Six new bilin chromophores of the plant photoreceptor phytochrome have been synthesized, carrying at the photoisomerizing ring D an oxygen or a sulfur atom or a methylene group instead of the pyrrole nitrogen atom. These furanone-, thiophenone- or cyclopentenone-containing compounds bound covalently to the recombinant apophytochrome phyA of Avena sativa. The novel chromoproteins showed hypsochromically shifted absorption spectra with respect to native phytochrome and a strongly diminished photochemical activity, but a three- to four-fold higher fluorescence quantum yield. These results demonstrate that, on the one hand, also ring D-modified chromophores can be forced into a partially extended structure, required for incorporation into the apoprotein binding pocket and covalent binding. On the other hand, the modifications introduced into ring D of the chromophores strongly impede the formation of stable far red-absorbing forms of plant photoreceptor phytochrome (P_fr-form) of the chromoproteins, highlighting especially the role of the pyrrole nitrogen atom and hydrogen bonding for the precise interactions between that part of the chromophore and the protein for the P_fr-formation.

Introduction

Phytochromes are master regulators for plant photomorphogenesis (1). They gain their capability as signal transducing proteins from a tight interaction between the protein moiety and their covalently bound chromophore, a bilin compound that undergoes a photoisomerization of one of its double bonds connecting the four pyrrole rings [C(15)–C(16), Fig. 1]. In most plant species investigated so far, phytochromobilin (PΦB, #1), is found as chromophore, except in two algal species, where phycocyanobilin (PCB, #2), has been identified (2,3). The finding of phytochrome-like proteins also in a large number of prokaryotic species (4), and a recently presented three-dimensional structure of a chromophore-binding domain from the phytochrome of Deinococcus radiodurans have unveiled quite a number of interactions between the chromophore and the surrounding protein. In the prokaryotic phytochromes, two classes of chromoproteins can be discerned: one using PCB (2) as chromophore, which binds covalently to a conserved cysteine in the cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase and E. coli transcription factor FhlA protein domain (GAF) as in the plant phytochromes, the other class employing biliverdin (BV) IXα, which is attached to a cysteine at the front end in the Per-Arnt-Sim (PAS, GAF, abundantly found protein folding motifs) domain. Although the phytochrome from D. radiodurans binds BV, the crystal structure revealed quite a number of hydrogen bonds and electrostatic interactions between the chromophore and many amino acids that are highly conserved in most other phytochromes, allowing to propose a generalized mechanism of chromophore fixation and directed photoisomerization. The stabilization of the far red-absorbing forms of plant photoreceptor phytochrome (P_fr-state), however, remains uncertain: the protein pocket around the photoisomerizing ring D of the chromophore is of fairly hydrophobic structure, and also ring D does not exhibit significantly functionalized substituents that might exert specific interactions to stabilize the P_fr-form after its formation. We thus proposed that it might be the pyrrole nitrogen atom itself and the attached proton that play an important role for the P_fr-stability. Based on a recent synthetic approach (5), we here present the six novel phytochrome chromophores 4–9 which instead of the photoisomerizing pyrrole ring D carry a furanone, a thiophenone, or a cyclopentenone ring, in each case substituted with either 18-ethyl-17-methyl or 17,18-dimethyl substituents (Fig. 1). The role of the modified ring D structure in chromoprotein generation and in their spectral properties is discussed.

Details are in the caption following the image — **Figure 1**
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The upper Kekulé formula shows PΦB (1) and PCB (2) in the *ZZZ*,*asa* state. Both chromophores vary from each other exclusively in their substitution pattern at position C(18) (vinyl vs ethyl) and are covalently bound to a conserved cysteine (position 321 of oat phytochrome) *via* a thioether linkage. The photoisomerization of the C(15)–C(16) double bond is indicated by an arrow. The lower Kekulé formula shows the structure of the PCB-analogous 4–9 which differ from the native chromophores 1 and 2 in their substitution pattern at ring D. For reasons of clarity they are shown with the same stereochemistry as the protein-bound chromophores 1 and 2.

Materials and methods

Biochemical section.

General methods: Generation of recombinant apophytochrome (N-terminal 65 kDa domain of oat phyA) followed essentially formerly reported literature protocols (6). Sample treatment was at ambient temperature and spectroscopic measurements were performed under temperature-controlled conditions at 20°C. Assembly of chromoproteins was accomplished by addition of 4–9 to an aqueous, buffered solution (50 mm Tris, 300 mm NaCl and 5% glycerol, pH 7.5) of the recombinant apophytochrome. Absorption spectra were recorded after the addition and purification of the respective chromoprotein at short time intervals for a period of 72 h. Measurements were done in the same buffer as used for the assembly reaction. After assembly, samples were handled under safe light (450–500 nm). Irradiation of the novel phytochromes was performed with light from a 100 W slide-projector bulb, equipped with appropriate interference filters [IF; irradiation of the P_r-, P_fr-form: 4- and 5-containing phytochromes: 616 nm (IF), 655 nm (IF); 6- and 7-containing phytochromes: 578 nm (IF), 715 nm (cut-off filter; CF); and 8- and 9-containing phytochromes: 578 nm (IF), 654 nm (CF); all IFs had a half-bandwidth of ±7 nm]. Samples were irradiated at a distance of 16 cm. A sample of PCB-assembled phytochrome was always used as control. Chromophore competition experiments were performed such that 2 μL of a concentrated solution of PCB (2) in DMSO was added to the fully assembled analog chromoprotein. UV/Vis: Shimadzu UV-2401 PC spectrometer. Fluorescence: Varian Cary Eclipse spectrometer. Data-acquisition and -treatment was performed with the OriginPro 7.5 software.

Chemical section.

General methods:

Column chromatography: Purifications were performed on Merck silica gel 60 (230–400 mesh) under slightly increased pressure.

HPLC: Purifications were carried out on the following columns: Kromasil C18 ODS-5-100 (RP-C₁₈, 5 μm, 250 × 21 mm, flow rate 0.8 mL min⁻¹) (column A), Phenomenex Luna 5μ C18 (RP-C₁₈, 5 μm, 125 × 4.6 mm, flow rate 1.0 mL min⁻¹) (column B), Phenomenex Gemini 5μ C18 (RP-C₁₈, 5 μm, 150 × 4.6 mm, flow rate 1.2 mL min⁻¹) (column C) or GLS Sciences, Inc. Inertsil C8-3 (RP-C₈, 5 μm, 125 × 4.6 mm, flow rate 0.8 mL min⁻¹) (column D).

NMR: Spectra were recorded on Bruker ARX 250, DRX 400 or DRX 500 spectrometers in the solvents indicated; chemical shifts (δ) are given in ppm relative to TMS, coupling constants (J) in Hz. The solvent signals were used as references and the chemical shifts were converted to the TMS scale (CDCl₃: δ_C = 77.0 ppm; residual CHCl₃ in CDCl₃: δ_H = 7.24 ppm; pyridine-d₅: δ_C = 123.5 ppm; residual pyridine in pyridine-d₅: δ_H = 7.19 ppm). IR: Perkin-Elmer System 2000 spectrometer; wavenumbers inline image in cm⁻¹.

UV/Vis: Shimadzu UV-2401 PC spectrometers; the solvents applied were of appropriate quality (Uvasol). MS (EI): Finnigan MAT 8200 or MAT 8400 (70 eV); ESI-MS: Bruker Esquire 3000 or HP Quadrupol MS Engine; accurate mass determinations (HRMS): Finnigan MAT 95; MALDI-TOF-MS: Applied Biosystems Voyager DE-Pro spectrometer connected with a LeCroy high speed digitizer (20 kV acceleration voltage, 130 ns delay time). Melting points: Reichert melting point microscope (uncorrected).

Although the nomination of the aldehydes 16–21 correlates with the IUPAC rules, for prudential reasons, the assignment of the hydrogen and carbon atoms in the case of NMR-analysis follows the Kekulé formula displayed in Fig. 2.

More detailed information concerning the reaction conditions are available directly from the authors.

