Sustainable production of catechol derivatives from waste tung nutshell C/G-type lignin via heterogeneous Cu-NC catalytic oxidation.

The sustainable production of catechol derivatives is a challenging task. Catechyl (C) and guaiacyl (G) lignins coexisting in waste tung nutshells are promising feedstocks to form valuable catechol derivatives, but the depolymerization of C/G lignin typically involves a catalytic reductive process that cannot produce these oxidized aromatic chemicals. Herein, we demonstrated that the sustainable production of catechol derivative aldehydes and acids from C/G lignin could be achieved through a heterogeneous copper-catalyzed oxidative process. Under optimized conditions, the Cu-NC-800 catalyst affords a 43.5 mg g-1 yield (8.9 wt%, based on Klason lignin) of aromatic aldehydes (protocatechuic aldehyde, vanillin) and acids (protocatechuic acid, vanillic acid). XRD and XPS analyses showed that CuO and Cu2O may be the active species during the heterogeneous oxidation of the Cu-NC-800 catalyst. This study opens new opportunities for the sustainable production of catechol derivatives from C/G-type lignin.

Lignin-derived polyphenols with enhanced antioxidant activities by chemical demethylation and their structure-activity relationship.

Lignin valorization to biobased polyphenols antioxidants is increasingly attractive in the modern industry due to their inherent phenolic structures. Herein, lignin-derived polyphenols with enhanced antioxidant activities were prepared from the most available technical lignin including organosolv lignin (OL), alkali lignin (AL), and enzyme lignin (EL) by iodocyclohexane (ICH) chemical demethylation. The structural evolution of lignin indicated that the CAr-OCH3 group and the CAr-O-Calkyl side-chain could be effectively transformed into the CAr-OH group, resulting in a significant increase of the phenolic-OH content and a slight decrease of the molecular weight. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenging activity was in the order of ICHOL-24 > ICHAL-24 > ICHEL-24 ≈ FA > BHT, and the IC50 value of ICHOL-24 was 0.56 times lower than that of BHT. The structure-activity relationship demonstrated the activities were quasi-linearly related to phenolic-OH contents and could be affected by molecular weights. The H/G/S proportions of lignin could be an indicator for accurate screening of efficient lignin-derived polyphenols antioxidants (LPA). It was preliminarily estimated to have economic feasibility for producing LPA from technical lignin by demethylation compared with synthetic or natural antioxidants. This work will help to develop efficient biobased antioxidants for lignin valorization.

Switching Amine Oxidation from Imines to Nitriles by Carbon-Hydrogen Bond Activation via Strong Base Modified Strategy.

Precisely controlling the product selectivity from the complex reaction is always an attractive topic in the catalysis field. In this paper, the Ru/strong base junction formed by the redox strategy was demonstrated as an efficient catalyst to switch the selectivity in aerobic oxidation of benzylamines. The zirconia-supported ruthenium (Ru-ZrO2) catalyst could catalyze benzylamine oxidation coupling to imines; however, the potassium oxide strong base modified zirconia-supported ruthenium (Ru-K-ZrO2) catalyst could catalyze benzylamine oxidation dehydrogenation to nitriles. Insight into the mechanism showed that the base modified catalyst had excellent dehydrogenation ability which was assisted by the C-H bond activation and changed the reaction pathway. The strong base modified strategy may provide a new approach for controlling the performance of heterogeneous catalysts and product selectivity.

Multi-functional porous organic polymers for highly-efficient solid-phase extraction of ß-agonists and ß-blockers in milk.

A simple, accurate, and highly sensitive analytical method was developed in this study for the determination of ten ß-agonists and five ß-blockers in milk. In this method, new adsorbent phosphonic acid-functionalized porous organic polymers were synthesized through a direct knitting method. The synthesis procedure of the materials and the extraction conditions (such as the composition of loading buffer and eluent) were optimized. Benefitting from the high surface area (545-804 m2 g-1), multiple functional framework and good porosity, the phosphonic acid-functionalized porous organic polymers showed a high adsorption rate and high adsorption capacity for ß-agonists (224 mg g-1 and 171 mg g-1 for clenbuterol and ractopamine, respectively). The analytes were quantified by ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. It showed a good linearity (with R 2 ranging from 0.9950 to 0.9991 in the linear range of 3-5 orders of magnitude), with low limits of quantification ranging from 0.05 to 0.25 ng g-1. The limits of detection of the method for the analytes were measured to be in the range of 0.02 to 0.1 ng g-1. The recoveries of target analytes from real samples on the material were in the range of 62.4-119.4% with relative standard deviations of 0.6-12.1% (n = 4). Moreover, good reproducibility of the method was obtained with the interday RSD being lower than 11.7% (n = 5) and intraday RSD lower than 12.2% (n = 4). The proposed method was accurate, reliable and convenient for the simultaneous analysis of multiple ß-agonists and ß-blockers. Finally, the method was successfully applied for the analysis of such compounds in milk samples.

