Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Production of Biomass-Derived p-Hydroxybenzamide: Synthesis of p-Aminophenol and Paracetamol.

As we work to transition the modern society that is based on non-renewable chemical feedstocks to a post-modern society built around renewable sources of energy, fuels, and chemicals, there is a need to identify the renewable resources and processes for converting them to platform chemicals. Herein, we explore a strategy for utilizing the p-hydroxybenzoate in biomass feedstocks (e. g., poplar and palm trees) and converting it into a portfolio of commodity chemicals. The targeted bio-derived product in the first processing stage is p-hydroxybenzamide produced from p-hydroxybenzoate esters found in the plant. In the second stage a continuous reaction process converts the p-hydroxybenzamide to p-aminophenol via the Hofmann rearrangement and recovers the unreacted p-hydroxybenzamide. In the third stage the p-aminophenol can be acetylated to form paracetamol, which is readily isolated by liquid/liquid extraction at >95 % purity and an overall p-hydroxybenzamide-to-paracetamol process yield of ~90 %. We explore how utilization of protecting groups alters the challenges in this process and expands the portfolio of possible products to include p-(methoxymethoxy)aniline and N-acetyl-p-(methoxymethoxy)aniline. These target compounds could become value-added renewably-sourced platform chemicals that could be used to produce biodegradable plastics, pigments, and pharmaceuticals.

QT-GWAS: A novel method for unveiling biosynthetic loci affecting qualitative metabolic traits.

Although the plant kingdom provides an enormous diversity of metabolites with potentially beneficial applications for humankind, a large fraction of these metabolites and their biosynthetic pathways remain unknown. Resolving metabolite structures and their biosynthetic pathways is key to gaining biological understanding and to allow metabolic engineering. In order to retrieve novel biosynthetic genes involved in specialized metabolism, we developed a novel untargeted method designated as qualitative trait GWAS (QT-GWAS) that subjects qualitative metabolic traits to a genome-wide association study, while the conventional metabolite GWAS (mGWAS) mainly considers the quantitative variation of metabolites. As a proof of the validity of QT-GWAS, 23 and 15 of the retrieved associations identified in Arabidopsis thaliana by QT-GWAS and mGWAS, respectively, were supported by previous research. Furthermore, seven gene-metabolite associations retrieved by QT-GWAS were confirmed in this study through reverse genetics combined with metabolomics and/or in vitro enzyme assays. As such, we established that CYTOCHROME P450 706A5 (CYP706A5) is involved in the biosynthesis of chroman derivatives, UDP-GLYCOSYLTRANSFERASE 76C3 (UGT76C3) is able to hexosylate guanine in vitro and in planta, and SULFOTRANSFERASE 202B1 (SULT202B1) catalyzes the sulfation of neolignans in vitro. Collectively, our study demonstrates that the untargeted QT-GWAS method can retrieve valid gene-metabolite associations at the level of enzyme-encoding genes, even new associations that cannot be found by the conventional mGWAS, providing a new approach for dissecting qualitative metabolic traits.

Contrasting geochemical and fungal controls on decomposition of lignin and soil carbon at continental scale.

Lignin is an abundant and complex plant polymer that may limit litter decomposition, yet lignin is sometimes a minor constituent of soil organic carbon (SOC). Accounting for diversity in soil characteristics might reconcile this apparent contradiction. Tracking decomposition of a lignin/litter mixture and SOC across different North American mineral soils using lab and field incubations, here we show that cumulative lignin decomposition varies 18-fold among soils and is strongly correlated with bulk litter decomposition, but not SOC decomposition. Climate legacy predicts decomposition in the lab, and impacts of nitrogen availability are minor compared with geochemical and microbial properties. Lignin decomposition increases with some metals and fungal taxa, whereas SOC decomposition decreases with metals and is weakly related with fungi. Decoupling of lignin and SOC decomposition and their contrasting biogeochemical drivers indicate that lignin is not necessarily a bottleneck for SOC decomposition and can explain variable contributions of lignin to SOC among ecosystems.

Lignin deconstruction by anaerobic fungi.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition.

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3'H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.

Flavonoids naringenin chalcone, naringenin, dihydrotricin, and tricin are lignin monomers in papyrus.

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the ß-ether linkages, indicating that at least a fraction of each was integrated into the lignin as ß-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3' and C-5' positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4'-O-ß bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon-carbon linkages, including 3'- or 5'-ß linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

Lignin lags, leads, or limits the decomposition of litter and soil organic carbon.