Methyl 3-{5-[(4-ethyl-3-methyl-5-oxofuran-2(5H)-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (16). A solution of the CD building block 10 (118 mg, 0.29 mmol) in TFA (8.4 mL, 108 mmol) was stirred for 3 min at ambient temperature in the dark and under argon. After addition of orthoformic trimethyl ester (8.4 mL, 75.2 mmol), the solution was stirred for additional 30 min and subsequently absorbed in methylene chloride (450 mL). This mixture was washed with H₂O (2 × 200 mL) and with a saturated solution of NaHCO₃ (2 × 200 mL). Afterwards, the organic layer was dried over Na₂SO₄ and the solvent was evaporated in vacuo. HPLC (column A) of the remaining green crude product with methanol and H₂O (5:1) as eluent yielded the expected reaction product 16 (52 mg, 0.16 mmol, 54%) as a yellow-colored solid. m.p. 129°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.13 (t, ³J = 7.59 Hz, 3H, 9²-CH₃), 2.11 (s, 3H, 4¹-CH₃), 2.13 (s, 3H, 8¹-CH₃), 2.39 (q, ³J = 7.47 Hz, 3H, 9¹-CH₂), 2.56 (t, ³J = 7.63 Hz, 2H, 3²-CH₂), 3.03 (t, ³J = 7.63 Hz, 2H, 3¹-CH₂), 3.64 (s, 3H, CO₂CH₃), 5.89 (s, 1H, 6-CH), 9.69 (s, 1H, CHO), 9.75 (br s, 1H, NH); ¹³C NMR (101 MHz, ¹H/¹³C-COSY, CDCl₃) δ = 8.75 (C-8¹), 9.67 (C-4¹), 12.73 (C-9²), 17.36 (C-9¹), 19.11 (C-3¹), 35.25 (C-3²), 51.75 (CO₂CH₃), 95.07 (C-6), 123.35 (C-3), 129.72, 130.53, 130.68, 132.02 (C-2, -4, -5, -9), 146.54 (C-8), 148.46 (C-7), 169.07 (C-10), 172.78 (CO₂CH₃), 177.47 (CHO); IR (KBr): inline image = 3439, 3070, 2965, 2928, 2852, 1759, 1734, 1637, 1444, 1384, 1213, 1164, 1100, 1008, 941, 574 and 458 cm⁻¹; UV/Vis [methanol + H₂O (5:1)]: λ_max = 266, 394 and 409 nm (ε not determined); MS (EI, 135°C): m/z (%): 331 (100) [M⁺], 330 (2), 303 (19), 302 (33), 300 (12), 299 (2), 272 (34), 271 (11), 270 (7), 260 (7), 243 (5), 230 (13), 228 (6), 217 (2), 203 (3), 186 (3), 176 (3), 162 (3), 158 (2), 146 (2), 136 (2), 134 (3), 119 (2), 118 (4), 117 (2), 107 (2), 106 (3), 104 (3), 91 (4), 79 (2), 77 (4), 67 (2), 65 (2), 53 (2), 41 (2); HRMS (ESI pos): m/z: calcd for C₁₈H₂₂NO₅ [M + H]⁺: 332.149799; found: 332.14999.

Additionally, the dimerization product 29 (16 mg, 0.022 mmol, 18%) was isolated in the form of a dark blue solid. m.p 178°C; ¹H NMR (500 MHz, CDCl₃) δ = 1.11 (t, ³J = 7.57 Hz, 6H, 2²-, 18²-CH₃), 2.14 (s, 6H, 7¹-, 13¹-CH₃), 2.18 (s, 6H, 3¹-, 17¹-CH₃), 2.35 (q, ³J = 7.50 Hz, 4H, 2¹-, 18¹-CH₂), 2.53 (t, ³J = 7.48 Hz, 4H, 8²-, 12²-CH₂), 2.92 (t, ³J = 6.46 Hz, 4H, 8¹-, 12¹-CH₂), 3.64 (s, 6H, CO₂CH₃), 6.21 (br s, 2H, 5-, 15-CH), 6.83 (br s, 1H, 10-CH); ¹³C NMR (126 MHz, BB, ¹H/¹³C-COSY, CDCl₃) δ = 9.53 (C-7¹, -13¹), 10.12 (C-3¹, -17¹), 12.41 (C-2², -18²), 17.10 (C-2¹, -18¹), 19.63 (C-8¹, -12¹), 34.90 (C-8², -12²), 51.45 (CO₂CH₃), 98.71 (C-5, -15), 116.14 (C-10), 128.04 (C-7, -13), 137.97 (C-9, -11), 140.76 (C-6), 146.98 (C-3, -17), 148.24 (C-4,- 16), 150.21 (C-14), 169.43 (C-1, -19), 172.83 (CO₂CH₃); IR (KBr): inline image = 3448, 2930, 2874, 1757, 1743, 1640, 1594, 1458, 1437, 1385, 1357, 1260, 1236, 1170, 1097, 1057, 1014, 950, 906, 883, 801, 775, 736, 705, 673 and 613 cm⁻¹; UV/Vis (diethyl ether): λ_max (ɛ) = 307 (12 500), 358 (22 900), 596 nm (12 400); MS (EI, 260°C): m/z (%): 616 (100) [M⁺], 557 (29), 529 (47), 497 (4), 469 (33), 392 (5), 316 (10), 242 (8) and 180 (3); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₅H₄₁N₂O₈ [M + H]⁺: 617.285743; found: 617.286033.

Methyl 3-{5-[(3,4-dimethyl-5-oxofuran-2(5H)-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (17): See synthesis of 16. Yellow colored solid (48 mg, 0.15 mmol, 52%). m.p. 152°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.95 (s, 3H, 9¹-CH₃), 2.11 (s, 3H, 4¹-CH₃), 2.12 (s, 3H, 8¹-CH₃), 2.56 (t, ³J = 7.62 Hz, 2H, 3²-CH₂), 3.03 (t, ³J = 7.62 Hz, 2H, 3¹-CH₂), 3.64 (s, 3H, CO₂CH₃), 5.89 (s, 1H, 6-CH), 9.69 (s, 1H, CHO), 9.72 (br s, 1H, NH); ¹³C NMR (101 MHz, CDCl₃) δ = 8.75 (C-8¹), 8.97 (C-9¹), 9.89 (C-4¹), 19.12 (C-3¹), 35.26 (C-3²), 51.71 (CO₂CH₃), 94.97 (C-6), 123.34 (C-3), 124.27, 126.79, 130.56, 132.41 (C-2, -4, -5, -9), 147.06 (C-8), 148.50 (C-7), 169.17 (C-10), 172.78 (CO₂CH₃), 177.32 (CHO); IR (KBr): inline image , 3064, 2921, 2854, 1767, 1730, 1633, 1540, 1439, 1396, 1219, 1164, 1094, 1023, 985, 907, 747, 594 cm⁻¹; UV/Vis [methanol + H₂O (5:1)]: λ_max = 266, 394, 408 nm (ε not determined); MS (EI, 150°C): m/z (%): 317 (100) [M⁺], 316 (3), 289 (21), 288 (33), 286 (12), 285 (2), 258 (40), 257 (14), 256 (10), 246 (10), 244 (21), 230 (18), 229 (9), 228 (5), 216 (21), 215 (5), 214 (6), 203 (3), 189 (6), 172 (2), 162 (4), 144 (4), 120 (3), 79 (3), 77 (6), 53 (4); HRMS (ESI pos, methanol): m/z: calcd for C₁₇H₂₀NO₅ [M + H]⁺: 318.134149; found: 318.13427.

Additionally, the dimerization product 30 (13 mg, 0.022 mmol, 16%) was isolated in the form of a dark blue solid. m.p 211°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.88 (s, 6H, 2¹-, 18¹-CH₃), 2.11 (s, 6H, 7¹-, 13¹-CH₃), 2.17 (s, 6H, 3¹-, 17¹-CH₃), 2.52 (t, ³J = 7.40 Hz, 4H, 8²-, 12²-CH₂), 2.89 (t, ³J = 7.40 Hz, 4H, 8¹-, 12¹-CH₂), 3.64 (s, 6H, CO₂CH₃), 6.15 (s, 2H, 5-, 15-CH), 6.75 (s, 1H, 10-CH); ¹³C NMR (101 MHz, CDCl₃) 8.89 (C-2¹, -18¹), 9.99 (C-7¹, 13¹), 19.87 (C-3¹, -17¹), 26.40 (C-8¹), 29.69 (C-12¹), 32.20 (C-8²), 34.33 (C-12²), 51.68 (CO₂CH₃), 97.20 (C-15), 99.27 (C-5), 115.94 (C-10), 124.00 (C-7), 125.04 (C-2, -18), 128.15 (C-13), 129.65 (C-6, -14), 135.21 (C-9, -11), 141.67, 150.11 (C-8, -12), 147.62 (C-3, -17), 148.57 (C-4, -16), 169.44 (C-1, -19), 173.16 (CO₂CH₃); IR (KBr): inline image = 3441, 3065, 2920, 2850, 1744, 1645, 1596, 1459, 1437, 1396, 1350, 1263, 1235, 1171, 1093, 1014, 964, 909, 875, 801, 750, 706, 678 cm⁻¹; UV/Vis (diethyl ether): λ_max (ɛ) = 304 (10 300), 358 (20 900), 595 nm (12 400); MS (EI, 235°C): m/z (%): 588 (100) [M⁺], 587 (5), 573 (5), 557 (16), 556 (19), 529 (36), 514 (2), 501 (46), 482 (3), 469 (5), 465 (2), 441 (44), 428 (33), 405 (2), 383 (2), 359 (3), 357 (2), 332 (3), 331 (4), 319 (5), 294 (4), 289 (2), 287 (2), 274 (3), 262 (2), 257 (3), 241 (4), 240 (3), 228 (4), 227 (5), 221 (3), 220 (2), 57 (2), 43 (2); HRMS (ESI pos, methanol): m/z: calcd for C₃₃H₃₇N₂O₈ [M + H]⁺: 589.254992; found: 589.25477.

