Chlorophyll Breakdown – On a Nonfluorescent Chlorophyll Catabolite from Spinach

In extracts of senescent leaves of spinach (Spinacia oleracea) that had degreened naturally after the onset of flowering, four colorless compounds, which had characteristic UV/VIS properties of nonfluorescent chlorophyll catabolites (NCCs), were detected by HPLC. From the extracts of 58.7 g of senescent leaves of Sp. oleracea, a two‐stage HPLC purification procedure provided ca. 15 μmol of So‐NCC‐2, the most abundant polar NCC in the leaves of this vegetable. So‐NCC‐2 was isolated as a slightly yellow powder and analyzed by spectroscopic means. The high‐resolution mass spectra indicated that So‐NCC‐2 has the same molecular formula as Hv‐NCC‐1 from barley (Hordeum vulgare), the first non‐green chlorophyll catabolite from a higher plant to be structurally analyzed. Homo‐ and hetero‐nuclear NMR spectroscopy indicated So‐NCC‐2 to have the same constitution as its epimer Hv‐NCC‐1, and to differ from the latter by the configuration at C(1). The catabolite from spinach could be identified with one of the products from OsO₄ dihydroxylation at the vinyl group of the main NCC from Cercidiphyllum japonicum. Chlorophyll breakdown in spinach and in C. japonicum apparently involves an enzyme‐catalyzed reduction that occurs with the same stereochemical sense at C(1), but opposite to that in barley.     

Hydroxymethylated Dioxobilins in Senescent Arabidopsis thaliana Leaves: Sign of a Puzzling Biosynthetic Intermezzo of Chlorophyll Breakdown

1‐Formyl‐19‐oxobilin‐type tetrapyrroles are characteristic, abundant products of chlorophyll breakdown in senescent leaves. However, in some leaves, 1,19‐dioxobilin‐type chlorophyll catabolites (DCCs) lacking the formyl group accumulate instead. A P450 enzyme was identified in in vitro studies that removed the formyl group of a primary fluorescent chlorophyll catabolite (pFCC) and generated fluorescent DCCs. These DCCs are precursors of isomeric nonfluorescent DCCs (NDCCs). Here, we report a structural investigation of the NDCCs in senescent leaves of wild‐type Arabidopsis thaliana. Four new NDCCs were characterized, two of which carried a stereoselectively added hydroxymethyl group. Such formal DCC hydroxymethylations were previously found in DCCs in leaves of a mutant of A. thaliana. They are now indicated to be a feature of chlorophyll breakdown in A. thaliana, associated with the specific in vivo deformylation of pFCC en route to NDCCs.     

Breakdown of Chlorophyll: A Tetrapyrrolic Chlorophyll Catabolite from Senescent Rape Leaves. Preliminary communication

The experiments leading to the isolation and to the elucidation of the constitution of Bn-NCC-1, a colourless non-fluorescent chlorophyll catabolite from senescent cotyledons of rape (Brassica napus L.), are described. A series of fast-atom-bombardment (FAB) mass and ¹H- and ¹³C-NMR spectral experiments are used to determine the constitution of the catabolite Bn-NCC-1. The structural information available indicates Bn-NCC-1 to be a 1-formyl-19-oxobilane, structurally related to ‘RP 14’, isolated earlier from artificially aged primary leaves of barley. The major differences between the constitution of the metal-free chlorophyll pheophorbide a and that of Bn-NCC-1 concern oxygenolytic opening of the porphinoid macrocycle at C(4)? C(5), saturation at the other meso positions, hydrolysis of the methyl-ester function, and functionalization by a malonic-acid unit of the side chain at C(8). This work provides for the first time the structural data of a chlorophyll-degradation product from senescent plant leaves formed under normal growth conditions.     