Wettability Control of Co-SiO2@Ti-Si Core-Shell Catalyst to Enhance the Oxidation Activity with the In Situ Generated Hydroperoxide.

With the aim of utilizing O2 as an oxidant, cascade reaction strategy was usually employed by first transforming O2 into the in situ generated hydroperoxide and then oxidized the substrate. To combine the two steps more efficiently to get a higher reaction rate, a series of core-shell catalysts with core and shell having different wettabilities were designed. The catalysts were characterized by transmission electron microscopy, UV-vis spectroscopy, Fourier transform infrared, sessile water contact angle, among other methods. These catalysts were applied in the research of the diphenyl sulfide oxidation by the in situ generated hydroperoxide derived from ethylbenzene oxidation. Through control experiments, the hydrophobic modification in the shell and core will influence different steps of the overall cascade reaction. Further insight into the reaction illustrated that the overall reaction rate was not simply an adduct of the promotion effects from the two steps, which was mainly attributed to the inhibition effect for the co-oxidation of ethylbenzene with diphenyl sulfide. Through the guidance of the relationship, a rationally designed core-shell catalyst with appropriate modifying organic groups showed an enhanced performance of the overall cascade reaction. The rational design of the catalysts would provide a reference for other cascade reactions.

Formation of Strong Basicity on Covalent Triazine Frameworks as Catalysts for the Oxidation of Methylene Compounds.

Porous solid bases are increasingly attractive for applications in green chemistry and heterogeneous catalysis under relatively mild conditions. Here, covalent triazine frameworks (CTFs) were first applied as a support for the porous solid strong bases through a redox process between the base precursor KNO3 and CTFs, leading to a relatively low calcination temperature (400 °C). As a result, porous organic frameworks possessing ordered microstructures as well as strong basic sites were successfully synthesized. The materials were characterized by X-ray diffraction, Fourier transform infrared, high-resolution transmission electron microscopy, temperature programmed desorption of CO2, and so forth. The obtained solid bases displayed remarkable catalytic activity in the aerobic oxidation of methylene compounds, and the yield of fluorenones could reach 93.6% at 120 °C, which was nearly 3 times higher than that of the control catalyst. The current research may present a new idea for the construction of porous organic polymers with strong basicity.

Oral Delivery of Propofol with Methoxymethylphosphonic Acid as the Delivery Vehicle.

Phosphonamidate 3a of methoxymethylphosphonic acid (MMPA) with propofol (1) and l-alanine ethyl ester was found to be an efficient scaffold for the oral delivery of compound 1. The synthesis and evaluation of MMPA based phosphonamidates of compound 1, HSK3486 (2), and other phenolic drugs revealed the general application of MMPA as the effective delivery vehicle for phenolic drugs. On the basis of plasma concentrations of compound 1 and SN38 (14), the oral bioavailability of compound 3a and 15 in beagle dogs was found to be 97.6% and 34.1%, respectively.

The TBK1-binding domain of optineurin promotes type I interferon responses.

Pathogen-associated molecular pattern (PAMP) recognition leads to TANK-binding kinase (TBK1) polyubiquitination and activation by transautophosphorylation, resulting in IFN-ß production. Here, we describe a mouse model of optineurin insufficiency (OptnΔ(157) ) in which the TBK1-interacting N-terminus of optineurin was deleted. PAMP-stimulated cells from OptnΔ(157) mice had reduced TBK1 activity, no phosphorylation of optineurin Ser(187) , and mounted low IFN-ß responses. In contrast to pull-down assays where the presence of N-terminus was sufficient for TBK1 binding, both the N-terminal and the ubiquitin-binding regions of optineurin were needed for PAMP-induced binding. This report establishes optineurin as a positive regulator TBK1 via a bipartite interaction between these molecules.