Methyl 3-{5-[(4-ethyl-3-methyl-5-oxothiophen-2(5H)-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (18): See synthesis of 16. Yellow colored solid (32 mg, 0.09 mmol, 34%). m.p. 151°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.07 (t, ³J = 7.55 Hz, 3H, 9²-CH₃), 2.14 (s, 3H, 4¹-CH₃), 2.29 (s, 3H, 8¹-CH₃), 2.44 (q, ³J = 7.52 Hz, 2H, 9¹-CH₂), 2.57 (t, ³J = 7.55 Hz, 2H, 3²-CH₂), 3.04 (t, ³J = 7.55 Hz, 2H, 3¹-CH₂), 3.65 (s, 3H, CO₂CH₃), 6.93 (s, 1H, 6-CH), 9.04 (br s, 1H, NH), 9.71 (s, 1H, CHO); ¹³C NMR (101 MHz, CDCl₃) δ = 8.96 (C-4¹), 12.66 (C-9²), 13.06 (C-9¹), 18.96 (C-8¹), 19.07 (C-3¹), 35.20 (C-3²), 51.75 (CO₂CH₃), 113.39 (C-6), 125.31, 131.61, 131.77, 132.40, 134.28 (C-2, -3, -4, -5, -7), 140.77 (C-9), 152.67 (C-8), 172.58 (CO₂CH₃), 177.65 (CHO), 193.00 (C-10); IR (KBr): inline image = 3428, 2972, 2934, 2861, 1742, 1673, 1643, 1608, 1584, 1439, 1412, 1385, 1368, 1225, 1203, 1167, 1059, 867, 852, 838, 715, 519, 471 cm⁻¹; UV/Vis [methanol + H₂O (5:1)]: λ_max = 241, 279, 407 nm (ε not determined); MS (EI, 155°C): m/z (%): 347 (100) [M⁺], 332 (4), 318 (32), 288 (20), 246 (9), 230 (8), 184 (2), 166 (3), 144 (3), 115 (2), 91 (2), 77 (3), 65 (1), 41 (1); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₁₈H₂₁NNaO₄S [M + Na]⁺: 370.108349; found: 370.108433.

Additionally, the dimerization product 31 (14 mg, 0.022 mmol, 8%) was isolated in the form of a dark blue solid. m.p. 209–211°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.04 (t, ³J = 7.54 Hz, 6H, 2²-, 18²-CH₃), 2.11 (s, 6H, 7¹-, 13¹-CH₃), 2.32 (s, 6H, 3¹-, 17¹-CH₃), 2.39 (q, ³J = 7.52 Hz, 4H, 2¹-, 18¹-CH₂), 2.55 (t, ³J = 7.55 Hz, 4H, 8²-CH₂), 2.93 (t, ³J = 7.41 Hz, 4H, 8¹-CH₂), 6.93 (br s, 1H, 10-CH), 7.02 (br s, 2H, 5-, 15-CH); ¹³C NMR (101 MHz, BB, CDCl₃) δ = 11.18 (C-7¹, -13¹), 12.76 (C-3¹, -17¹), 12.94 (C-2², -18²), 18.81 (C2¹, -18¹), 19.84 (C-8¹, -12¹), 35.10 (C-8², -12²), 51.72 (CO₂CH₃), 115.56 (C-5, -15), 117.26 (C-10), 128.53, 128.79, 130.86, 138.21, 139.31 (C-4, -6, -7, -8, -9, -11, -12, -13, -14, -16), 141.88 (C-2, -18), 152.26 (C-3, -17), 173.03 (CO₂CH₃), 195.15 (C-1, -19); IR (KBr): inline image = 3449, 2965, 2931, 2872, 1736, 1666, 1652, 1609, 1458, 1437, 1384, 1364, 1315, 1265, 1197, 1171, 1095, 966, 934, 860 cm⁻¹; UV/Vis (diethyl ether): λ_max (ε) = 261 (6700), 328 (13 500), 378 (24 000), 628 nm (13 000); MS (EI, 250°C): m/z (%): 648 (100) [M⁺], 561 (16), 501 (13), 408 (4), 349 (5), 271 (4), 199 (5), 139 (1), 97 (1), 55 (1).

Methyl 3-{5-[(3,4-dimethyl-5-oxothiophen-2(5H)-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (19): See synthesis of 16. Yellow colored solid (41 mg, 0.12 mmol, 36%). m.p. 182°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.98 (s, 3H, 9¹-CH₃), 2.14 (s, 3H, 4¹-CH₃), 2.28 (d, J = 0.80 Hz, 3H, 8¹-CH₃), 2.57 (t, ³J = 7.55 Hz, 2H, 3²-CH₂), 3.03 (t, ³J = 7.55 Hz, 2H, 3¹-CH₂), 3.66 (s, 3H, CO₂CH₃), 6.93 (s, 1H, 6-CH), 9.04 (br s, 1H, NH), 9.71 (s, 1H, CHO); ¹³C NMR (101 MHz, CDCl₃) δ = 8.99 (C-4¹), 10.99 (C-9¹), 13.01 (C-8¹), 18.97 (C-3¹), 35.20 (C-3²), 51.77 (CO₂CH₃), 113.32 (C-6), 125.36, 131.60, 131.76, 132.45, 134.17 (C-2, -3, -4, -5, -7), 135.20 (C-9), 152.96 (C-8), 172.59 (CO₂CH₃), 177.68 (CHO), 192.11 (C-10); IR (KBr): inline image = 3426, 2921, 2858, 1727, 1669, 1632, 1609, 1587, 1438, 1417, 1386, 1366, 1317, 1240, 1220, 1167, 1023, 957, 861, 842, 758, 724, 530, 485 cm⁻¹; UV/Vis [methanol + H₂O (5:1)]: λ_max = 240, 279, 407 nm (ε not determined); MS (EI, 160°C): m/z (%): 333 (100) [M⁺], 304 (24), 274 (21), 260 (10), 246 (10), 216 (4), 202 (5), 166 (4), 144 (4), 115 (6), 91 (3), 77 (5), 59 (5), 39 (2); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₁₇H₁₉NNaO₄S [M + Na]⁺: 356.092702; found 356.092725.

Methyl 3-{5-[(3-ethyl-2-methyl-4-oxocyclopent-2en-1-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (20): See synthesis of 16. Yellow colored spicular crystals (24 mg, 0.07 mmol, 41%). m.p. 186°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.04 (t, ³J = 7.59 Hz, 3H, 9²-CH₃), 2.11 (s, 3H, 4¹-CH₃), 2.18 (s, 3H, 8¹-CH₃), 2.34 (q, ³J = 7.57 Hz, 2H, 9¹-CH₂), 2.56 (t, ³J = 7.67 Hz, 2H, 3²-CH₂), 3.03 (t, ³J = 7.61 Hz, 2H, 3¹-CH₂), 3.20 (s, 2H, 11-CH₂), 3.65 (s, 3H, CO₂CH₃), 6.47 (s, 1H, 6-CH), 8.87 (br s, 1H, NH), 9.63 (s, 1H, CHO); ¹³C NMR (101 MHz, ¹H/¹³C-COSY, CDCl₃) δ = 8.83 (C-4¹), 11.69 (C-8¹), 12.90 (C-9²), 17.02 (C-9¹), 19.03 (C-3¹), 35.34 (C-3²), 38.62 (C-11), 51.74 (CO₂CH₃), 109.31 (C-6), 123.03 (C-4), 130.20 (C-2), 133.03 (C-3), 133.67 (C-5), 136.87 (C-7), 145.77 (C-9), 162.76 (C-8), 172.64 (CO₂CH₃), 177.00 (CHO), 202.42 (C-10); IR (KBr): inline image = 3341, 2960, 2934, 2873, 1725, 1677, 1663, 1588, 1552, 1479, 1445, 1395, 1334, 1305, 1273, 1248, 1192, 1168, 1064, 1004, 939, 886, 798, 768, 690, 648, 623 cm−1; UV/Vis [methanol + H₂O (5:1)]: λmax = 229, 273, 386 nm (ε not determined); MS (EI, 150°C): m/z (%): 329 (100) [M+], 314 (5), 300 (30), 270 (20), 256 (8), 242 (9), 226 (12), 212 (13), 198 (10), 170 (6), 144 (4), 130 (6), 106 (5), 91 (6), 77 (6), 65 (3), 53 (3), 41 (5); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₁₉H₂₃NNaO₄ [M + Na]⁺: 352.151930; found 352.151556.