Coordination‐Driven Dimerization of Zinc Chlorophyll Derivatives Possessing a Dialkylamino Group

Zinc chlorophyll derivatives Zn‐1 – 3 possessing a tertiary amino group at the C3¹ position have been synthesized through reductive amination of methyl pyropheophorbide‐d obtained from naturally occurring chlorophyll‐a . In a dilute CH₂Cl₂ solution as well as in a dilute 10 %(v/v) CH₂Cl₂/hexane solution, Zn‐1 possessing a dimethylamino group at the C3¹ position showed red‐shifted UV/Vis absorption and intensified exciton‐coupling circular dichroism (CD) spectra at room temperature owing to its dimer formation via coordination to the central zinc by the 3¹‐N atom of the dimethylamino group. However, Zn‐2/3 bearing 3¹‐ethylmethylamino/diethylamino groups did not. The difference was dependent on the steric factor of the substituents in the tertiary amino group, where an increase of the carbon numbers on the N atom reduced the intermolecular N⋅⋅⋅Zn coordination. UV/Vis, CD, and ¹H NMR spectroscopic analyses including DOSY measurements revealed that Zn‐1 formed closed‐type dimers via an opened dimer by single‐to‐double axial coordination with an increase in concentration and a temperature decrease in CH₂Cl₂, while Zn‐2/3 gave open and flexible dimers in a concentrated CH₂Cl₂ solution at low temperature. The supramolecular closed dimer structures of Zn‐1 were estimated by molecular modelling calculations, which showed these structures were promising models for the chlorophyll dimer in a photosynthetic reaction center.     

Chlorophyll Breakdown in Maize: On the Structure of Two Nonfluorescent Chlorophyll Catabolites

In extracts of senescent leaves of the maize plant Zea mays, two colorless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named Zm-NCCs. The constitution of the two polar Zm-NCCs was determined by spectroscopic means, which confirmed both of these tetrapyrroles to have the basic ligand structure typical of the NCCs from (other) senescent higher plants. In the less polar catabolite, named Zm-NCC-2, the core structure was conjugated at the 8²-position with a glucopyranose unit. Zm-NCC-2 had the same constitution as Nr-NCC-2, an NCC from tobacco (Nicotiana rustica). Indeed, the two NCCs were identified (further) based on their HPL-chromatographic and NMR-spectroscopic properties. The more polar NCC from maize, Zm-NCC-1, differed from Zm-NCC-2 by carrying a dihydroxyethyl side chain instead of a vinyl group at the 3-position. In earlier work on polar NCCs, only separate glucopyranosyl- and dihydroxyethyl-functionalities were detected. Zm-NCC-1 thus is a new constitutional variant of the structures of NCCs from senescent higher plants.     

A Dioxobilin‐Type Fluorescent Chlorophyll Catabolite as a Transient Early Intermediate of the Dioxobilin‐Branch of Chlorophyll Breakdown in Arabidopsis thaliana

Chlorophyll breakdown in higher plants occurs by the so called “PaO/phyllobilin” path. It generates two major types of phyllobilins, the characteristic 1‐formyl‐19‐oxobilins and the more recently discovered 1,19‐dioxobilins. The hypothetical branching point at which the original 1‐formyl‐19‐oxobilins are transformed into 1,19‐dioxobilins is still elusive. Here, we clarify this hypothetical crucial transition on the basis of the identification of the first natural 1,19‐dioxobilin‐type fluorescent chlorophyll catabolite (DFCC). This transient chlorophyll breakdown intermediate was isolated from leaf extracts of Arabidopsis thaliana at an early stage of senescence. The fleetingly existent DFCC was then shown to represent the direct precursor of the major nonfluorescent 1,19‐dioxobilin that accumulated in fully senescent leaves.     

Chlorophyll Breakdown in Maize: On the Structure of Two Nonfluorescent Chlorophyll Catabolites

Summary. In extracts of senescent leaves of the maize plant Zea mays, two colorless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named Zm-NCCs. The constitution of the two polar Zm-NCCs was determined by spectroscopic means, which confirmed both of these tetrapyrroles to have the basic ligand structure typical of the NCCs from (other) senescent higher plants. In the less polar catabolite, named Zm-NCC-2, the core structure was conjugated at the 8²-position with a glucopyranose unit. Zm-NCC-2 had the same constitution as Nr-NCC-2, an NCC from tobacco (Nicotiana rustica). Indeed, the two NCCs were identified (further) based on their HPL-chromatographic and NMR-spectroscopic properties. The more polar NCC from maize, Zm-NCC-1, differed from Zm-NCC-2 by carrying a dihydroxyethyl side chain instead of a vinyl group at the 3-position. In earlier work on polar NCCs, only separate glucopyranosyl- and dihydroxyethyl-functionalities were detected. Zm-NCC-1 thus is a new constitutional variant of the structures of NCCs from senescent higher plants.     

