Production Of Benzenoids Research Articles

Phosphorane-Promoted C-C Coupling during Aryne Annulations.

Arynes are fleeting, high-energy intermediates that undergo myriad trapping reactions by nucleophiles. Their unusual reactivity compared to other electrophiles can spur unexpected mechanistic pathways enroute to the formation of benzenoid products. Herein we explore a particularly unique case of thermally generated arynes reacting with phosphoranes to form helical dibenzothiophenes and -selenophenes. Multiple new helical polycyclic aromatic products are reported. DP4+ and X-ray crystallographic analysis were used in tandem to confirm the structural topologies of selected products and to demonstrate the utility of DP4+ for distinguishing between isomeric polycyclic aromatic compounds. Lastly, we discuss a plausible mechanism consistent with DFT computations that accounts for the product formation; namely, ligand coupling (i.e., reductive elimination) within a hypervalent, pentacarbon-ligated σ-phosphorane furnishes the dibenzothio- or dibenzoselenophene.

Phosphorane‐Promoted C−C Coupling during Aryne Annulations

AbstractArynes are fleeting, high‐energy intermediates that undergo myriad trapping reactions by nucleophiles. Their unusual reactivity compared to other electrophiles can spur unexpected mechanistic pathways enroute to the formation of benzenoid products. Herein we explore a particularly unique case of thermally generated arynes reacting with phosphoranes to form helical dibenzothiophenes and ‐selenophenes. Multiple new helical polycyclic aromatic products are reported. DP4+ and X‐ray crystallographic analysis were used in tandem to confirm the structural topologies of selected products and to demonstrate the utility of DP4+ for distinguishing between isomeric polycyclic aromatic compounds. Lastly, we discuss a plausible mechanism consistent with DFT computations that accounts for the product formation; namely, ligand coupling (i.e., reductive elimination) within a hypervalent, pentacarbon‐ligated σ‐phosphorane furnishes the dibenzothio‐ or dibenzoselenophene.

Hexadehydro-Diels-Alder Reaction: Benzyne Generation via Cycloisomerization of Tethered Triynes.

The hexadehydro-Diels-Alder (HDDA) reaction is the thermal cyclization of an alkyne and a 1,3-diyne to generate a benzyne intermediate. This is then rapidly trapped, in situ, by a variety of species to yield highly functionalized benzenoid products. In contrast to nearly all other methods of aryne generation, no other reagents are required to produce an HDDA benzyne. The versatile and customizable nature of the process has attracted much attention due not only to its synthetic potential but also because of the fundamental mechanistic insights the studies often afford. The authors have attempted to provide here a comprehensive compilation of publications appearing by mid-2020 that describe experimental results of HDDA reactions.

Oenological attributes of the yeast Hanseniaspora vineaeand its application for white and red winemaking

Flavour and some compounds associated with wine colour are known to be yeast strain-dependent. These metabolites are important for the sensory quality of wines, studies searching for increase aroma and color are a key area today in winemaking. The aim of this work was to study the oenological potential of the two main strains of Hanseniaspora vineae,native to Uruguay to better understand their successful application at winery level. It is known that these strains contribute with extracellular proteases and β-glucosidase enzyme activities that might increase cell lysis and flavor depending in grape varieties. Application and nutrient management of the process of these strains in production of white wines (Chardonnay, Macabeo and Petit Manseng) and red wine Tannat are discussed. Wines were evaluated to determine the volatile compounds composition and their effect compared to conventional processes. Low production of short and medium chain fatty acids and ethyl esters, and high production of acetate esters and isoprenoids are found compared to S. cerevisiaestrains. The most outstanding characteristic of the species H. vineaewas the production of benzenoids, phenylpropanoids and acetate esters. This behavior was reflected in the sensory evaluation, where all the fermentations performed with H. vineaewere considered superior compared to Saccharomyces cerevisiaewine strains.

Transcriptomic and proteomic approaches to explore the differences in monoterpene and benzenoid biosynthesis between scented and unscented genotypes of wintersweet.

Wintersweet (Chimonanthus praecox L.) is an important ornamental plant in China with a pleasant floral scent. To explore the potential mechanisms underlying differences in the fragrances among genotypes of this plant, we analyzed floral volatile organic compounds (VOCs) from two different genotypes: SW001, which has little to no fragrance, and the scented genotype H29. The major VOCs in H29 were linalool, trans-β-ocimene, benzyl acetate, methyl salicylate, benzyl alcohol (BAlc) and methyl benzoate. The most important aroma-active compound in H29, linalool, was emitted at a low concentration in SW001, which had markedly higher levels of trans-β-ocimene than H29. Next, to investigate scent biosynthesis, we analyzed the transcriptome and proteome of fully open flowers of the two genotypes. A total of 14 443 differentially expressed unigenes and 196 differentially expressed proteins were identified. Further analyses indicated that 56 differentially expressed genes involved in the terpenoid and benzenoid biosynthesis pathways might play critical roles in regulating floral fragrance difference. Disequilibrium expression of four terpene synthase genes resulted in diverse emission of linalool and trans-β-ocimene in both genotypes. In addition, the expressions of two CpMYC2 transcription factors were both upregulated in H29, implying that they may regulate linalool production. Notably, 16 of 20 genes in the benzenoid biosynthesis pathway were downregulated, corresponding to the relatively low level of benzenoid production in SW001. The lack of benzyl acetate might indicate that SW001 may lack substrate BAlc or functional acetyl-CoA:benzylalcohol acetyltransferase.

Mechanism of the reactions of alcohols with o-benzynes.