Methyl 3-{5-[(2,3-dimethyl-4-oxocyclopent-2en-1-ylidene)methyl]-2-formyl-4-methyl-1H-pyrrol-3-yl}propanoate (21): See synthesis of 16. Yellow colored spicular crystals (30 mg, 0.09 mmol, 33%). m.p. 196°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.87 (s, 3H, 9¹-CH₃), 2.11 (s, 3H, 4¹-CH₃), 2.17 (s, 3H, 8¹-CH₃), 2.56 (t, ³J = 7.60 Hz, 2H, 3²-CH₂), 3.03 (t, ³J = 7.59 Hz, 2H, 3¹-CH₂), 3.20 (s, 2H, 11-CH₂), 3.65 (s, 3H, CO₂CH₃), 6.47 (s, 1H, 6-CH), 8.84 (br s, 1H, NH), 9.63 (s, 1H, CHO); ¹³C NMR (101 MHz, ¹H/¹³C-COSY, CDCl₃) δ = 8.84 (C-4¹), 8.88 (C-9¹), 11.96 (C-8¹), 19.04 (C-3¹), 35.31 (C-3²), 38.46 (C-11), 51.76 (CO₂CH₃), 109.05 (C-6), 122.89 (C-4), 129.75 (C-2), 132.35 (C-3), 133.43 (C-5), 136.35 (C-7), 140.05 (C-9), 162.89 (C-8), 172.14 (CO₂CH₃), 176.27 (CHO), 202.23 (C-10); IR (KBr): inline image = 3340, 2953, 2917, 2858, 1725, 1682, 1656, 1596, 1550, 1479, 1445, 1395, 1347, 1306, 1262, 1236, 1192, 1167, 1093, 1065, 1012, 938, 881, 783, 690, 657, 625 cm−1; UV/Vis [methanol + H₂O (5:1)]: λ^max = 277, 386 nm (ε not determined); MS (EI, 160°C): m/z (%): 315 (100) [M+], 300 (2), 286 (30), 256 (24), 242 (10), 228 (10), 212 (14), 198 (9), 184 (8), 170 (6), 144 (3), 130 (4), 106 (3), 91 (3), 77 (3), 65 (1), 55 (1), 41 (1); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₁₈H₂₁NNaO₄ [M + Na]⁺: 338.136279; found 338.136020.

18-Ethyl-17-methyl-oxa-PCB-dimethyl ester (23). The AB building block 22 (18.4 mg, 0.04 mmol), dissolved in TFA (0.9 mL, 11.6 mmol), was stirred for 30 min at ambient temperature in the dark and under argon. After cooling down to −12°C within 5 min a solution of the aldehyde 16 (16.0 mg, 0.05 mmol) in TFA (4.2 mL, 54.0 mmol) was added. The resulting mixture was stirred for 8 h at −12°C. Methanol (1.9 mL, 46.9 mmol) was added and the solution was stirred for additional 30 min at ambient temperature. After evaporation of the solvents in a stream of argon, the remaining solid was dissolved in methylene chloride (30 mL) and washed with a saturated solution of NaHCO₃ (30 mL). Afterwards, the organic layer was dried over Na₂SO₄, the solvent was evaporated in vacuo, and the remaining crude product was purified by HPLC (column A) with methanol and H₂O (5:1) as eluent. Evaporation of methanol, followed by extraction of the aqueous dispersion with methylene chloride (3 × 30 mL), drying of the combined organic layers over Na₂SO₄, and evaporation of the solvent in vacuo yielded the expected reaction product 23 (15.8 mg, 0.03 mmol, 63%) as a dark blue solid. m.p. 189–191°C; ¹H NMR (400 MHz, pyridine-d₅) δ = 1.06 (t, ³J = 8.61 Hz, 3H, 18²-CH₃), 1.41 (d, ³J = 7.52 Hz, 3H, 2¹-CH₃), 1.64 (dd, ³J_d = 7.28 Hz, J_d = 0.76 Hz, 3H, 3²-CH₃), 1.87 (s, 3H, 7¹-CH₃), 1.97 (s, 3H, 17¹-CH₃), 1.99 (s, 3H, 13¹-CH₃), 2.25 (q, ³J = 7.57 Hz, 2H, 18¹-CH₂), 2.62 (t, ³J = 7.42 Hz, 2H, 8²-, 12²-CH₂), 2.66 (t, ³J = 7.53 Hz, 2H, 8²-, 12²-CH₂), 2.96 (t, ³J = 7.44 Hz, 2H, 8¹-, 12¹-CH₂), 2.98 (t, ³J = 7.42 Hz, 2H, 8¹-, 12¹-CH₂), 3.22 (q, ³J = 7.63 Hz, 1H, 2-CH), 3.53 (s, 3H, CO₂CH₃), 3.54 (s, 3H, CO₂CH₃), 5.94 (s, 1H, 5-CH), 5.99 (s, 1H, 15-CH), 6.31 (dq, J_d = 1.95 Hz, ³J_q = 6.93 Hz, 1H, 3¹-CH), 7.02 (s, 1H, 10-CH); IR (KBr): inline image = 2925, 2857, 1737, 1638, 1622, 1592, 1457, 1384, 1315, 1230, 1168, 1129, 1100, 945, 800, 699 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 279 (6400), 363 (15 200), 603 nm (4800); MS (EI, 235°C): m/z (%): 615 (50) [M⁺], 600 (100), 584 (4), 556 (2), 528 (8), 468 (3), 440 (2), 316 (7), 255 (2), 227 (4), 158 (1); MS (ESI pos, methanol): m/z (%): 242 (2), 300 (3), 413 (4), 525 (5), 616 (71) [M + H]⁺, 638 (100) [M + Na]⁺, 654 (20) [M + K]⁺, 700 (27); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₅H₄₁N₃NaO₇ [M + Na]⁺: 638.283672; found 638.283886.

17,18-Dimethyl-oxa-PCB-dimethyl ester (24): See synthesis of 23. Dark blue solid (11.5 mg, 0.02 mmol, 71%). m.p. 228°C; ¹H NMR (400 MHz, pyridine-d₅) δ = 1.47 (d, ³J = 7.51 Hz, 3H, 2¹-CH₃), 1.68 (d, ³J = 7.28 Hz, 3H, 3²-CH₃), 1.81 (s, 3H, 18¹-CH₃), 1.90 (s, 3H, 7¹-CH₃), 2.02 (s, 3H, 17¹-CH₃), 2.04 (s, 3H, 13¹-CH₃), 2.67 (t, ³J = 7.53 Hz, 2H, 8²-, 12²-CH₂), 2.71 (t, ³J = 7.72 Hz, 2H, 8²-, 12²-CH₂), 3.02 (t, ³J = 6.84 Hz, 4H, 8¹-, 12¹-CH₂), 3.25 (q, ³J = 7.34 Hz, 1H, 2-CH), 3.58 (s, 3H,CO₂CH₃), 3.59 (s, 3H, CO₂CH₃), 5.99 (s, 1H, 5-CH), 6.05 (s, 1H, 15-CH), 6.35 (dq, J_d = 2.26 Hz, ³J_q = 7.21 Hz, 1H, 3¹-CH), 7.08 (s, 1H, 10-CH); IR (KBr): inline image = 2922, 2855, 1740, 1724, 1643, 1622, 1591, 1457, 1411, 1384, 1347, 1314, 1229, 1166, 1129, 1095, 998, 908, 798, 751, 699 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 275 (5800), 361 (14 200), 605 nm (4400); MS (EI, 240°C): m/z (%): 601 (49) [M⁺], 586 (100), 570 (4), 514 (9), 482 (2), 454 (3), 289 (15), 216 (12), 158 (1), 106 (1); MS (ESI pos, methanol): m/z (%): 153 (1), 360 (1), 489 (1), 602 (9) [M + H]⁺, 624 (100) [M + Na]⁺, 646 (5), 722 (1); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₄H₄₀N₃O₇ [M + H]⁺: 602.286079; found 602.285919.

18-Ethyl-17-methyl-thia-PCB-dimethyl ester (25): See synthesis of 23. Purified by HPLC (column D) with a mixture of KH₂PO₄ buffer (7 mm, pH 7) and acetonitrile (gradient 7:3–1:4) as eluent. Dark blue solid (16.5 mg, 0.03 mmol, 65%). m.p. 143°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.07 (t, ³J = 7.31 Hz, 3H, 18²-CH₃), 1.41–1.46 (m, 3H, 2¹-CH₃), 1.69 (d, ³J = 6.48 Hz, 3H, 3²-CH₃), 1.91–1.95 (m, 3H, 7¹-CH₃), 2.14 (s, 3H, 13¹-CH₃), 2.29 (s, 3H, 17¹-CH₃), 2.44 (q, ³J = 6.40 Hz, 2H, 18¹-CH₂), 2.57 (t, ³J = 7.53 Hz, 4H, 8²-, 12²-CH₂), 3.04 (t, ³J = 7.55 Hz, 4H, 8¹-, 12¹-CH₂), 3.31–3.36 (m, 1H, 2-CH), 3.63 (d, J = 2.68 Hz, 3H, CO₂CH₃), 3.65 (d, J = 1.49 Hz, 3H, CO₂CH₃), 5.87 (s, 1H, 5-CH), 6.43 (s, 1H, 15-CH), 6.51–6.58 (m, 1H, 3¹-CH), 6.98 (s, 1H, 10-CH); IR (KBr): inline image = 3443, 2925, 2855, 1734, 1637, 1560, 1507, 1458, 1438, 1384, 1339, 1261, 1211, 1168, 1130, 1107, 1038, 984, 854, 805, 774, 527, 498 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 279 (9800), 379 (13 100), 629 nm (5500); MS (EI, 250°C): m/z (%): 631 (52) [M⁺], 616 (100), 600 (5), 544 (7), 492 (5), 376 (6), 319 (7), 300 (20), 241 (37), 227 (6), 180 (3), 150 (26), 135 (8), 93 (15), 43 (4); MS (ESI pos, methanol + methylene chloride): m/z (%):370 (16), 632 (61) [M + H]⁺, 654 (100) [M + Na]⁺, 1001 (10), 1264 (14) [2 M + H]⁺, 1286 (27) [2 M + Na]⁺; HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₅H₄₂N₃O₆S [M + H]⁺: 632.278887; found 632.278375.