Hypervalent radical formation probed by electron transfer dissociation of zwitterionic tryptophan and tryptophan‐containing dipeptides complexed with Ca2+ and 18‐crown‐6 in the gas phase

The relationship between peptide structure and electron transfer dissociation (ETD) is important for structural analysis by mass spectrometry. In the present study, the formation, structure and reactivity of the reaction intermediate in the ETD process were examined using a quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. ETD product ions of zwitterionic tryptophan (Trp) and Trp‐containing dipeptides (Trp‐Gly and Gly‐Trp) were detected without reionization using non‐covalent analyte complexes with Ca²⁺ and 18‐crown‐6 (18C6). In the collision‐induced dissociation, NH₃ loss was the main dissociation pathway, and loss related to the dissociation of the carboxyl group was not observed. This indicated that Trp and its dipeptides on Ca²⁺(18C6) adopted a zwitterionic structure with an NH₃⁺ group and bonded to Ca²⁺(18C6) through the COO^? group. Hydrogen atom loss observed in the ETD spectra indicated that intermolecular electron transfer from a molecular anion to the NH₃⁺ group formed a hypervalent ammonium radical, R‐NH₃, as a reaction intermediate, which was unstable and dissociated rapidly through N–H bond cleavage. In addition, N–C_α bond cleavage forming the z₁ ion was observed in the ETD spectra of Trp‐GlyCa²⁺(18C6) and Gly‐TrpCa²⁺(18C6). This dissociation was induced by transfer of a hydrogen atom in the cluster formed via an N–H bond cleavage of the hypervalent ammonium radical and was in competition with the hydrogen atom loss. The results showed that a hypervalent radical intermediate, forming a delocalized hydrogen atom, contributes to the backbone cleavages of peptides in ETD. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the Stereoselective Synthesis of Deuterated D‐Ribose Derivatives

In view of the importance of the site‐specific substitution of the H‐atom by its stable isotope ²H in a stereoselective/stereospecific manner at the pentose sugar residue, decreasing the spectral overcrowding in various regions of 1D and 2D homo‐ and heteronuclear correlation spectra of oligo‐DNA and ‐RNA, there is always a need for the development of new methods for the incorporation of ²H at different sites of a ribose. High‐yielding multistep syntheses of C(2)‐, and (5R)‐ and (5S)‐3,5‐deuterated ribose derivatives have been envisaged for the application of site‐specific incorporation of multilabeled nucleosides into oligomers to facilitate their structure elucidation by NMR spectroscopy. All syntheses started from D ‐glucose after proper derivatization. In the case of C(2), >97 atom‐% isotope was incorporated, employing an inversion of the configuration at C(2) as the key reaction. For C(5), two different routes were envisaged: on the one hand, deuterated achiral reagent was treated with a conformationally locked sugar moiety 15 , while, on the other, chiral protonated sources were used to transfer the H‐atom to a C(5)‐deuterated aldehyde 18 . In all cases, enantiomeric and isotopic purities were found to be as high as >97% as determined by NMR spectroscopy.     

Stereo‐ and Regioselective Phyllobilane Oxidation in Leaf Homogenates of the Peace Lily (Spathiphyllum wallisii): Hypothetical Endogenous Path to Yellow Chlorophyll Catabolites