Lignin's role in litter and soil organic carbon (SOC) decomposition remains contentious. Lignin decomposition was traditionally thought to increase during midstage litter decomposition, when cellulose occlusion by lignin began to limit mass loss. Alternatively, lignin decomposition could be greatest in fresh litter as a consequence of co-metabolism, and lignin might decompose faster than bulk SOC. To test these competing hypotheses, we incubated 10 forest soils with C4 grass litter (amended with 13 C-labeled or unlabeled lignin) over 2 yr and measured soil respiration and its isotope composition. Early lignin decomposition was greatest in 5 of 10 soils, consistent with the co-metabolism hypothesis. However, lignin decomposition peaked 6-24 months later in the other five soils, consistent with the substrate-limitation hypothesis; these soils were highly acidic. Rates of lignin, litter, and SOC decomposition tended to converge over time. Cumulative lignin decomposition was never greater than SOC decomposition; lignin decomposition was significantly lower than SOC decomposition in six soils. Net nitrogen mineralization predicted lignin decomposition ratios relative to litter and SOC. Although the onset of lignin decomposition can indeed be rapid, lignin still presents a likely bottleneck in litter and SOC decomposition, meriting a reconsideration of lignin's role in modern decomposition paradigms.

Enrichment of Lignin-Derived Carbon in Mineral-Associated Soil Organic Matter.

A modern paradigm of soil organic matter proposes that persistent carbon (C) derives primarily from microbial residues interacting with minerals, challenging older ideas that lignin moieties contribute to soil C because of inherent recalcitrance. We proposed that aspects of these old and new paradigms can be partially reconciled by considering interactions between lignin decomposition products and redox-sensitive iron (Fe) minerals. An Fe-rich tropical soil (with C4 litter and either 13C-labeled or unlabeled lignin) was pretreated with different durations of anaerobiosis (0-12 days) and incubated aerobically for 317 days. Only 5.7 ± 0.2% of lignin 13C was mineralized to CO2 versus 51.2 ± 0.4% of litter C. More added lignin-derived C (48.2 ± 0.9%) than bulk litter-derived C (30.6 ± 0.7%) was retained in mineral-associated organic matter (MAOM; density >1.8 g cm-3), and 12.2 ± 0.3% of lignin-derived C vs 6.4 ± 0.1% of litter C accrued in clay-sized (<2 µm) MAOM. Longer anaerobic pretreatments increased added lignin-derived C associated with Fe, according to extractions and nanoscale secondary ion mass spectrometry (NanoSIMS). Microbial residues are important, but lignin-derived C may also contribute disproportionately to MAOM relative to bulk litter-derived C, especially following redox-sensitive biogeochemical interactions.

Thirty Years of (TMS)3SiH: A Milestone in Radical-Based Synthetic Chemistry.

This review is an update on tris(trimethylsilyl)silane, TTMSS, in organic chemistry, focusing on the advancements of the past decade. The overview includes a wide range of chemical processes and synthetic strategies under different experimental conditions, including functional group insertion and transformations, as well as preparation of complex molecules, natural products, polymers, surfaces, and new materials. These results reveal how TTMSS has matured over the past 30 years, and they further establish its value as a free radical reagent with widespread academic and industrial applications.

Lignin decomposition is sustained under fluctuating redox conditions in humid tropical forest soils.

Lignin mineralization represents a critical flux in the terrestrial carbon (C) cycle, yet little is known about mechanisms and environmental factors controlling lignin breakdown in mineral soils. Hypoxia is thought to suppress lignin decomposition, yet potential effects of oxygen (O2 ) variability in surface soils have not been explored. Here, we tested the impact of redox fluctuations on lignin breakdown in humid tropical forest soils during ten-week laboratory incubations. We used synthetic lignins labeled with 13 C in either of two positions (aromatic methoxyl or propyl side chain Cß ) to provide highly sensitive and specific measures of lignin mineralization seldom employed in soils. Four-day redox fluctuations increased the percent contribution of methoxyl C to soil respiration relative to static aerobic conditions, and cumulative methoxyl-C mineralization was statistically equivalent under static aerobic and fluctuating redox conditions despite lower soil respiration in the latter treatment. Contributions of the less labile lignin Cß to soil respiration were equivalent in the static aerobic and fluctuating redox treatments during periods of O2 exposure, and tended to decline during periods of O2 limitation, resulting in lower cumulative Cß mineralization in the fluctuating treatment relative to the static aerobic treatment. However, cumulative mineralization of both the Cß - and methoxyl-labeled lignins nearly doubled in the fluctuating treatment relative to the static aerobic treatment when total lignin mineralization was normalized to total O2 exposure. Oxygen fluctuations are thought to be suboptimal for canonical lignin-degrading microorganisms. However, O2 fluctuations drove substantial Fe reduction and oxidation, and reactive oxygen species generated during abiotic Fe oxidation might explain the elevated contribution of lignin to C mineralization. Iron redox cycling provides a potential mechanism for lignin depletion in soil organic matter. Couplings between soil moisture, redox fluctuations, and lignin breakdown provide a potential link between climate variability and the biochemical composition of soil organic matter.