17,18-Dimethyl-thia-PCB-dimethyl ester (26): See synthesis of 25. Dark blue solid (13.7 mg, 0.02 mmol, 54%). m.p. 188°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.37 (dd, J_d = 0.91, ³J_d = 6.56 Hz, 3H, 2¹-CH₃), 1.92 (d, ³J = 7.71 Hz, 3H, 3²-CH₃), 1.94 (s, 3H, 7¹-CH₃), 2.07 (s, 3H, 18¹-CH₃), 2.15 (dd, J_d =2.66 Hz, J_d = 6.87 Hz, 3H, 13¹-CH₃), 2.29 (d, J = 2.32 Hz, 3H, 17¹-CH₃), 2.55 (t, ³J = 8.25 Hz, 4H, 8²-, 12²-CH₂), 2.96 (t, ³J = 7.74 Hz, 4H, 8¹-, 12¹-CH₂), 3.16–3.23 (m, 1H, 2-CH), 3.64 (s, 3H, CO₂CH₃), 3.66 (, 3H, CO₂CH₃), 5.86 (s, 1H, 5-CH), 6.36–6.54 (m, 1H, 3¹-CH), 5.95 (s, 1H, 15-CH), 6.98 (s, 1H, 10-CH); IR (KBr): inline image = 3449, 2930, 1734, 1664, 1594, 1523, 1458, 1439, 1384, 1319, 1209, 1168, 1122, 1109, 1039, 931, 864, 806, 683, 636 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 279 (7000), 380 (13 300), 595 (3600), 620 nm (3600); MS (EI, 275°C): m/z (%): 617 (62) [M⁺], 602 (100), 530 (6), 470 (3), 307 (14), 268 (2), 227 (3), 202 (3), 132 (3), 77 (2), 57 (2); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₄H₄₀N₃O₆S [M + H]⁺: 618.263234; found 618.263043.

18-Ethyl-17-methyl-carba-PCB-dimethyl ester (27): See synthesis of 23. Dark blue solid (15.1 mg, 0.03 mmol, 65%). m.p. 198°C; ¹H NMR (400 MHz, CDCl₃) δ = 1.07 (t, ³J = 7.54 Hz, 3H, 18²-CH₃), 1.42 (d, ³J = 7.45 Hz, 3H, 2¹-CH₃), 1.93 (d, ³J = 7.56 Hz, 3H, 3²-CH₃), 2.10 (s, 3H, 7¹-CH₃), 2.13 (s, 3H, 13¹-CH₃), 2.21 (s, 3H, 17¹-CH₃), 2.35 (q, ³J = 7.42 Hz, 2H, 18¹-CH₂), 2.56 (t, ³J = 7.63 Hz, 2H, 8²-, 12²-CH₂), 2.59 (t, ³J = 7.55 Hz, 2H, 8²-, 12²-CH₂), 3.01 (t, ³J = 7.11 Hz, 2H, 8¹-, 12¹-CH₂), 3.04 (t, ³J = 7.46 Hz, 2H, 8¹-, 12¹-CH₂), 3.22 (q, ³J = 8.28 Hz, 1H, 2-CH), 3.55 (s, 1H, 20-CH₂), 3.59 (s, 1H, 20-CH₂), 3.63 (s, 3H, CO₂CH₃), 3.65 (s, 3H, CO₂CH₃), 5.28 (s, 1H, 15-CH), 5.86 (s, 1H, 5-CH), 6.50 (dq, J_d = 1.86 Hz, ³J_q = 6.64 Hz, 1H, 3¹-CH), 6.67 (s, 1H, 10-CH); IR (KBr): inline image = 2974, 2937, 2872, 1733, 1722, 1687, 1622, 1594, 1452, 1439, 1405, 1384, 1317, 1282, 1244, 1226, 1210, 1190, 1168, 1098, 1057, 966, 938, 832, 746, 690 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 267 (8600), 291 (7700), 361 (17 200), 604 nm (7900); MS (EI, 240°C): m/z (%): 613 (79) [M⁺], 598 (100), 526 (9), 492 (2), 466 (3), 300 (6), 254 (2), 226 (1), 55 (1); MS (ESI pos, methanol): m/z (%): 304 (2), 367 (2), 457 (2), 529 (2), 614 (20) [M + H]⁺, 636 (100) [M + Na]⁺, 652 (15) [M + K]⁺, 675 (68), 766 (4); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₆H₄₃N₃NaO₆ [M + Na]⁺: 636.304403; found 636.303838.

17,18-Dimethyl-carba-PCB-dimethyl ester (28): See synthesis of 23. Dark blue solid (11.8 mg, 0.02 mmol, 48%). m.p. 196°C; ¹H NMR (400 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.48 (d, ³J = 7.54 Hz, 3H, 2¹-CH₃), 1.70 (d, ³J = 7.28 Hz, 3H, 3²-CH₃), 1.78 (s, 3H, 18¹-CH₃), 1.99 (s, 3H, 7¹-CH₃), 2.11 (s, 6H, 13¹-, 17¹-CH₃), 2.72 (t, ³J = 7.13 Hz, 2H, 8²-, 12²-CH₂), 2.75 (t, ³J = 7.03 Hz, 2H, 8²-, 12²-CH₂), 3.10 (t, ³J = 7.33 Hz, 4H, 8¹-, 12¹-CH₂), 3.44–3.47 (m, 1H, 2-CH), 3.48 (s, 1H, 20-CH₂), 3.59 (s, 3H, CO₂CH₃), 3.60 (s, 3H, CO₂CH₃), 3.63 (s, 1H, 20-CH₂), 6.08 (s, 1H, 5-CH), 6.36 (q, ³J = 6.86 Hz, 1H, 3¹-CH), 6.55 (s, 1H, 15-CH), 7.13 (s, 1H, 10-CH); IR (KBr): inline image = 2923, 2855, 1728, 1687, 1621, 1589, 1441, 1406, 1384, 1317, 1213, 1169, 1089, 1056, 1041, 965, 879, 837, 808, 746, 693, 651, 617, 441 cm⁻¹; UV/Vis (methanol): λ_max (ε) = 266 (8200), 293 (7300), 362 (16 800), 603 nm (7900); MS (EI, 240°C): m/z (%): 601 (13), 600 (36), 599 (92) [M⁺], 586 (9), 585 (38), 584 (100), 512 (9), 452 (5), 300 (19), 214 (6), 180 (3), 105 (6); MS (ESI pos, methanol + methylene chloride): m/z (%): 600 (22) [M + H]⁺, 601 (8), 621 (3), 622 (100) [M + Na]⁺, 623 (38), 624 (8), 644 (7), 720 (4), 1199 (21) [2M + H]⁺, 1200 (15), 1201 (62), 1221 (58) [2M + Na]⁺, 1222 (59), 1223 (18); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₅H₄₁N₃NaO₆ [M + Na]⁺: 622.288753; found 622.288616.

18-Ethyl-17-methyl-oxa-PCB (4). The ester 23 (11.1 mg, 0.02 mmol) was dissolved in a mixture of TFA and H₂O (30 mL, 1:1, vol/vol). Subsequently, acidic ion exchange resin DOWEX 50W×8−200 (12.4 g) which was washed three times with H₂O and dried in a stream of air before it was added and the dispersion was stirred for 48 h at ambient temperature in the dark. The ion exchange resin was filtered off (sintered glass filter, size 4) and washed alternately with H₂O (5 × 40 mL) and a solution of chloroform and methanol (49:1, 5 × 40 mL). The aqueous layer of the combined filtrates was separated and extracted with a solution of chloroform and methanol (49:1, 4 × 40 mL). Afterwards the combined organic phases were washed with brine (50 mL portions) until pH 6 and dried over Na₂SO₄. Evaporation of the solvent in vacuo yielded the crude product (17.6 mg) which was purified by HPLC (column B) with a mixture of ammonium acetate buffer (10 mm, pH 7) and methanol (gradient 1:1–1:8) as eluent. Evaporation of the methanol followed by extraction of the aqueous dispersion with methylene chloride (5 × 30 mL), drying of the combined organic layers over Na₂SO₄, and evaporation of the solvent in vacuo yielded the expected reaction product 4 (7.8 mg, 0.01 mmol, 74%) as a dark blue glowing solid. Decomposition until 335°C; ¹H NMR (400 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.11 (t, ³J = 7.55 Hz, 3H, 18²-CH₃), 1.46 (d, ³J = 7.52 Hz, 3H, 2¹-CH₃), 1.68 (dd, ³J_d = 7.35 Hz, J_d = 0.64 Hz, 3H, 3²-CH₃), 1.91 (s, 3H, 7¹-CH₃), 2.06 (s, 3H, 17¹-CH₃), 2.07 (s, 3H, 13¹-CH₃), 2.29 (q, ³J = 7.45 Hz, 2H, 18¹-CH₂), 2.84 (t, ³J = 7.35 Hz, 2H, 8²-, 12²-CH₂), 2.89 (t, ³J = 7.54 Hz, 2H, 8²-, 12²-CH₂), 3.15 (t, ³J = 7.32 Hz, 2H, 8¹-, 12¹-CH₂), 3.15 (t, ³J = 7.21 Hz, 2H, 8¹-, 12¹-CH₂), 3.26 (dq, J_d = 0.65 Hz, ³J_q = 7.52 Hz, 1H, 2-CH), 5.97 (s, 1H, 5-CH), 6.02 (s, 1H, 15-CH), 6.33 (dq, J_d = 2.31 Hz, ³J_q = 7.33 Hz, 1H, 3¹-CH), 7.30 (s, 1H, 10-CH); UV/Vis [methanol + H₂O (3:1)]: λ_max = 279, 367, 611 nm (ε not determined); MS (ESI pos, methanol + methylene chloride): m/z (%): 441 (28), 491 (100), 553 (22), 588 (59) [M + H]⁺, 589 (20), 590 (5), 610 (37) [M + Na]⁺, 611 (15), 632 (18), 654 (19); MS (MALDI-TOF pos, α-CHCA): m/z (%): 573 (19) [M – 15]⁺, 574 (41) [M – 14]⁺, 575 (19), 576 (5), 587 (22), 588 (82) [M⁺], 589 (100) [M + H]⁺, 590 (91), 610 (9), 611 (35) [M + Na]⁺, 612 (16), 613 (6), 627 (6) [M + K]⁺; HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₃H₃₈N₃O₇ [M + H]⁺: 588.270429; found 588.271108.

