2-vinyl Group Research Articles

Photoredox-Catalyzed C-Indolyl/Quinolyl Glycosylation from 2-Styrylisocyanides and Glycosyl Bromides.

Indole and quinoline structures are present in numerous biologically active molecules, making the synthesis of their glycosylation products a subject of extensive research and interest in drug development. Here, we report a photoredox strategy for the synthesis of C-indolyl and C-quinolyl glycosides using 2-styrylisocyanides and glycosyl bromides as building blocks. This approach offers mild reaction conditions, high α-selectivity, and scalability for large-scale reactions. The radical cyclization mode switching from 5-exo-trig to 6-endo-trig is achieved by selecting the substituents on the 2-vinyl group. This strategy enriches the toolbox of heterocyclic glycosylation methods and benefits advances in research on heteroaryl-based pharmaceuticals.

Rhodium-Catalyzed Regioselective Formal Hydroacylation of Vinyl Epoxides toward Esters Involving β-Carbon Cleavage.

Herein we disclose the first example of the formal hydroacylation reactions of vinyl epoxides with chelating aldehydes enabled by rhodium catalysis for the efficient construction of functionalized esters. Detailed investigations of the mechanistic pathway reveal that the presence of a 2-vinyl group is essential in contributing to the success of this regioselective reaction, which might proceed through β-carbon cleavage as the key procedure.

Styrene-containing Phosphine-sulfonate Ligands for Nickel- and Palladium-catalyzed Ethylene Polymerization

A series of phosphine-sulfonate ligands bearing 2-, 3- and 4-vinylphenyl on the phosphorus atom were designed, synthesized, characterized and investigated in Ni- and Pd-catalyzed ethylene polymerization. The structure of the phosphine-sulfonate Pd complex bearing 2-vinylphenyl on the phosphorus atom showed 2,1-insertion for the 2-vinyl group. The phosphine-sulfonate Ni complex bearing 2-vinylphenyl resulted in significantly increased thermal stability and polyethylene molecular weights (Mn = 3.69×104 g·mol−1 at 80 °C) versus the counterparts bearing 3-/4-vinyl groups as well as previously reported phosphine-sulfonate Ni complexes bearing bulky biaryl substituents.

Unique Tyr-heme double cross-links in F43Y/T67R myoglobin: an artificial enzyme with a peroxidase activity comparable to that of native peroxidases.

The X-ray crystal structure of F43Y/T67R myoglobin revealed unique Tyr-heme double cross-links between Tyr43 and the heme 4-vinyl group, which represents a novel post-translational modification of heme proteins. Moreover, with the feature of a distal His-Arg pair, the designed artificial enzyme exhibited a peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase.

The sensing property of charge-transfer chemosensors tuned by acceptors for colorimetric and fluorometric detection of CN−/HCN in solutions and in gas phase

Cyanide ion (CN−) is highly toxic, and its wide use in industry poses an immediate threat to public safety. Consequently, it is desired to develop reliable and facile methods to detect this dangerous chemical. In this work, four diethylaminoquinoline derivatives (3a–3d) with different 2-vinyl substituents have been constructed for the detection of CN−/HCN via a Michael addition. The electrophilicity of the β-carbon of 2-vinyl group depends on the electron-withdrawing ability of the substituents, which tune the photophysical properties of sensors and sensing products by varying intramolecular charge transfer (ICT). Among these sensors, 3a displays the best sensing behavior towards CN−, that is, rapid colorimetric and ratiometric fluorescent response (∼10 s), high sensitivity (LOD = 15 nM) and high selectivity in solutions. The sensing mechanism of a two-step reaction has been clearly demonstrated by 1H NMR, HRMS and tracking and titration experiments. Moreover, the test strip with 3a has been fabricated with a polyethyl oxide and successfully applied to detect HCN gas (60 ppm) visually and selectively.

