Dietmar A. Plattner

Electrospray mass spectrometry beyond analytical chemistry: studies of organometallic catalysis in the gas phase

Abstract Electrospray tandem mass spectrometry, one of the most important techniques for the characterization of biological macromolecules, has become increasingly popular as an analytical tool in inorganic/organometallic chemistry. Going one step further, we have shown that the coupling of electrospray ionization to ion-molecule techniques in the gas phase can yield detailed information about elementary reaction steps of transition-metal compounds with fully intact coordination spheres. This method opens a door to the study of extremely reactive intermediates that have previously not been within reach of condensed-phase techniques. Moreover, working in the gas phase, information about the intrinsic reactivity of the complex itself is obtained, thus excluding solvent effects, aggregation phenomena etc. We have demonstrated the usefulness of this method for various important transition-metal mediated reactions such as Cue5f8H activation, oxidation, and olefin polymerization. Through the utilization of collision-induced dissociation (CID) threshold methodology, the quantitative measurement of thermochemical data for metal-ligand bond energies and elemental reaction steps is possible. In several instances, we have demonstrated that the CID threshold methodology can be applied to molecules with relatively many degrees of freedom, yielding experimental thermochemical data of high quality. Both the qualitative and quantitative reaction studies of organometallic intermediates will contribute to deepen our mechanistic understanding of important catalytic reactions.

Structural Basis of Substrate Conversion in a New Aromatic Peroxygenase CYTOCHROME P450 FUNCTIONALITY WITH BENEFITS

Background: Aromatic peroxygenases (APOs) are the “missing link” between heme peroxidases and P450-monooxygenases. Results: Based on two crystal structures the substrate conversion of APOs is elucidated. Conclusion: The specific design of the heme cavity and the distal heme access channel govern substrate specificity. Significance: APOs can be utilized in biotechnology and organic synthesis having significant advantages when compared with cytochrome P450 enzymes. Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.

First Crystal Structure of a Fungal High-Redox Potential Dye-decolorizing Peroxidase: Substrate Interaction Sites and Long-Range Electron Transfer

Background: DyP-type peroxidases catalyze biotechnologically important reactions. Results: Based on the crystal structure of a fungal DyP, the conformational flexibility of Asp-168 is elucidated. Tyr-337 is identified as a surface-exposed substrate interaction site. Conclusion: Asp-168 and Tyr-337 are key residues directly involved in AauDyPI-catalysis. Significance: Peroxidases are biocatalysts, much sought after and ubiquitous enzymes in nature. Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a β-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

[P9]+[Al(ORF)4]−, the Salt of a Homopolyatomic Phosphorus Cation†

Positive at last: The first condensed-phase homopolyatomic phosphorus cation [P(9)](+) was prepared using a combination of the oxidant [NO](+) and weakly coordinating anion, [Al{OC(CF(3))(3)}(4)](-). [P(9)](+) consists of two P(5) cages linked by a phosphonium atom to give a D(2d)-symmetric Zintl cluster. NMR (see picture), Raman, and IR spectroscopy, mass spectrometry, and quantum-chemical calculations confirmed the structure.

Coordination chemistry of manganese–salen complexes studied by electrospray tandem mass spectrometry: the significance of axial ligands

Abstract Tandem mass spectrometric techniques in combination with high-level quantum chemical calculations have been employed to study the coordination chemistry of manganese– and oxomanganese–salen complexes in the gas phase. Electrospray ionization was used to transfer the ionic complexes from solution to the gas phase. The formation of five- versus six-coordinate manganese(III) species was subsequently probed by ion–molecule reactions with neutral ligands, e.g. acetonitrile, pyridine, alcohols, etc. The reactivity of the so far elusive oxomanganese(V)–salen complexes, readily accessible by fragmentation of μ-oxomanganese(IV) dimers, and their coordination chemistry was studied in the same way. Hybrid Hartree-Fock/density functional calculations have been performed to assess the geometries and energies of the triplet and quintet states of the manganese complexes in question. The effects of axial ligation on the geometry and reactivity of the oxo complex were found to be quite drastic. Finally, the epoxidation of olefins by oxomanganese(V)–salen was studied intramolecularly by tethering the substrate to the metal center. No indication for precoordination of the substrate as prerequisite for oxidation was found.