17,18-Dimethyl-oxa-PCB (5): See synthesis of 4. Dark blue solid (13.3 mg, 0.02 mmol, 70%). Decomposition until 335°C; ¹H NMR (400 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.47 (d, ³J = 7.21 Hz, 3H, 2¹-CH₃), 1.68 (d, ³J = 7.21 Hz, 3H, 3²-CH₃), 1.81 (s, 3H, 18¹-CH₃), 1.89 (s, 3H, 7¹-CH₃), 2.06 (s, 3H, 17¹-CH₃), 2.07 (s, 3H, 13¹-CH₃), 2.85 (t, ³J = 7.28 Hz, 2H, 8²-, 12²-CH₂), 2.89 (t, ³J = 7.43 Hz, 2H, 8²-, 12²-CH₂), 3.15 (t, ³J = 6.87 Hz, 2H, 8¹-, 12¹-CH₂), 3.16 (t, ³J = 7.50 Hz, 2H, 8¹-, 12¹-CH₂), 3.24 (q, ³J = 7.74 Hz, 1H, 2-CH), 5.98 (s, 1H, 5-CH), 6.04 (s, 1H, 15-CH), 6.35 (dq, J_d = 1.82 Hz, ³J_q = 6.88 Hz, 1H, 3¹-CH), 7.32 (s, 1H, 10-CH); UV/Vis [methanol + H₂O (3:1)]: λ_max = 279, 366, 613 nm (ε not determined); MS (ESI pos, methanol): m/z (%): 357 (10), 393 (10), 413 (25), 489 (11), 574 (100) [M + H]⁺, 575 (37), 576 (10), 577 (5), 596 (73) [M + Na]⁺, 597 (30), 598 (6), 599 (5), 618 (33), 640 (27); MS (ESI neg, methanol): m/z (%): 468 (11), 528 (42), 529 (15), 572 (83) [M − H]⁻, 573 (31), 574 (7), 575 (2), 594 (100), 595 (38), 596 (7), 597 (3), 758 (3), 813 (47), 814 (24); MS (MALDI-TOF pos, α-CHCA): m/z (%): 523 (11), 524 (5), 551 (12), 552 (6), 559 (4) [M − 15]⁺, 560 (15) [M − 14]⁺, 561 (7), 573 (10), 574 (78) [M]⁺, 575 (100) [M + H]⁺, 576 (68), 577 (21), 597 (5) [M + Na]⁺, 612 (3), 613 (10) [M + K]⁺ 614 (4); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₂H₃₆N₃O₇ [M + H]⁺: 574.254778; found 574.254557.

18-Ethyl-17-methyl-thia-PCB (6): See synthesis of 4. Purified by HPLC (column B) with a mixture of KH₂PO₄ buffer (7 mm, pH 7) and acetonitrile (gradient 7:3–1:4) as eluent. Dark blue solid (16.9 mg, 0.03 mmol, 69%). Decomposition until 335°C; ¹H NMR (400 MHz, pyridine-d₅) δ = 1.06–1.10 (m, 3H, 18²-CH₃), 1.49 (d, ³J = 7.48 Hz, 3H, 2¹-CH₃), 1.69 (d, ³J = 7.09 Hz, 3H, 3²-CH₃), 1.94 (s, 3H, 7¹-CH₃), 2.14 (s, 3H, 17¹-CH₃), 2.15 (d, J = 2.27 Hz, 3H, 13¹-CH₃), 2.41 (q, ³J = 7.47 Hz, 2H, 18¹-CH₂), 2.87 (t, ³J = 7.26 Hz, 4H, 8²-, 12²-CH₂), 3.18 (t, ³J = 7.58 Hz, 2H, 8¹-, 12¹-CH₂), 3.20 (t, ³J = 7.52 Hz, 2H, 8¹-, 12¹-CH₂), 3.36 (q, ³J = 7.97 Hz, 1H, 2-CH), 5.67 (s, 1H, 5-CH), 6.05 (s, 1H, 15-CH), 6.35 (q, ³J = 6.85 Hz, 1H, 3¹-CH), 7.24 (s, 1H, 10-CH); UV/Vis [acetonitrile + H₂O (1:1)]: λ_max = 271, 294, 382, 609, 630 nm (ε not determined); MS (ESI neg, methanol): m/z (%): 147 (4), 255 (4), 339 (5), 397 (1), 513 (1), 558 (6), 602 (100) [M − H]⁻, 663 (7), 723 (3); MS (MALDI-TOF pos, α-CHCA): m/z (%): 589 (2) [M − 15]⁺, 590 (12) [M − 14]⁺, 591 (5), 603 (6), 604 (35) [M⁺], 605 (100) [M + H]⁺, 606 (55), 607 (27), 608 (9), 626 (4), 627 (15) [M + Na]⁺, 628 (10); HRMS (EI): m/z: calcd for C₃₃H₃₈N₃O₆S [M + H]⁺: 604.247585; found 604.247875.

17,18-Dimethyl-thia-PCB (7): See synthesis of 6. Dark blue solid (12.0 mg, 0.02 mmol, 92%). Decomposition until 335°C; ¹H NMR (500 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.48 (d, ³J = 7.54 Hz, 3H, 2¹-CH₃), 1.69 (d, ³J = 7.26 Hz, 3H, 3²-CH₃), 1.88 (s, 3H, 7¹-CH₃), 2.11 (s, 3H, 18¹-CH₃), 2.14 (s, 3H, 17¹-CH₃), 2.14 (s, 3H, 13¹-CH₃), 2.87 (t, ³J = 7.29 Hz, 2H, 8²-, 12²-CH₂), 2.91 (t, ³J = 7.50 Hz, 2H, 8²-, 12²-CH₂), 3.18 (t, ³J = 7.27 Hz, 2H, 8¹-, 12¹-CH₂), 3.20 (t, ³J = 7.38 Hz, 2H, 8¹-, 12¹-CH₂), 3.37 (q, ³J = 7.57 Hz, 1H, 2-CH), 5.66 (s, 1H, 5-CH), 6.06 (s, 1H, 15-CH), 6.35 (dq, J_d = 2.03 Hz, ³J_q = 7.12 Hz, 1H, 3¹-CH), 7.23 (s, 1H, 10-CH); UV/Vis [acetonitrile + H₂O (1:1)]: λ_max = 271, 294, 382, 609, 629 nm (ε not determined); UV/Vis [H₂O + methanol (4:1)]: λ_max = 294, 382, 629 nm (ε not determined); MS (ESI neg, methanol): m/z (%): 265 (3), 339 (18), 397 (1), 441 (1), 485 (1), 544 (9), 588 (100) [M − H]^–, 649 (3), 708 (1); MS (MALDI-TOF pos, α-CHCA): m/z (%): 575 (5) [M − 15]⁺, 576 (9) [M − 14]⁺, 578 (5), 589 (12), 590 (33) [M⁺], 591 (100) [M + H]⁺, 592 (34), 593 (7), 613 (4) [M + Na]⁺, 614 (4); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₂H₃₅N₃NaO₆S [M + Na]⁺: 612.213878; found 612.213153.