A highly diastereoselective oxidant contributes to Ligninolysis by the white rot basidiomycete Ceriporiopsis subvermispora.

The white rot basidiomycete Ceriporiopsis subvermispora delignifies wood selectively and has potential biotechnological applications. Its ability to remove lignin before the substrate porosity has increased enough to admit enzymes suggests that small diffusible oxidants contribute to delignification. A key question is whether these unidentified oxidants attack lignin via single-electron transfer (SET), in which case they are expected to cleave its propyl side chains between Cα and Cß and to oxidize the threo-diastereomer of its predominating ß-O-4-linked structures more extensively than the corresponding erythro-diastereomer. We used two-dimensional solution-state nuclear magnetic resonance (NMR) techniques to look for changes in partially biodegraded lignin extracted from spruce wood after white rot caused by C. subvermispora. The results showed that (i) benzoic acid residues indicative of Cα-Cß cleavage were the major identifiable truncated structures in lignin after decay and (ii) depletion of ß-O-4-linked units was markedly diastereoselective with a threo preference. The less selective delignifier Phanerochaete chrysosporium also exhibited this diastereoselectivity on spruce, and a P. chrysosporium lignin peroxidase operating in conjunction with the P. chrysosporium metabolite veratryl alcohol did likewise when cleaving synthetic lignin in vitro. However, C. subvermispora was significantly more diastereoselective than P. chrysosporium or lignin peroxidase-veratryl alcohol. Our results show that the ligninolytic oxidants of C. subvermispora are collectively more diastereoselective than currently known fungal ligninolytic oxidants and suggest that SET oxidation is one of the chemical mechanisms involved.

Evidence from Serpula lacrymans that 2,5-dimethoxyhydroquinone Is a lignocellulolytic agent of divergent brown rot basidiomycetes.

Basidiomycetes that cause brown rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of brown rot fungi have arisen independently from ligninolytic white rot ancestors via loss of lignocellulolytic enzymes. Brown rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe(3+) in colonized wood. Since there is little evidence that white rot fungi produce these metabolites, one question is the extent to which independent lineages of brown rot fungi may have evolved different Fe(3+) reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry in Serpula lacrymans, a brown rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay by S. lacrymans. We found also that variegatic acid failed to reduce in vitro the Fe(3+) oxalate chelates that predominate in brown-rotting wood and that it did not drive Fenton chemistry in vitro under physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain brown rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.

Kinetic studies on the formation of sulfonyl radicals and their addition to carbon-carbon multiple bonds.

The reactions of α-hydroxyl and α-alkoxyl alkyl radicals with methanesulfonyl chloride (MeSO(2)Cl) have been studied by pulse radiolysis at room temperature. The alkyl radicals were produced by ionizing radiation of N(2)O-saturated aqueous solution containing methanol, ethanol, isopropanol, or tetrahydrofuran. The transient optical absorption spectrum consisted of a broad band in the region 280-380 nm with a maximum at 320 nm typical of the MeSO(2)(•) radical. The rate constants in the interval of 1.7 × 10(7)-2.2 × 10(8) M(-1) s(-1) were assigned to an electron-transfer process that leads to MeSO(2)Cl(•-), subsequently decaying into MeSO(2)(•) radical and Cl(-). The rate constants for the addition of CH(3)SO(2)(•) to acrolein and propiolic acid were found to be 4.9 × 10(9) M(-1) s(-1) and 5.9 × 10(7) M(-1) s(-1), respectively, in aqueous solutions and reversible. The reactivity of tosyl radical (p-CH(3)C(6)H(4)SO(2)(•)) toward a series of alkenes bearing various functional groups was also determined by competition kinetics in benzene. The rate constants for the addition of tosyl radical to alkenes vary in a much narrower range than the rate constants for the reverse reaction. The stabilization of the adduct radical substantially contributes to the increase of the rate constant for the addition of tosyl radical to alkenes and, conversely, retards the ß-elimination of tosyl radical.

In search of efficient 5-endo-dig cyclization of a carbon-centered radical: 40 years from a prediction to another success for the Baldwin rules.