Optineurin insufficiency impairs IRF3 but not NF-κB activation in immune cells.

Optineurin is a widely expressed polyubiquitin-binding protein that has been implicated in regulating cell signaling via its NF-κB essential modulator-homologous C-terminal ubiquitin (Ub)-binding region. Its functions are controversial, with in vitro studies finding that optineurin suppressed TNF-mediated NF-κB activation and virus-induced activation of IFN regulatory factor 3 (IRF3), whereas bone marrow-derived macrophages (BMDMs) from mice carrying an optineurin Ub-binding point mutation had normal TLR-mediated NF-κB activation and diminished IRF3 activation. We have generated a mouse model in which the entire Ub-binding C-terminal region is deleted (Optn(470T)). Akin to C-terminal optineurin mutations found in patients with certain neurodegenerative diseases, Optn(470T) was expressed at substantially lower levels than the native protein, allowing assessment not only of the lack of Ub binding, but also of protein insufficiency. Embryonic lethality with incomplete penetrance was observed for 129 × C57BL/6 Optn(470T/470T) mice, but after further backcrossing to C57BL/6, offspring viability was restored. Moreover, the mice that survived were indistinguishable from wild type littermates and had normal immune cell distributions. Activation of NF-κB in Optn(470T) BMDM and BM-derived dendritic cells with TNF or via TLR4, T cells via the TCR, and B cells with LPS or anti-CD40 was normal. In contrast, optineurin and/or its Ub-binding function was necessary for optimal TANK binding kinase 1 and IRF3 activation, and both Optn(470T) BMDMs and bone marrow-derived dendritic cells had diminished IFN-ß production upon LPS stimulation. Importantly, Optn(470T) mice produced less IFN-ß upon LPS challenge. Therefore, endogenous optineurin is dispensable for NF-κB activation but necessary for optimal IRF3 activation in immune cells.

Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP.

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.

A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families.

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-theta and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.

Protein kinase specificity. A strategic collaboration between kinase peptide specificity and substrate recruitment.

Specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. Specificity is determined by two critical elements: (1) peptide specificity of the kinase, i. e., preferential phosphorylation of S/T/Y residues surrounded by particular patterns of amino acids; and (2) recruitment, i. e., increasing the frequency of encounter between kinase and substrate. Historically, the importance of peptide specificity was studied first, but it has been somewhat overshadowed by emerging emphasis on the importance of recruitment. Recent studies confirm and extend understanding of the relative contribution of these two elements. Peptide specificity always constrains the range of sites that can be phosphorylated by a kinase. Only when recruitment is very strong, as in the case with autophosphorylation, can markedly suboptimal substrates be phosphorylated.

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

Kinase peptide specificity: improved determination and relevance to protein phosphorylation.

Specificity of phosphorylation is critical to signal transduction. Recent emphasis on colocalization of substrate and kinase has eclipsed emphasis on peptide specificity, i.e., kinase preference for particular amino acids surrounding the phosphorylation site. We describe an approach to determining peptide specificity by using positional scanning of biotinylated oriented peptide libraries and insights emerging from those determinations. We accurately determine preference (or disfavor) for residues at a given substrate position (such as P+2) by comparison of in vitro phosphorylation of peptide libraries differing by a single residue at that position. By analysis of all positions near the phosphorylation site, position-specific scoring matrices are generated and used both to understand the basis of specificity and to predict phosphorylation. PKC-delta and -zeta predictions have been validated rigorously by comparisons with measured phosphorylation. The results demonstrate specificity and sensitivity (80-90%) much better than the previous predictive method. These predictions can be accessed at http://mpr.nci.nih.gov. The accuracy of the specificity determination allows identification of an important difference in peptide specificity between these closely related kinases; Ile/Leu at the P-1 position is disfavored by PKC-zeta but not PKC-delta. Our findings and visual representation of peptide specificity highlight the importance of disfavored residues. Finally, analysis of 124 experimentally determined PKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. Thus, position-specific scoring matrices generated by this method provide a foundation for quantitative analyses of kinase specificity and improved predictions of previously determined physiologically relevant phosphorylation sites.