assuming that the saturation value of the fractional fast phase represents a 1:1 complex.
Figure 4
Figure 4
Structural model of wire 6 docked into iNOSheme.
Figure 5
Figure 5
Photoreduction of iNOSheme using wire 6. Illumination of the iNOSheme:wire complex under an atmosphere of CO causes an increase in absorption at 420 nm, consistent with the formation of the reduced CO-bound P-420 species (black line, 0 min, blue line, 30 min). 1 mM TMPD was used for the experiment; no spectral change was observed without TMPD, or without light. Chemical reduction with Na2S2O4 in the dark results in production of a combination of P-450 and P-420 species (red line).
Scheme 1
Scheme 1
Synthesis of pterin probes. Reagents and conditions: a) DMF-acetal (10 eq.), DMF, 50° C, 2 h, 92%; b) ethyl 6-bromohexanoate (5 eq.), K2CO3 (2 eq.), DMF, 4 h, 68%; c) 1 M NaOH/DMF, 1 h, 82%; d) NH3/MeOH, 18 h, 85%; e) 4, EDCI (1.5 eq.), DMAP (1.5 eq), pyridine, 12 h, 68%.
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