Formation of Cys-heme cross-link in K42C myoglobin under reductive conditions with molecular oxygen

The structure and function of heme proteins are regulated by diverse post-translational modifications including heme-protein cross-links, with the underlying mechanisms not well understood. In this study, we introduced a Cys (K42C) close to the heme 4-vinyl group in sperm whale myoglobin (Mb) and solved its X-ray crystal structure. Interestingly, we found that K42C Mb can partially form a Cys-heme cross-link (termed K42C Mb-X) under dithiothreitol-induced reductive conditions in presence of O2, as suggested by guanidine hydrochloride-induced unfolding and heme extraction studies. Mass spectrometry (MS) studies, together with trypsin digestion studies, further indicated that a thioether bond is formed between Cys42 and the heme 4-vinyl group with an additional mass of 16 Da, likely due to hydroxylation of the α‑carbon. We then proposed a plausible mechanism for the formation of the novel Cys-heme cross-link based on MS, kinetic UV–vis and electron paramagnetic resonance (EPR) studies. Moreover, the Cys-heme cross-link was shown to fine-tune the protein reactivity toward activation of H2O2. This study provides valuable insights into the post-translational modification of heme proteins, and also suggests that the Cys-heme cross-link can be induced to form in vitro, making it useful for design of new heme proteins with a non-dissociable heme and improved functions.

Coupling of helix E-F motion with the O-nitrito and 2-nitrovinyl coordination in myoglobin

Myoglobin (Mb) is known to react slowly with nitirite to form the green pigment by NO2− cordination to the heme Fe in the O-binding nitrito (O1NO2) mode and to the heme 2-vinyl position. Nitrite is a powerful oxidizing agent and a biological reservoir for NO that has been implicated in a variety of aerobic biological systems. Accordingly, it is important to elucidate the nature and variety of NO2− reaction mechanisms with Mb. We have performed principal component analysis (PCA, or essential dynamics) on Molecular Dynamics trajectories of all MbNO2 coordination states to resolve the most important motions in the protein at 298K. We show that the coordination or removal of NO2− to/from the heme iron is associated mainly with a motion of helix E and the coordination of NO2− to the 2-vinyl is associated with a motion of helix F and a correlated motion of helices E-F. This latter correlated motion can be attributed to the interaction of Val68 and Ile107 with the 2-nitrovinyl moiety. The resonance Raman results show that coordination of NO2− to the 2-vinyl is increased at pH6.0 demonstrating that the amide protons in the F helix are not protected from access of solvent water and the helix F motion allows solvent access to the 2-vinyl group, without affecting the coordination to the heme Fe.

Enhanced Dehaloperoxidase Activity of F43Y Myoglobin with a Novel Thyrosine–Heme Crosslink

Sperm whale myoglobin mutant, F43Y Mb, with Tyr43 forming a novel crosslink to the heme 4-vinyl group, was found to exhibit dramatically enhanced dehaloperoxidase activity compared to that of wild-type Mb (ca. 115-fold), which is even beyond the catalytic efficiency of the native dehaloperoxidase from Amphitrite ornata (ca. 9-fold), indicating a potential application for bioremediation.

A novel tyrosine-heme C−O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins.

Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins.

Preparation of amino-substituted indenes and 1,4-dihydronaphthalenes using a one-pot multireaction approach: total synthesis of oxybenzo[c]phenanthridine alkaloids.

Allylic trichloroacetimidates bearing a 2-vinyl or 2-allylaryl group have been designed as substrates for a one-pot, two-step multi-bond-forming process leading to the general preparation of aminoindenes and amino-substituted 1,4-dihydronaphthalenes. The synthetic utility of the privileged structures formed from this one-pot process was demonstrated with the total synthesis of four oxybenzo[c]phenanthridine alkaloids, oxychelerythrine, oxysanguinarine, oxynitidine, and oxyavicine. An intramolecular biaryl Heck coupling reaction, catalyzed using the Hermann-Beller palladacycle was used to effect the key step during the synthesis of the natural products.

Horseradish Peroxidase Inactivation: Heme Destruction and Influence of Polyethylene Glycol

Horseradish peroxidase (HRP) mediates efficient conversion of many phenolic contaminants and thus has potential applications for pollution control. Such potentially important applications suffer however from the fact that the enzyme becomes quickly inactivated during phenol oxidation and polymerization. The work here provides the first experimental data of heme consumption and iron releases to support the hypothesis that HRP is inactivated by heme destruction. Product of heme destruction is identified using liquid chromatography with mass spectrometry. The heme macrocycle destruction involving deprivation of the heme iron and oxidation of the 4-vinyl group in heme occurs as a result of the reaction. We also demonstrated that heme consumption and iron releases resulting from HRP destruction are largely reduced in the presence of polyethylene glycol (PEG), providing the first evidence to indicate that heme destruction is effectively suppressed by co-dissolved PEG. These findings advance a better understanding of the mechanisms of HRP inactivation.