18-Ethyl-17-methyl-carba-PCB (8): See synthesis of 4. Dark blue solid (7.3 mg, 0.01 mmol, 51%). Decomposition until 335°C; ¹H NMR (400 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.04 (t, ³J = 7.53 Hz, 3H, 18²-CH₃), 1.49 (d, ³J = 7.54 Hz, 3H, 2¹-CH₃), 1.70 (d, ³J = 7.20 Hz, 3H, 3²-CH₃), 2.02 (s, 3H, 7¹-CH₃), 2.15 (s, 3H, 17¹-CH₃), 2.15 (s, 3H, 13¹-CH₃), 2.32 (dq, J_d = 1.78 Hz, ³J_q = 7.56 Hz, 2H, 18¹-CH₂), 2.89 (t, ³J = 7.32 Hz, 2H, 8²-, 12²-CH₂), 2.93 (t, ³J = 7.58 Hz, 2H, 8²-, 12²-CH₂), 3.21 (t, ³J = 6.27 Hz, 2H, 8¹-, 12¹-CH₂), 3.23 (t, ³J = 6.35 Hz, 2H, 8¹-, 12¹-CH₂), 3.44–3.47 (m, 1H, 2-CH), 3.49 (s, 1H, 20-CH₂), 3.56 (s, 1H, 20-CH₂), 6.06 (s, 1H, 5-CH), 6.34 (dq, J_d = 2.22 Hz, ³J_q = 7.33 Hz, 1H, 3¹-CH), 6.54 (s, 1H, 15-CH), 7.23 (s, 1H, 10-CH); UV/Vis [methanol + H₂O (7:2)]: λ_max = 268, 294, 366, 608 nm (ε not determined); MS (ESI pos, methanol + methylene chloride): m/z (%): 586 (100) [M + H]⁺, 587 (35), 588 (8), 608 (65) [M + Na]⁺, 609 (25), 610 (5); MS (MALDI-TOF pos, α-CHCA): m/z (%): 571 (4) [M − 15]⁺, 572 (11) [M − 14]⁺, 573 (5), 585 (11), 586 (55) M⁺, 587 (100) [M + H]⁺, 588 (52), 589 (16), 590 (5), 608 (6), 609 (23) [M + Na]⁺, 610 (9), 625 (5) [M + K]⁺, 631 (7), 651 (4), 657 (4); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₄H₄₀N₃O₆ [M + H]⁺: 586.291161; found 586.291504.

17,18-Dimethyl-carba-PCB (9): See synthesis of 4. Purified by HPLC (column B + C) with a mixture of ammonium acetate buffer (10 mm, pH 7) and methanol (gradient 1:1–1:8) as eluent. Dark blue solid (6.1 mg, 0.01 mmol, 66%). Decomposition until 335°C; ¹H NMR (400 MHz, ¹H/¹H-COSY, pyridine-d₅) δ = 1.48 (d, ³J = 7.54 Hz, 3H, 2¹-CH₃), 1.69 (dd, J_d = 0.90 Hz, ³J_d = 7.05 Hz, 3H, 3²-CH₃), 1.78 (s, 3H, 18¹-CH₃), 1.99 (s, 3H, 7¹-CH₃), 2.14 (s, 3H, 17¹-CH₃), 2.15 (s, 3H, 13¹-CH₃), 2.89 (t, ³J = 7.18 Hz, 2H, 8²-, 12²-CH₂), 2.94 (t, ³J = 7.54 Hz, 2H, 8²-, 12²-CH₂), 3.22 (t, ³J = 6.98 Hz, 2H, 8¹-, 12¹-CH₂), 3.23 (t, ³J = 7.32 Hz, 2H, 8¹-, 12¹-CH₂), 3.44–3.48 (m, 1H, 2-CH), 3.50 (s, 1H, 20-CH₂), 3.60 (s, 1H, 20-CH₂), 6.07 (s, 1H, 5-CH), 6.33 (dq, J_d = 2.50 Hz, ³J_q = 7.34 Hz, 1H, 3¹-CH), 6.54 (s, 1H, 15-CH), 7.29 (s, 1H, 10-CH); UV/Vis [methanol + H₂O (4:1)]: λ_max = 268, 294, 365, 606 nm (ε not determined); MS (ESI pos, methanol): m/z (%): 242 (5), 572 (100) [M + H]⁺, 573 (36), 574 (7), 594 (6), 1143 (18) [2M + H]⁺, 1144 (13), 1145 (5); MS (ESI neg, methanol): m/z (%):284 (4), 339 (2), 570 (100) [M − H]⁻, 571 (22), 572 (7), 573 (1), 592 (1), 606 (1); MS (MALDI-TOF pos, α-CHCA): m/z (%): 523 (3), 558 (7) [M − 14]⁺, 559 (4), 571 (5), 572 (64) [M⁺], 573 (100) [M + H]⁺, 574 (56), 575 (14), 595 (3) [M + Na]⁺, 612 (4); HRMS (ESI pos, methanol + methylene chloride): m/z: calcd for C₃₃H₃₈N₃O₆[M + H]⁺: 572.275511; found 572.275946.

Results

Synthesis of novel oxa-, thia- and carba-“bilins” 4–9

A recently reported synthetic approach yielded six pyrryl-furanones, -thiophenones and -cyclopentenones (10–15, Fig. 2) (5) which represent the “right halves” of the convergent bilin synthesis, introduced by Gossauer et al. (7). These compounds were always generated as pairs in 9-ethyl-8-methyl and in 8,9-dimethyl substituted form (the substitution pattern referring to positions 17 and 18 of ring D in the native chromophore, Fig. 1). Systematic improvement of the condensation with the “left half” of PCB as well as of the workup procedure, with respect to the recently published method (8), yielded six PCB-analog compounds, each carrying an oxygen or a sulfur atom, or a methylene group at the position of the pyrrole nitrogen atom in ring D, still in form of their methyl propionates 23–28 (Fig. 3). The coupling reaction takes place in two steps (indicated as a and b in Fig. 3) such that an 8 h treatment of 22 with TFA leads to the removal of its t-butyl ester and a decarboxylation of both free carboxyl functions, followed by formation of the central carbon–carbon bond during reaction with the compounds 16–21. Subsequent addition of methanol allows the generation of the double bond. Saponification employing an ion exchange resin (9) then yielded the final analog chromophores 4–9 in a futher improved yield compared with the literature. Besides the target compounds, dimers of 10–12 formed unintended via their formylation (introduction of a formyl group, finally serving as the central methine bridge is a necessary intermediate step in bilin synthesis, see Fig. 2). These side products (29–31) were easily separated from the target compounds by chromatography and chemically characterized. Although they showed absorbance spectra akin to tetrapyrroles (Fig. 4), due to the small quantity they were not employed for any further studies. The absorbance spectra of compounds 4–9 showed the bilin-typical features such that the most intense absorption was between 363 and 382 nm, and a broad absorption band was found around 620 nm (see Fig. 5 and Table 1; only the spectra of the 18-ethyl-17-methyl compounds are shown, their dimethyl analogs absorb virtually identical). This long-wavelength band usually consists of two closely lying absorption bands with one or the other being of slightly higher intensity. The long-wavelength absorbance maxima of the six novel “PCB-analogs”4–9, together with those of PCB (2) and 17,18-dimethyl PCB (3) (10) can be arranged into two groups: The carba- and the oxa-derivatives 4–7 showed relatively blueshifted absorption maxima of 606 and 608 nm (9, 8), and of 611 and 613 nm (4, 5), whereas the thia- (6, 7) and aza-derivatives PCB (2) and 17,18-dimethyl PCB (3) show absorption maxima with a redshift of 20 and more nm compared with the former ones: 629, 630 nm for 7 and 6, respectively, and 627 and 630 nm for PCB (2) itself and for 17,18-dimethyl PCB (3) (see Table 1).

Table 1. UV/Vis absorption parameters of the PCB analogs in form of their dimethyl esters 23–28 (all values were determined in methanol), and the free acids 4–9 [all values were determined in methanol/H₂O (3:1–4:1, vol/vol)].

	λ _max [nm] (ε [L mol⁻¹ cm⁻¹])
23	–	279 (6400)	363 (15 200)	603 (4800)
24	–	275 (5800)	361 (14 200)	605 (4400)
25	–	279 (9800)	379 (13 100)	629 (5500)
26	–	279 (7000)	380 (13 300)	620 (3600)
27	267 (8600)	291 (7700)	361 (17 200)	604 (7900)
28	266 (8200)	293 (7300)	362 (16 800)	603 (7900)
2	270	279	363	603/627
3	–	n.d.	363	587/630
4	–	279	367	611
5	–	279	366	613
6	271	294	382	630
7	271	294	382	629
8	268	294	366	608
9	268	294	365	606

For reasons of limited material availability, extinction coefficients were not determined for the free acids. n.d., not determined.

