New syntheses of the C,D-ring pyrromethenones of phytochrome and phycocyanin

Pyrromethenone 7, the C,D-ring segment of phytochrome (Pr, 4), has been prepared in an efficient fashion employing three new strategies. Each of these has potential advantages for the synthesis of labeled material. Our first approach is related to the Gossauer synthesis, with the difference that strong alkali is avoided in the condensation of the C- and D-ring components 8 and 17. The key silyloxypyrrole 17 was readily prepared on multigram scales beginning with inexpensive butyrolactone (10). A second synthesis began with 2-acetylbutyrolactone (41). The key steps involved conversion of 41 to the Z-enoltriflate 42, followed by Pd(0)-catalyzed coupling with trimethylsilylacetylene, p-chlorophenylselenide ring opening, and finally, amidation to afford the ring-D synthon 45 having the proper geometry and oxidation state for conversion to 7. Sonogashira coupling of 45 with the iodopyrrole 22, followed by oxidative elimination, and F(-)-induced 5-exo-dig cyclization of the resultant pyrroloalkyne 47, then completed the synthesis. In similar fashion, we have also prepared pyrromethenone 6, the C,D-ring segment of phycocyanin (2).     

The molecular topography of phytochrome: chromophore and apoprotein

Phytochrome serves as the photochromic receptor for a number of morphogenic and developmental responses to red light in higher plants. The photoreversible phototransformation of 124 kDa oat phytochrome involves several structural changes in the chromophore and the apoprotein, including a configurational/conformational isomerization and secondary/tertiary structural changes respectively. For example, there appears to be a specific interaction between the chromophore and the amino terminus segment in the Pfr form of phytochrome, which results in a photoreversible peptide folding of the amino terminus peptide chain. Other structural changes also accompany the phototransformation, as has been probed by peptide mapping, phosphorylation, and monoclonal antibodies.     

Determination of the chromophore structures in the photoinduced reaction cycle of phytochrome

The chromophore structures in the parent states Pr and Pfr as well as in the photocycle intermediate Lumi-R of oat phytochrome phyA are determined by comparison of the experimental resonance Raman spectra with calculated Raman spectra that have been obtained by density functional theory calculations (B3LYP) using scaled force fields. The spectra were calculated for various tetrapyrrole geometries including more than twenty different methine bridge isomers. For the parent states Pr and Pfr the best agreement in terms of vibrational frequencies, isotopic shifts, and Raman intensities was achieved with the ZZZasa and ZZEssa geometry, respectively. For the first intermediate Lumi-R, the chromophore geometry is concluded to be the ZZEasa configuration. These finding imply that the primary step of the photoactivation of phytochrome is the Z/E isomerization of the C-D methine bridge double bond, whereas the single bond remains in the anti conformation. The subsequent transition to the physiologically active state Pfr includes a (partial) single bond rotation of the A-B methine bridge.     

Interactions between chromophore and protein in phytochrome identified by novel oxa-, thia- and carba-chromophores

Abstract 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.     

On the molecular structure of the phytochrome chromophore: X-ray analysis of two 2,3-dihydrobilatriene-abc derivatives

The molecular and crystal structures of the two 2,3-dihydrobilatrienes-abc1 and2, representing model compounds for the phytochrome chromophore, were determined by X-ray crystallography at 97 K. Crystals of the racemate1 contain disordered regions. Both molecules are found to be ofall-(Z) configuration, assuming a helical conformation. The acidic hydrogen atoms are localized at the nitrogen atoms of rings A, C and D. A summary of the geometries of unsaturated five-membered rings as observed in four accurate low-temperature crystal structures of bilatrienes-abc is given.Herrn Prof.Josef Schurz zum 60. Geburtstag gewidmet.     

Heteronuclear NMR investigation on the structure and dynamics of the chromophore binding pocket of the cyanobacterial phytochrome Cph1

Structural changes of the chromophore in phytochrome proteins associated with its photocycle are still not fully understood. We use heteronuclear NMR to investigate the conformation and dynamics of the chromophore in the binding pocket of the cyanobacterial phytochrome Cph1. On the basis of distance information obtained from three-dimensional nuclear Overhauser enhancement (3D-NOESY) spectra using the photochemically intact photosensory module of Cph1 we demonstrate that the chromophore is in the ZZZssa form in the P(r) (red absorbing form) state and the ZZEssa form in the P(fr) (far-red absorbing form) state of the protein. While ZZZssa for the P(r) state is in agreement with a recently determined X-ray structure, no comparable information for the P(fr) state of photochemically intact phytochrome has been available up to now. In addition, the chromophore in the binding pocket of Cph1 exhibits a notable mobility, which is distinctly different in the two photostates.     

Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade

The pyoverdine chromophore is formed in a reaction involving a two-electron oxidation, a conjugate addition, and a second two-electron oxidation. This oxidative cascade can be carried out with polyphenol oxidase (PPO), MnO(2), and cell-free extracts from Pseudomonas aeruginosa. [reaction: see text]     

Assignments of the Pfr-Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using 15N and 13C isotopically labeled phycocyanobilin chromophore

The reversible red and far-red light-induced transitions of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 were investigated by Fourier transform infrared (FTIR) difference spectroscopy. High-quality light-induced Pfr-Pr difference FTIR spectra were recorded for the 58 kDa N-terminal domain of Cph1 by repetitive photochemical cycling and signal averaging. The Pfr-Pr difference spectra in H(2)O and D(2)O were very similar to those previously reported for full-length 85 kDa Cph1.(1) Published assignments were extended by analysis of the effects of (13)C and (15)N isotope substitutions at selected sites in the phycocyanobilin chromophore and by (15)N global labeling of the protein. The Pfr-Pr difference spectra were dominated by an amide I peak/trough at 1653 cm(-1)(+)/1631 cm(-1)(-) and a smaller amide II band at 1554 cm(-1). Labeling effects allowed specific chromophore assignments for the C(1)=O (1736 cm(-1)(-)/1724 cm(-1)(+)) and C(19)=O (1704 cm(-1)(-)) carbonyl vibrations, C=C vibrations at 1589 cm(-1)(+), and bands at 1537(-), 1512(+), 1491(-), 1163(+), 1151(-), 1134(+), 1109(-), and 1072(-) cm(-1) that must involve chromophore C-N bonds. A variety of additional changes were insensitive to isotope labeling of the chromophore. Effects of (15)N labeling of the protein were used to tentatively assign some of these to specific amino acid changes. Those insensitive to (15)N labeling included a protonated aspartic or glutamic acid at 1734 cm(-1)(-)/1722 cm(-1)(+) and a cysteine at 2575 cm(-1)(+)/2557 cm(-1)(-). Bands sensitive to (15)N protein labeling at 1487 cm(-1)(+)/1502 cm(-1)(-) might arise from trytophan and bands at 1261 cm(-1)(+)/1244 cm(-1)(-) and 1107 cm(-1)(-)/1095 cm(-1)(+) might arise from a histidine environment or protonation change. These assignments are discussed in light of the 15Z-E photoisomerization model of phototransformation and the associated protein conformational changes.     

Absorption and fluorescence spectra of open-chain tetrapyrrole pigments–bilirubins,biliverdins, phycobilins,and synthetic analogues

Open-chain tetrapyrroles are ubiquitous and abundant in living organisms (algae, animals, bacteria, and plants), including examples such as bilirubin, biliverdin, phycocyanobilin, phycoerythrobilin, and urobilin. The open-chain tetrapyrroles, collectively termed bilins, arise from biosynthesis or degradation of tetrapyrrole macrocycles. Bilins are now known to play a wide variety of biological roles encompassing light-harvesting (in phycobiliproteins), photomorphogenesis, signaling, and redox chemistry. The absorption spectra of bilins spans the ultraviolet (UV), visible, to near-infrared (NIR) regions depending on the degree of conjugation, thereby providing a wide range of colors from red/orange to blue/green. The fluorescence intensity of bilins is often quite low and hence fewer spectra are available, but can be increased substantially by structural rigidification, as evidenced by the wide use of biliproteins as fluorescent labels. The present article describes a database of absorption and fluorescence spectra of bilins from natural and synthetic origins for 220 compounds (270 absorption and 13 fluorescence spectral traces). Spectral traces of bilins published over the past ∼50 years have been digitized and assembled along with information concerning solvent, photochemical properties (molar absorption coefficient and fluorescence quantum yield), and literature references. The spectral traces (xy-coordinate data files) can be viewed, downloaded, and accessed at www.photochemcad.com. The accessibility of spectral traces in digital format should facilitate identification and quantitative calculations of interest in diverse scientific areas.     

Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved α,β-hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the α,β-hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.     

Carrier Protein Mediated Formation of the Dihydropyridazinone Ring in Actinopyridazinone Biosynthesis

Heterocycles with nitrogen-nitrogen (N−N) bonds are privileged building blocks of synthetic drugs. They are also found in natural products, although the biosynthetic logic behind them is poorly understood. Actinopyridazinones produced by Streptomyces sp. MSD090630SC-05 possess unique dihydropyridazinone rings that have been studied as core nuclei in several approved synthetic therapeutics. Herein, we performed gene knockouts and in vitro biochemical experiments to elucidate the major steps in actinopyridazinone biosynthesis, including the unprecedented carrier protein mediated machinery for dihydropyridazinone formation.     

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H⁺-ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.     

Biosynthesis and Ether‐Bridge Formation in Nargenicin Macrolides

The nargenicin family of antibiotics are macrolides containing a rare ether‐bridged cis‐decalin motif. Several of these compounds are highly active against multi‐drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron‐α‐ketoglutarate‐dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13‐deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides.     

Tricarbocyanines with dihydropyran,dihydrothiapyran, and N-methyltetrahydropyridine rings in the chromophore

Glutaconic dialdehyde dianil salts, the ,-carbon atoms of which are included in dihydropyran, dihydrothiapyran, or N-methyltetrahydropyridine rings, were synthesized. Tricarbocyanine dyes with epoxydimethylene, epithiodimethylene, or N-methylepiminodimethylene groupings were synthesized by condensation of the dianil salts with a 2-methylbenzothiazole quaternary salt. The color of the dyes is discussed.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 175–178, February, 1976.     

Formation of the early photoproduct lumi-R of cyanobacterial phytochrome cph1 observed by ultrafast mid-infrared spectroscopy

The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.     

Pyrylocyanines. 31. Benzopyrylocarbocyanines with bridged groupings in the chromophore

4,4-Tetramethylene- and 4,4-pentamethylenebisflavylium salts and their sulfur- and selenium-containing analogs were synthesized. Trimethinecyanine dyes with ethylene and trimethylene bridged groupings in the chromophore were obtained from these salts. The factors that affect the color of these dyes were analyzed thoroughly.For Communication 30 see [1].Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1319–1323, October, 1993.