Modulation of Nitric Oxide Reactivity by Heme Posttranslational Modification in the Cyanobacterial Hemoglobin, GlbN

Synechococcus sp. PCC 7002 is a model cyanobacterium capable of thriving under conditions that promote the buildup of reactive oxygen and nitrogen species (ROS/RNS). This organism harbors a hemoglobin (GlbN) that is thought to aid in the detoxification of RNS including NO.GlbN achieves hexacoordinate heme (FeIII/FeII) binding using His70 (proximal) and His46 (distal). In vitro, this coordination scheme protects against H2O2-induced damage, facilitates electron transfer (ET), and lowers redox potential. Under reducing conditions, His117 attacks the 2-vinyl group to form a covalent crosslink. The irreversible posttranslational modification (PTM) of GlbN yields GlbN-A. Ligands such as CO, O2, and NO inhibit the facile PTM. This and other observations suggest that both GlbN and GlbN-A are active in the cell. How does the His-heme PTM influence GlbN reactivity towards NO?NMR and optical spectroscopies were used to study the differences in NO binding, NO oxidation, ET, and NO reduction. We observed that GlbN and GlbN-A can form unusually stable diamagnetic FeIII-NO complexes. Both FeII-O2 proteins appear capable of NO dioxygenation, where ET is typically rate-determining. Each GlbN exhibits facile ET, with measured self-exchange rates slightly slower than cytochrome b5. A difference in NO reactivity is observed under strongly reducing conditions: surprisingly, unmodified GlbN is capable of reducing NO to nitrosyl hydride (HNO). Additionally, FeII-NO binding in GlbN results in immediate heme loss.The results provide some insight into the ability of GlbN to protect the cyanobacterium from RNS/ROS. The data suggest that GlbNs can serve as NO dioxygenases, but may not require a dedicated reductase because of their propensity for facile ET. Additionally, an unusual NO reductase-like activity may also exist for GlbN, and the His-heme PTM appears to eliminate this chemistry.Supported by NSF MCB 0843439

P51 Activation of hydrogen sulfide by heme-superoxide limits triggering heme proteins oxidative products

Hydrogen sulfide (H2S) has been regarded as a poisonous gas with a wide spectrum of cytotoxic effects. However, a new controversial role is emerging for H2S in the chemistry of biological systems. It has been found that H2S is synthesized endogenously in mammalian tissues and that it functions as a neuromodulator, and a smooth muscle relaxant [1] . However, the reaction of H2S with Hb and Mb, in the presence of H2O2or O2, results in covalent modification of the heme pyrrole ring bearing the 4-vinyl group, generating the so-called sulfmyoglobin and sulfhemoglobin derivatives. These sulfheme derivatives have lower O2 affinity affecting the heme protein functionality, resulting in a rare blood disease called sulfhemoglobinemia. For the formation of the sulfheme complex, the presence of a heme-ferryl and RSH species are needed, in addition to the adequate oriented histidine in the heme distal site [2] , [3] . However, the structural transient species are not known. Analysis of the sulfheme complex to further comprehend the mechanism and action mode of H2S in the human body, as well as in other organisms is essential. Therefore, it is important to define the relationship between H2S and hemeproteins since these proteins are prime targets. Here, we focus on determining the key intermediates controlling the appropriate interactions that lead to the formation of the sulfheme protein. To accomplish this aim, EPR analysis on Myoglobin (Mb) in the presence of H2S and H2O2 or O2 was performed. The data showed bands associated with ferric low spin species. Thus, the results indicate that the final sulfheme complex is a six coordinated low spin specie. Also, the data showed the development of bands associated characteristic thiol radical bands around 2.051 g and 2.032 g values. Interestingly, the formation of the thiol radicals was not observed upon the reaction of Mb with H2S, in the absence of O2 or H2O2. These data suggests that a thiol radical is a key intermediate in the mechanism that leads to the sulfMb complex formation. As a consequence, the results produced insight into the chemistry of H2S with hemeproteins where H2S reacts as an anti-oxidant, limiting the availability of the heme ferryls species to react with nearby amino acids. Therefore, the evidence shows that a single Histidine E7 amino acid controls the selective toxicity and reactivity of H2S with different hemoglobins, limiting ROS activity with heme proteins.

Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

Bovine liver cytochrome b (5) (cyt b (5)), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b (5) N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b (5) N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b (5) family, domestic silkworm cyt b (5) (DS cyt b (5)), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b (5) N57C, and Cys56, a naturally occurring cysteine in DS cyt b (5), have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b (5) maturation involves a b-type intermediate.

Chemical reactivity of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H(2)O(2).

Over-Expression and Characterization of Bacillus subtilis Heme O Synthase

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme O synthase in Bacillus subtilis (CtaB) has eight transmembrane helices and catalyses the transfer of a farnesyl group from farnesyl diphosphate to the 2-vinyl group on pyrrole ring A of ferrous heme B. In this study, we constructed the overproduction system for the B. subtilis CtaB in Escherichia coli. We isolated His(7)-CtaB by affinity chromatography and demonstrated the presence of the heme-binding site in heme O synthase. His(7)-CtaB binds substoichiometric amounts of heme B and O, substrate and unreleased product, respectively. Mutagenesis studies suggest that strictly conserved His199 present at the extra-cellular side of helix 5 would serve as the heme-binding site. We are hoping that the overproducing system for heme O synthase would help understanding of detailed mechanism on heme O biosynthesis and X-ray crystallographic studies.

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C–N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme–protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron.

On the Functional Role of a Water Molecule in Clade 3 Catalases: A Proposal for the Mechanism by Which NADPH Prevents the Formation of Compound II

X-ray structures of the 13 different monofunctional heme catalases published to date were scrutinized in order to gain insight in the mechanism by which NADPH in Clade 3 catalases may protect the reactive ferryloxo intermediate Compound I (Cpd I; por (*+)Fe (IV)O) against deactivation to the catalytically inactive intermediate Compound II (Cpd II; porFe (IV)O). Striking similarities in the molecular network of the protein subunits encompassing the heme center and the surface-bound NADPH were found for all of the Clade 3 catalases. Unique features in this region are the presence of a water molecule (W1) adjacent to the 4-vinyl group of heme and a serine residue or a second water molecule hydrogen-bonded to both W1 and the carbonyl group of a threonine-proline linkage, with the proline in van der Waals contact with the dihydronicotinamide group of NADPH. A mechanism is proposed in which a hydroxyl anion released from W1 undergoes reversible nucleophilic addition to the terminal carbon of the 4-vinyl group of Cpd I, thereby producing a neutral porphyrin pi-radical ferryloxo (HO-por (*)Fe (IV)O) species of reduced reactivity. This structure is suggested to be the elusive Cpd II' intermediate proposed in previous studies. An accompanying proton-shifting process along the hydrogen-bonded network is believed to facilitate the NADPH-mediated reduction of Cpd I to ferricatalase and to serve as a funnel for electron transfer from NADPH to the heme center to restore the catalase Fe (III) resting state. The proposed reaction paths were fully supported as chemically reasonable and energetically feasible by means of density functional theory calculations at the (U)B3LYP/6-31G* level. A particularly attractive feature of the present mechanism is that the previously discussed formation of protein-derived radicals is avoided.

Structural chemistry involved in information detection and transmission by gas sensor heme proteins: Resonance Raman investigation

Abstract A variety of heme-containing gas sensor proteins have been discovered by gene analysis from bacteria to mammals. In general, these proteins are composed of an N-terminal heme-containing sensor domain and a C-terminal catalytic domain. Binding of O2, CO, or NO to the heme causes a change in the structure of heme, which alters the protein conformation in the vicinity of the heme, and the conformational change is propagated to the catalytic domain, leading to regulation of the protein activity. This mini-review summarizes the recent resonance Raman studies obtained with both visible and UV excitation sources for two O2 sensor proteins, EcDOS and HemAT-Bs. These investigations have shown the role of heme propionate hydrogen-bonding interactions in communicating the heme structural changes, which occur upon ligand binding, from heme to the protein moiety. Furthermore, it is deduced that the contact interactions between the heme 2-vinyl group and the surrounding residues are also important for signal transmission from heme to protein in EcDOS.

Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242-16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem-methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.