The Reaction of White Phosphorus with NO+/NO2+[Al(ORF)4]−: The [P4NO]+ Cluster Formed by an Unexpected Nitrosonium Insertion†

Despite decades of intense research into polyphosphorus chemistry, our knowledge of homoleptic polyphosphorus cations is still limited to the results of mass spectrometry and quantum chemical calculations. In general, the diamagnetic cage cations with an odd number of phosphorus atoms are more stable, with P9 , composed of two C2v symmetric P5 cages joined by a common phosphonium atom having special stability. This cage was found in one of the few types of simple inorganic phosphorus cluster cations that are known, that is, [P5R2] + (R = Cl, Br, I, Ph, DippN(Cl)NDipp (Dipp = 2,6-diisopropylphenyl)). Those P5 cages are formed by the formal insertion of carbene-analogous PR2 + fragments into the P P bond of P4 (see Ref. [9, 10] for Reviews on P4 activation). Stable carbenes also interact with P4, leading to compounds including P1 up to P12 moieties, depending on the electronic nature of the carbene. Larger cationic P7 cages were recently prepared, but all preparative approaches to true Pn + ions remained futile. However, we expected that an appropriate one-electron oxidant should be able to oxidize P4 (ionization energy (IE) 9.34 eV) and lead to phosphorus cluster cations Pn . Herein we give an account of the reaction of P4 with the salts [NO] [Al(OC(CF3)3)4] [13] (1; IE NO = 9.26 eV) and [NO2] [Al(OC(CF3)3)4] (2 ; IE NO2 = 9.59 eV. At least 2 was expected to be a strong enough oxidant to yield Pn + cations. The novel salt 2 was synthesized in 94 % yield from NO2[BF4] and Li[Al(OC(CF3)3)4] in SO2 solution with precipitation of insoluble Li[BF4]; it was fully characterized by X-ray diffraction and vibrational and NMR spectroscopy (for details, see the Supporting Information). Unexpectedly, the reactions of 1 and 2 with P4 in CH2Cl2 show an analogous process, regardless of the ratios of phosphorus to oxidant employed (between 3P:1 NOx + and 9P:1 NOx ). They form a red intermediate and yield the same yellow final product ([P4NO] [Al(OC(CF3)3)4] (3 ; Scheme 1). Compound 3 may be viewed as the insertion

Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H(2)O(2) for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 A resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 A resolution by molecular replacement.

Enhanced Reactivity of 2‐Rhodaoxetanes through a Labile Acetonitrile Ligand

New cationic, square-planar, ethene complexes [(Rbpa)RhI(C2H4)]+ [2a]--[2c]+ (Rbpa = N-alkyl-N,N-di(2-pyridylmethyl)amine; [2a]+: alkyl =R=Me; [2b]+: R = Bu; [2c]+: R = Bz) have been selectively oxygenated in acetonitrile by aqueous hydrogen peroxide to 2-rhoda(III)oxetanes with a labile acetonitrile ligand, [(Rbpa)RhIII(kappa2-C,O-CH2CH2O-)(MeCN)]+, [3a]+-[3c]+. The rate of elimination of acetaldehyde from [(Rbpa)RhIII(kappa2-C,O-CH2CH2O-)(MeCN)]+ increases in the order R = Me< R = Bu< R = Bz. Elimination of acetaldehyde from [(Bzbpa)RhIII(kappa2-C,O-CH2CH2O)(MeCN)]+ [3c]+, in the presence of ethene results in regeneration of ethene complex [(Bzbpa)RhI(C2H4)]+ [2c]+, and closes a catalytic cycle. In the presence of Z,Z-1,5-cyclooctadiene (cod) the corresponding cod complex [(Bzbpa)RhI(cod)]+ [6c]+ is formed. Further oxidation of [3c]+ by H2O2 results in the transient formylmethyl-hydroxy complex [(Bzbpa)RhIII(OH)[kappa1-C-CH2C(O)H]]+ [5c]+.

Radical formation on a conserved tyrosine residue is crucial for DyP activity

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates. This amino acid is conserved in eucaryotic DyPs but is missing in the structurally related chlorite dismutases (Clds). Dye-decolorizing peroxidases of procaryotic origin equally possess a conserved tyrosine in the same region of the polypeptide albeit not at the homologous position.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase – Identification of three new potential substrate-interaction sites

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450. Imidazole is confirmed as a competitive inhibitor with regard to peroxide binding. In contrast, bulky substrates such as anthraquinone dyes are converted at the enzyme surface. In the crystal structure a substrate analog, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), binds to a tyrosine-rich hollow harboring Y25, Y147, and Y337. Spin trapping with a nitric oxide donor uncovers Y229 as an additional tyrosine-based radical center in AauDyPI. Multi-frequency EPR spectroscopy further reveals the presence of at least one intermediate tryptophanyl radical center in activated AauDyPI with W377 as the most likely candidate.