In senescent leaves, chlorophyll typically is broken down to colorless and essentially photo‐inactive phyllobilanes, which are linear tetrapyrroles classified as “nonfluorescent” chlorophyll catabolites (NCCs) and dioxobilane‐type NCCs (DNCCs). In homogenates of senescent leaves of the tropical evergreen Spathiphyllum wallisii, when left at room temperature and extracted with methanol, the major endogenous, naturally formed NCC was regio‐ and stereoselectively oxidized (in part) to a mixture of its 15‐hydroxy and 15‐methoxy derivative. In the absence of methanol in the extract, only the 15‐OH‐NCC was observed. The endogenous oxidation process depended upon molecular oxygen. It was inhibited by carbon monoxide, as well as by keeping the leaf homogenate and extract at low temperatures. The remarkable “oxidative activity” was inactivated by heating the homogenate for 10 min at 70 °C. Upon addition of a natural epimeric NCC (epiNCC) to the homogenate of senescent or green Sp. wallisii leaves at room temperature, the exogenous epiNCC was oxidized regio‐ and stereoselectively to 15‐OH‐epiNCC and 15‐OMe‐epiNCC. The identical two oxidized epiNCCs were also obtained as products of the oxidation of epiNCC with dicyanodichlorobenzoquinone (DDQ). Water elimination from 15‐OH‐epiNCC occurred readily and gave a known “yellow” chlorophyll catabolite (YCC). The endogenous oxidation process, described here, may represent the elusive natural path from the colorless NCCs to yellow and pink coloured phyllobilins, which were found in (extracts of) some senescent leaves.     

(1S)‐1‐Phenylethanaminium 4‐{[(1S,2S)‐1‐hydroxy‐2,3‐dihydro‐1H,1′H‐[2,2′‐biinden]‐2‐yl]methyl}benzoate

The title molecular salt, C₈H₁₂N⁺·C₂₆H₂₁O₃⁻, contains a dimeric indane pharmacophore that demonstrates potent anti‐inflammatory activity. The indane group of the anion exhibits some disorder about the α‐C atom, which appears common to many structures containing this group. A model to account for the slight disorder was attempted, but this was deemed unsuccessful because applying bond‐length constraints to all the bonds about the α‐C atom led to instability in the refinement. The absolute configuration was determined crystallographically as S,S,S by anomalous dispersion methods with reference to both the Flack parameter and Bayesian statistics on Bijvoet differences. The configuration was also determined by an a priori knowledge of the absolute configuration of the (1S)‐1‐phenylethanaminium counter‐ion. The molecules pack in the crystal structure to form an infinite two‐dimensional hydrogen‐bond network in the (100) plane of the unit cell.     

Toluene Dioxygenase‐Catalyzed Synthesis of cis‐Dihydrodiol Metabolites from 2‐Substituted Naphthalene Substrates: Assignments of Absolute Configurations and Conformations from Circular Dichroism and Optical Rotation Measurements

cis‐Dihydrodiol metabolites have been isolated from naphthalene and six 2‐substituted naphthalene substrates. Their structures and absolute configurations have been determined by a combination of calculated (TDDFT) and experimentally based circular dichroism (CD) and optical rotation (OR) methods. The “inverse” styrene helicity rule is shown to be incorrect for the interpretation of the CD spectra of cis‐dihydrodiols. A striking conclusion is that CD spectra correlate directly with the helicity of the styrene chromophore: that is, the sign of the long‐wavelength Cotton effect is identical with the sign of styrene torsion angle, whereas the OR sign is dependent on the absolute configuration of the allylic carbon atom. The results demonstrate that a predictive model previously used for the determination of preferred regio‐ and stereoselectivity associated with TDO‐catalyzed cis‐dihydroxylation of substituted benzene substrates can now be successfully extended to substituted naphthalene substrates.     

Formation of Diastereoisomerically Pure Oxazaphospholes and Their Reaction to Chiral Phosphane‐Borane Adducts

Starting with achiral phosphines and (1S,2S)‐2‐(methylamino)‐1‐phenylpropan‐1‐ol ((+)‐pseudoephedrine) or (1R,2S)‐2‐(methylamino)‐1‐phenylpropan‐1‐ol ((−)‐ephedrine), as chiral auxiliaries, diastereoisomerically pure oxazaphospholes were prepared (Scheme 1). The configuration at the P‐atom is controlled by the configuration at the Ph‐substituted C(1) of (+)‐pseudoephedrine or (−)‐ephedrine, respectively. This was confirmed by X‐ray crystal‐structure analyses of two intermediate compounds in the synthesis route to the chiral triarylborane‐phosphane adducts.     

