Probing the Photoreaction Mechanism of Phytochrome through Analysis of Resonance Raman Vibrational Spectra of Recombinant Analogues

Abstract

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.

Syntheses of Biliverdin Derivatives Sterically Locked at the CD‐Ring Components.

“…The crude product 13 was refluxed in DMF in the presence of pyridine to afford the desired AB-ring component 4 as only Z-isomer in 57% yield in three steps from compound 12. (5). The sterically locked CD-ring component 5 bearing Z-syn configuration and conformation was prepared via our Wittig-type coupling reaction between 3-ethyl-4-methyl-5-tosyl-1,5-dihydro-2H-pyrrol-2-one 8b (9a) and formylpyrrole 10a in the presence of a mixture of tributylphosphine and DBU in THF to afford a mixture of coupling products (Z)-and (E)-8a in 87% yield (Scheme 2).…”

“…When compound 20 was formylated by Vilsmeier reaction to give the formylated product in situ, chlorination of the hydroxy group proceeded simultaneously to afford the formylpyrrole 10b bearing 2-chloroethyl group in 94% yield. Preparation of Allyl (Z) 4,5,…”

“…Mp 70-71 C (from EtOAc/hexane). IR (neat) 2976,2933,1738,1691,1458,1425,1396,1370,1345,1294,1243,1167,1128,1047,1002,959,849,773 [1,4]diazepin-9-yl)propanoate (5). A solution of compound 14 (46 mg, 0.1 mmol) in TFA (1 mL) was allowed to stir for 30 min at room temperature under N 2 .…”

“…The solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was washed with both a saturated aqueous solution of NaHSO 3 and brine, and dried over t-Butyl (Z) 4,5, 2-carboxylate (21). To a solution of compound (Z)-8b (490 mg, 1.0 mmol) in THF (20 mL), DBU (0.46 mL, 3.0 mmol) was added under N 2 , and the mixture was allowed to stir at 50 C overnight.…”

“…10, 1561-1572(2006) 1561 the phytochrome chromophore in the P r , P fr , and intermediate states, but the data were ambiguous and have been interpreted in different ways. 2,[5][6][7] For example, it was proposed that the formation of P fr is accompanied by a syn/anti rotation around the C14-C15 single bond. 5 More recently, interpretation of resonance Raman spectra by density functional theory calculations proposed that the C14-C15 single bond is in an anti conformation throughout the entire photocycle and that the C5-C6 single bond rotates from anti to syn upon conversion from P r to P fr as shown in Fig.…”