Assembly of 4–9 with recombinant apophytochrome

All six analog chromophores formed stable chromoproteins, when incubated with the N-terminal 65 kDa domain of recombinant oat apophytochrome. The absorption spectra of their P_r-forms are much akin that of native phytochrome in showing an absorbance around 350–380 nm of lower intensity and a strong absorbance band in the long wavelength region of the spectrum (see Table 2, Fig. 6; note that the traces in the upper panel show also the spectra of the P_fr-forms after irradiation. In some cases, the changes between both forms are practically negligible, yielding nearly identical, coinciding absorption spectra.). The ratio between the absorbances in the 350–380 nm region and the P_r-peaks was not significantly different to PCB-assembled phytochrome (spectra not shown). Like for phytochrome, the absorbance bands showed a shoulder at the high energy part. This shoulder in the absorption band is most intense for the oxa-derivatives, whereas for the other four compounds, the thia- and the carba-derivatives, it is barely visible. For all six novel chromophores, the absorption bands of the P_r-forms are blueshifted compared with phytochrome or PCB-assembled phytochrome. Still, the binding with the apoprotein causes a bathochromic shift of the absorption of the free chromophores 2–5, 8, 9, except of the thia-derivatives 6, 7, which exhibit practically identical absorption maxima in free- and protein-bound form. Addition of PCB (2) to the novel phytochromes did not change their spectra nor their photochemical reactivity (see below), indicating that the hetero atom-substituted compounds 4–9 occupied the same binding pocket and formed stable covalent bonds to the canonical chromophore-binding cysteine residue of the apophytochrome. The spectral contributions from the analog-assembled phytochromes (structured, phytochrome-typical absorption bands) and from added PCB (2) (broad, unstructured absorption band) were clearly discernible. The absorption from unbound PCB (2) was lost after a gel filtration step, which left the analog-compound absorption bands undisturbed, giving further evidence for the covalently bound PCB-analogs.

Table 2. UV/Vis absorption and fluorescence emission parameters of the phytochromes, assembled with PCB (2) and its analogs 3–9.

	P_{r max} [nm]	P_{fr max} [nm]	Δ [cm⁻¹]	λ _ex [nm]	λ _em [nm]	Δ_St [cm⁻¹]	φ [× 10⁻³]
2	651	711	1296	656	675	429	3.7 ± 0.4*
3	655	714	1262	n.d.	n.d.	n.d.	n.d.
4	634	669	825	638	655	407	11.3 ± 0.8
5	635	670	823	638	653	360	8.6 ± 1.0
6	629	690	1405	632	656	579	14.2 ± 1.6
7	633	690	1305	637	660	547	9.3 ± 1.1
8	629	637†	199	622	648	645	11.4 ± 1.2
9	626	641†	373	628	647	468	12.4 ± 1.4

λ _ex, excitation wavelength; λ_ex, emission wavelength; Δ_St, Stokes’ shift (λ_em [cm⁻¹]–λ_ex [cm⁻¹]); n.d., not determined. *Reference (16); †Due to the low photochemical activity, these values are estimated, see text.

Steady state spectroscopy

Irradiation of phytochromes carrying chromophores 4–9 in the range of their long-wavelength P_r-band (616, 578 and 578 nm, respectively) revealed a very reduced photochemical reactivity. A P_fr-form as well as a difference spectrum between P_r and P_fr could be generated for the oxa- and the thia-derivatives, whereas the carba-compounds showed an extremely diminished photochemical reactivity making a determination of the P_r- and P_fr-maxima uncertain (Fig. 6). The hypsochromic shift of the P_r-maxima at ca. 20 nm, compared with PCB-assembled phytochrome (see previous paragraph), is also found for the P_fr-peaks. However, whereas the P_r-forms absorb in a fairly narrow spectral range, a discrimination is seen for the P_fr-forms: the longest absorption maximum is found for both thia-derivatives (690 nm), the oxa-derivatives generate P_fr-forms at 669 and 670 nm, respectively, and the most blueshifted P_fr-absorption was found for the carba-analogs at 637 and 641 nm, respectively (see Table 2, Fig. 6; the P_fr absorption maxima have been estimated as the centers of very shallow broad bands with 635–640 nm for 8 and 636–645 nm for 9).

Steady state fluorescence measurements showed an at least three-fold higher fluorescence quantum yield for any of the six novel chromoproteins (Table 2); however, the Stokes shifts were of similar size, ranging from 15 nm (360 cm⁻¹) in compound 5-assembled phytochrome up to 26 nm (645 cm⁻¹) in 8-assembled phytochrome (Fig. 7). The higher fluorescence quantum yield for the PCB analogs might be explained by a less intimate interaction with the protein, compared with PCB (2). This native chromophore is apparently optimally incorporated into the protein cavity, allowing a very efficient deactivation of the excited state by interactions with the protein surrounding.

Discussion

The light-induced switching between two thermally stable states (P_r and P_fr) is essential for the function of phytochromes. For the P_r-state, the three-dimensional structure of the chromophore-binding domain of D. radiodurans has revealed many precise interactions between chromophore and protein that stabilize the chromophore conformation in its dark state and direct the photochemical activity towards the CD ring region. An explanation of how the thermal stability of the P_fr-state is accomplished, however, remains still elusive, in particular, as the protein cavity around ring D is relatively unfunctionalized and is composed mostly of hydrophobic amino acid side chains. In addition, part of the chromophore binding site might be missing in the crystallized phytochrome domains, as the construct used for these studies consisted only of the PAS- and the GAF-domain. Yet, the crystal structure gives information on how the selectivity of double bond isomerization is directed towards the CD methine bridge. The rings A (by covalent attachment), B and C (by strong interactions of the propionate side chains with the protein) are so tightly fixed that only the CD methine bridge is allowed to isomerize. In particular the role of the propionate groups in dissociated state has been documented by employing a series of chemically synthesized, structurally modified tetrapyrroles (11), and also by the use of mono methyl esters of PCB (2) (12). In addition, the crystal structure of the chromophore-binding domain of the D. radiodurans phytochrome (13) revealed the strong hydrogen-bonding interaction between the protonated ring A, B and C nitrogens with the backbone carbonyl group of Asp207. A modification of the chromophore structure, as already documented by e.g. Inomata et al. (14), thus appeared as a valuable tool to study in greater detail the mechanisms which stabilize the bilin moiety in its photoisomerized P_fr-state. As it has already been shown (10,15), neither a change of the methyl substituent at position 17 nor of the vinyl- (PΦB, 1) or the ethyl-substituent (PCB, 2) at position 18 cause severe changes in the photochemistry of these modified phytochromes, except of a steric hindrance induced by an isopropyl group at position 17 (10). Besides interactions derived from the carbonyl function, which have not been addressed so far, the polar pyrrole ring with its hydrogen donor capacity might contribute to a stabilization of the P_fr-form of phytochrome.

In fact, the variation in polarity outlined here points to an involvement of the nitrogen atom and the attached proton of ring D within the chromophore-protein interactions. The situation is different in the nonprotein bound form: the spectral properties of all six novel compounds 4–7 are very similar to those of PCB (2), except of the fact that in free form the sulfur atom (compounds 6 and 7) and the nitrogen atom PCB (2), due to their greater polarizability, show a larger auxochromic effect than the oxygen atom (4 and 5) or the methylene group (8 and 9). This effect is leveled off in the protein-bound form, where, apparently, other factors govern the position of the P_r-absorption band. Here, the most redshifted absorption of the P_r-form is that of PCB-assembled phytochrome, indicating the importance of hydrogen bonds for the spectral properties of phytochromes. The difference in chemical reactivity becomes even more evident, when the photochemical properties were investigated. None of the three motifs, oxa-, thia- or carba-derivative, underwent a noticeable P_r-to-P_fr conversion, in fact, the diminished P_fr-formation of the carba-compounds 8 and 9 made a detection of the P_fr-maximum nearly impossible. The low photochemical reactivity does not concur with the wavelength difference between the P_r- and the P_fr-form. Although showing the most redshifted absorption maxima (P_r and P_fr), PCB-assembled phytochrome does not undergo the largest bathochromic shift upon P_fr-formation. The difference (in wavenumbers) between both forms of PCB-assembled phytochrome is 1296 and 1262 cm⁻¹ for the 17,18-dimethyl PCB derivative 3. Also the native chromophore PΦB (1) causes a similar P_r-P_fr shift of 1302 cm⁻¹. An even larger difference between the P_r- and P_fr-state is measured for both thia-substituted chromophores: 1405 cm⁻¹ for 6 and 1305 cm⁻¹ for its 17,18-dimethyl derivative 7. On the other hand, the oxa-substituted chromophores generate a significantly smaller shift: 825 and 823 cm⁻¹ for 4 and 5, respectively, and, although most restricted due to the uncertainty of detection, the carba-derivatives 8 and 9 show bathochromic shifts of only 199 and 373 cm⁻¹. Unfortunately, the low quantum yield of these phytochrome analogs impeded a time-resolved study of light-induced reactions which might have given information whether at all an isomerization took place which immediately reverted into the parent P_r-state.

Taken together, the results obtained with the hetero atom derivatives 4–9 of PCB (2) point to a strong contribution of the polarizable nitrogen atom in ring D of the chromophore. All six derivatives (4–9) show a lower polarity of the ring D than PCB (2), and no acidic hydrogen atom is present, giving a strong argument that the conformation of the chromophore in the P_fr-state is stabilized—amongst other forces—by hydrogen bonds and polar interactions between the nitrogen atom and its protein environment.

Acknowledgements— The excellent technical support by Manuela Trinoga in the HPLC purifications and by Helene Steffen in the protein preparations is gratefully acknowledged. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG, GA377/13) and by the Volkswagen-Foundation (I/82 627).

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Citing Literature

Volume84, Issue5

September/October 2008

Pages 1109-1117

This article also appears in:

Photosensory Biology

Interactions Between Chromophore and Protein in Phytochrome Identified by Novel Oxa-, Thia- and Carba-Chromophores

Abstract

Introduction