Reaction of Optically Active Oxiranes with Thiofenchone and 1‐Methylpyrrolidine‐2‐thione: Formation of 1,3‐Oxathiolanes and Thiiranes

The SnCl₄‐catalyzed reaction of (?)‐thiofenchone (=1,3,3‐trimethylbicyclo[2.2.1]heptane‐2‐thione; 10 ) with (R)‐2‐phenyloxirane ((R)‐ 11 ) in anhydrous CH₂Cl₂ at ?60° led to two spirocyclic, stereoisomeric 4‐phenyl‐1,3‐oxathiolanes 12 and 13 via a regioselective ring enlargement, in accordance with previously reported reactions of oxiranes with thioketones (Scheme 3). The structure and configuration of the major isomer 12 were determined by X‐ray crystallography. On the other hand, the reaction of 1‐methylpyrrolidine‐2‐thione ( 14a ) with (R)‐ 11 yielded stereoselectively (S)‐2‐phenylthiirane ((S)‐ 15 ) in 56% yield and 87–93% ee, together with 1‐methylpyrrolidin‐2‐one ( 14b ). This transformation occurs via an S_N2‐type attack of the S‐atom at C(2) of the aryl‐substituted oxirane and, therefore, with inversion of the configuration (Scheme 4). The analogous reaction of 14a with (R)‐2‐{[(triphenylmethyl)oxy]methyl}oxirane ((R)‐ 16b ) led to the corresponding (R)‐configured thiirane (R)‐ 17b (Scheme 5); its structure and configuration were also determined by X‐ray crystallography. A mechanism via initial ring opening by attack at C(3) of the alkyl‐substituted oxirane, with retention of the configuration, and subsequent decomposition of the formed 1,3‐oxathiolane with inversion of the configuration is proposed (Scheme 5).     

Stereochemistry of two new polyfunctionalized gem‐dihalo­cyclo­propanes

The two new gem‐dihalogeno­cyclo­propanes (1′S,3R)‐3‐(2′,2′‐di­chloro‐1′‐methyl­cyclo­propyl)‐6‐oxoheptanoic acid, C₁₁H₁₆­Cl₂O₃, (2), and (1′S,3R)‐3‐(2′,2′‐di­bromo‐1′‐methyl­cyclo­propyl)‐6‐oxoheptanoic acid, C₁₁H₁₆Br₂O₃, (3), are isostructural. Both present two stereogenic centers at C1′ and C3. The absolute configuration was determined by X‐ray methods. The cyclo­propyl rings are unsymmetrical, the shortest bond being distal with respect to the alkyl‐substituted C atom.     

Enhancement of Light Absorption Ability of Synthetic Chlorophyll Derivatives by Conjugation with a Difluoroboron Diketonate Group

The enhancement of the light absorption ability of synthetic chlorophyll derivatives is demonstrated. Chlorophyll derivatives directly conjugated with a difluoroboron 1,3‐diketonate group at the C3 position were synthesized from methyl pyropheophorbide‐d through Barbier acylmethylation of the C3‐formyl moiety, oxidation of the C3‐carbinol, and difluoroboron complexation of the diketonate. Electronic absorption spectra in a diluted solution showed that the synthetic conjugates gave an absorption band at λ=400–500 nm, with a Qy band shifted to a longer wavelength of λ≈700 nm. DFT calculations demonstrated that the absorption bands and redshifts were ascribable to the coupling of the LUMO of chlorin with that of the difluoroboron diketonate moiety. The introduction of a pyrenyl group at the C3³‐position of the conjugate afforded an additional charge‐transfer band over λ=500 nm, producing a pigment that bridged the green gap in standard chlorophylls.     

Ethyl 3‐[1‐(5,5‐dimethyl‐2‐oxo‐1,3,2‐dioxaphosphorin‐2‐yl)propan‐2‐ylidene]carbazate: a combined X‐ray and density functional theory (DFT) study

In the title compound, C₁₁H₂₁N₂O₅P, one of the two carbazate N atoms is involved in the C=N double bond and the H atom of the second N atom is engaged in an intramolecular hydrogen bond with an O atom from the dimethylphosphorin‐2‐yl group, which is in an uncommon cis position with respect to the carbamate group. The cohesion of the crystal structure is also reinforced by weak intermolecular hydrogen bonds. Density functional theory (DFT) calculations at the B3LYP/6‐311++g(2d,2p) level revealed the lowest energy structure to have a Z configuration at the C=N bond, which is consistent with the configuration found in the X‐ray crystal structure, as well as a less stable E counterpart which lies 2.0 kcal mol⁻¹ higher in potential energy. Correlations between the experimental and computational studies are discussed.     

