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  • The use of deuterated camphor as a substrate in (1)H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound I.

The use of deuterated camphor as a substrate in (1)H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound I.

Biochemistry (2012-12-12)
Roman Davydov, John H Dawson, Roshan Perera, Brian M Hoffman
RESUMEN

Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OH(exo) and C5-H(endo)] from camphor. When D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, the magnitude of the C5-OH(exo) signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.

MATERIALES
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Sigma-Aldrich
(±)-Alcanfor, 96%
Sigma-Aldrich
(1R)-(+)-Camphor, 98%
Sigma-Aldrich
D-Camphor, ≥97%, FG
Sigma-Aldrich
(1S)-(−)-Camphor, 95%
Sigma-Aldrich
(±)-Alcanfor, ≥95.5%
Supelco
D-Camphor, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
(±)-Alcanfor, meets analytical specification of Ph. Eur., BP, racemic, ≥95% (GC)
Sigma-Aldrich
(±)-Alcanfor, purum, synthetic, ≥95.0% (GC)
Supelco
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(±)-Alcanfor, primary reference standard
Sigma-Aldrich
(±)-Alcanfor, SAJ first grade, ≥96.0%