Despite being predicted to be stereoelectronically favorable by the Baldwin rules, efficient formation of a C-C bond through a 5-endo-dig radical cyclization remained unknown for more than 40 years. This work reports a remarkable increase in the efficiency of this process upon beta-Ts substitution, which led to the development of an expedient approach to densely functionalized cyclic 1,3-dienes. Good qualitative agreement between the increased efficiency and stereoselectivity for the 5-endo-dig cyclization of Ts-substituted vinyl radicals and the results of density functional theory analysis further confirms the utility of computational methods in the design of new radical processes. Although reactions of Br atoms generated through photochemical Ts-Br bond homolysis lead to the formation of cyclic dibromide side products, the yields of target bromosulfones in the photochemically induced reactions can be increased by recycling the dibromide byproduct into the target bromosulfones through a sequence of addition/elimination reactions at the exocyclic double bond. Discovery of a relatively efficient radical 5-endo-dig closure, accompanied by a C-C bond formation, provides further support to stereoelectronic considerations at the heart of the Baldwin rules and fills one of the last remaining gaps in the arsenal of radical cyclizations.

[Bis(trimethylsilyl)amido-kappaN]{tert-butyl[(E)-2-(tert-butylimino)ethyl]amido-kappa(2)N,N'}tin(II), a key intermediate in the synthesis of 1,3-di-tert-butyl-2,3-dihydro-1H-1,3,2-diazastannole.

The title compound, [Sn(C10H21N2)(C6H18NSi2)], contains the Sn(II) centre in a trigonal-pyramidal geometry. The basal plane is formed by three N atoms and the fourth apical position is occupied by a stereoactive lone pair. The Sn atom is displaced from the plane of the three N atoms by 1.1968 (12) A. The Sn-N bonds are highly polarized toward the N atoms, as confirmed by natural bonding orbital analysis.

Brønsted base-modulated regioselectivity in the aerobic oxidative amination of styrene catalyzed by palladium.

Palladium(II)-catalyzed aerobic oxidative amination of styrene with oxazolidinone proceeds with catalyst-controlled regioselectivity: (CH3CN)2PdCl2 (1) and (Et3N)2PdCl2 (2) catalyze formation of the anti-Markovnikov and Markovnikov enecarbamate products, 3 and 4, respectively. Kinetic studies and deuterium kinetic isotope effects demonstrate that these two reactions possess different rate-limiting steps, and the data indicate that the product regiochemistry arises from the presence or absence of an effective Brønsted base in the reaction. In the presence of a Brønsted base such as triethylamine or acetate, the kinetically preferred Markovnikov aminopalladation adduct of styrene is trapped via rapid deprotonation of a zwitterionic intermediate and leads to formation of 4. In the absence of an effective Brønsted base, however, slow deprotonation of this adduct enables aminopalladation to be reversible, and product formation proceeds through the thermodynamically preferred anti-Markovnikov aminopalladation adduct to yield 3.

Aerobic oxidative amination of unactivated alkenes catalyzed by palladium.

The first examples of palladium-catalyzed oxidative amination of unactivated alkyl olefins have been identified. To be successful, these reactions must be conducted under cocatalyst-free conditions that involve direct dioxygen-coupled turnover of the palladium catalyst. The oxidative amination products of norbornene and other cyclic alkenes implicate a cis-aminopalladation mechanism.

Thiyl radical-mediated cleavage of allylic C-N bonds: scope, limitations, and theoretical support to the mechanism.

Thiols mediate the radical isomerization of allylic amines into enamines. The reaction results in the cleavage of the allylic C-N bond, after treatment with aqueous HCl. The mechanism involves the abstraction of an allylic hydrogen alpha to nitrogen by thiyl radical, followed by a return hydrogen transfer from the thiol to the carbon gamma to nitrogen in the intermediate allylic radical. The scope and limitations of the reaction with respect to the nature of the thiol, to the structure of the allylic chain, and to the nature of the substituents at nitrogen were investigated. The experimental results were interpreted on the ground of DFT calculations of the C-Halpha BDE in the starting allylic amines, and of the C-Hgamma BDE in the resulting enamines. The efficiency of the initial hydrogen transfer is the first requirement for the reaction to proceed. A balance must be found between the S-H BDE and the two above-mentioned C-H BDEs. The incidence of stereoelectronic factors was analyzed through NBO calculations performed on the optimized geometries of the starting allylic amines. Additional calculations of the transition structures and subsequent tracing of the reaction profiles were performed for the abstraction of Halpha from both the allyl and the prenyl derivatives by p-TolS(*). The latter allowed us to estimate the rate constant for the abstraction of hydrogen by thiyl radical from an N-prenylamine and an N-allylamine.