Synthesis and Spectroscopic Characterization of the Benzaldehyde Schiff Base of S‐Allyldithiocarbazate and its Copper(II) Complex and the X‐ray Crystal Structure of Bis[S‐allyl‐β‐N‐benzylidene)dithiocarbazato]Copper(II)

S‐allyl‐β‐N‐(benzylidene)dithiocarbazate (sabdtc, 1 ), prepared from hydrazine hydrate, carbon disulfide, allyl bromide and benzaldehyde, reacts with copper(II) nitrate in ethanol solution to form the complex Cu(sabdtc)₂ ( 2 ). 1 function as a single negatively charged bidentate chelating ligand and coordinates via the azomethine nitrogen atom and thioenolate sulfur atom to the metal ion. 1 and 2 were characterized by elemental analyses, ¹H NMR, ¹³C NMR, UV/Vis, IR, and mass spectra. Spectroscopic evidences suggest a four‐coordinate distorted square planar structure for 2 . The molecular and crystal structure of 2 was determined by single crystal X‐ray analysis. Crystallographic data for 2 at 291(2) K: space group C2/c, a = 20.340(3), b = 15.503(3), c = 7.659(1) Å, β = 95.96(2)°, Z = 4, R₁ = 0.044 and wR2 = 0.0689.     

Synthesis,Structure, and Properties of a New Calcium(II) Complex of 1,10‐Phenanthroline, 1,2,4,5‐Benzenetetracarboxylic Acid,and a New Precursor to Produce Pure Phase Micro‐crystalline Calcite Particles

The self‐assembly of 1, 0‐phenanthroline (phen) and 1,2,4,5‐benzenetetracarboxylic acid(H₄btc) with Ca(NO₃)₂ gives rise to a two‐dimensional network structure coordination polymer, [Ca(phen)(btc)_0.5(H₂O)] ( 1 ), which was characterized by elemental analysis, IR spectroscopy, and single‐crystal X‐ray diffraction. This compound is monoclinic, space group C2/c, with Z = 8 in a unit cell with dimensions a = 21.744(3) Å, b = 10.0151(12) Å, c = 14.7122(17) Å, β = 110.2850(10)°. The structure contains one crystallographic unique Ca^II atom, one phen coordinated molecule and a half of btc^4– anion. The phen molecule acts as a didentate ligand chelating with a Ca^II atom, whereas the btc^4– anion acts as a μ₆‐bridge linking six different Ca^II atoms to form a two‐dimensional network with (4, 4) topological structure. The three dimensional stacking structures are formed by C–H ··· O hydrogen bonding and π–π interaction. The thermal stability and fluorescent properties of 1 were investigated. Calcite particles are produced by calcination of compound 1 at 580 °C. The obtained calcite was characterized by XRD and SEM analyses.     

Uncovering stereochemical relations in a compound with a stereogenic N—O axis: methyl 2‐(4‐methyl‐2‐thio­xo‐2,3‐di­hydro­thia­zol‐3‐yl­oxy)­propanoate

The geometry of racemic methyl 2‐(4‐methyl‐2‐thio­xo‐2,3‐di­hydro­thia­zol‐3‐yl­oxy)­propanoate, C₈H₁₁NO₃S₂, (I), is characterized by a distorted heterocyclic five‐membered ring and an enantiomorphic N‐alkoxy substituent, which is inclined at an angle of −68.8° to the thia­zole­thione plane in (M)‐(I). The unit cell consists of a 1:1 ratio of R,P‐ and S,M‐configured mol­ecules of (I). The combination of a P configuration at the N—O axis and an R configuration at the asymmetric propanoate C_β atom on one side, and an S,M configuration on the other side, is considered to originate from steric interactions. The largest substituent at the asymmetric propanoate C_β atom, i.e. the methoxycarbonyl group, resides above the methyl substituent; the medium‐sized propanoate γ‐methyl substituent points in the opposite direction with respect to the N—O bond, whereas the H atom is located above the C=S double bond of the thiazolethione subunit.