We have studied reactions of secondary and primary alcohols with benzynes generated by the hexadehydro-Diels–Alder (HDDA) reaction. These alcohols undergo competitive addition vs dihydrogen transfer to produce aryl ethers vs reduced benzenoid products, respectively. During the latter process, an equivalent amount of oxidized ketone (or aldehyde) is formed. Using deuterium labeling studies, we determined that (i) it is the carbinol C–H and adjacent O–H hydrogen atoms that are transferred during this process and (ii) the mechanism is consistent with a hydride-like transfer of the C–H. Substrates bearing an internal trap attached to the reactive, HDDA-derived benzyne intermediate were used to probe the kinetic order of the alcohol trapping agent in the H2-transfer as well as in the alcohol addition process. The H2-transfer reaction is first order in alcohol. Our results are suggestive of a concerted H2-transfer process, which is further supported by density functional theory (DFT) computational studies and results of a kinetic isotope effect experiment. In contrast, alcohol addition to the benzyne is second order in alcohol, a previously unrecognized phenomenon. Additional DFT studies were used to further probe the mechanistic aspects of the alcohol addition process.

Catalytic, formal homo-Nazarov-type cyclizations of alkylidene cyclopropane-1,1-ketoesters: access to functionalized arenes and heteroaromatics.

A catalytic, formal homo-Nazarov-type cyclization of alkylidene cyclopropanes (ACPs) to give functionalized arenes and heteroaromatics is reported. In the presence of a Lewis acid catalyst, the ACP 1,1-ketoesters undergo distal bond cleavage to afford an allyl cation intermediate. Adjacent π-attack on the allyl cation then provides a six-membered ring that undergoes rapid aromatization. In these cases, benzenoid products are formed in up to 98% yield. Strategic choice of the substitution about the ACP allows for the generation of other useful isomeric products in good yields.

Diverging pathways: Differential benzenoid and phenylpropanoid volatile production in Phlox subulata L. cultivars

Previous research into the genetic mechanisms of benzenoid and phenylpropanoid volatile biosynthesis has suggested the potential for metabolic flux, in which phenylalanine substrate dedicated to one pathway branch (i.e., benzenoid production) could alter volatile production in other pathways (i.e., phenylpropanoid production). However, little research has been conducted in planta to verify the validity of this hypothesis. We examined the emission rates of representative benzenoid and phenylpropanoid volatiles from seven cultivars of Phlox subulata L. to determine if cultivars had metabolic flux differences in terms of these two compound categories. Cultivars that produced large quantities of methyl benzoate and benzaldehyde were found to emit little or no phenylacetaldehyde and 2-phenylethanol, and vice versa. These results suggest that P. subulata cultivars experience phenylalanine substrate flux directed toward one pathway and away from the alternate branch. Such a pattern may be the result of differential selection pressures, in which gene expression has been altered to direct flux away from either benzenoid or phenylpropanoid production. Moreover, if these patterns hold true in wild populations, metabolic flux may lead to differential pollinator behavior and further phenotypic evolution. Future research using molecular tools could verify the role of metabolic flux in determining scent phenotypes and pinpoint the exact nature of the genetic mutations leading to phenotypic differences in odor. Simplified metabolic flux pattern found in Phlox subulata ; phenylalanine substrate can be directed toward phenylpropanoid (phenylacetaldehyde and 2-phenylethanol) or benzenoid (benzaldehyde and methyl benzoate) production. • Phlox subulata cultivars emit benzenoid and phenylopropanoid volatiles. • Plants with high benzenoid emission emit little/no phenylpropanoids and vice versa. • This pattern suggests differential phenylalanine flux through biochemical pathways. • Such a pattern may result from differential selection and gene expression.

New Synthesis—Regulatory Evolution, the Veiled World of Chemical Diversification

Chemical Ecology focuses on how chemicals mediate interactions among organisms. This role of chemistry in organismal interactions generates an evolutionary pressure of pests and hosts to enter into a repetitive cycle of defense novelization and counter-response. This cycle provides the central evolutionary hypothesis about the pressure to derive novel chemicals and thus generate a massive chemical diversification among and within plant and fungal lineages. In the past decades, significant efforts from a large research community have made great progress in cloning numerous enzymes from diverse biochemical pathways leading to a general genomic thesis for this chemical diversification. This thesis can be stated as follows: genes duplicate either individually or as whole genomes. These duplicated genes can either “neo-functionalize” (evolve completely new biochemical functions) or “sub-functionalize” (evolve a fraction of the original function). It is the presence of these changed duplicated genes that provides new enzymatic capacity that can somehow be coordinated into a single pathway to produce a single compound. However, this gene evolution model and the data supporting it focus almost entirely on how the enzymatic capacity to make a new compound arises, while saying little about the regulatory processes that may have accompanied this evolution. This is despite the fact that such regulatory processes are essential to coordinate the pathway and allow it to function. It is possible that part of this lack of enquiry into regulatory evolution comes from the common observation that most secondary metabolite pathways show similar inductive regimes in response to pathogens or herbivores, generating the illusion that they may involve conserved regulators that trap the biochemical pathway promoters. However, recent work is suggesting that there is as much regulatory evolution at play in chemical diversification as there is enzyme evolution. A common observation in secondary metabolism is that biosynthetic pathways frequently are controlled by bHLH andMYB transcription factors. A recent multi-species genomic phylogenetic analysis of the bHLH and MYB families showed that like secondary metabolite enzymes, these families have undergone a massive expansion within the vascular plants (Feller et al., 2011). There were similar levels of gene numbers as for key secondary metabolite enzymes like CYP450s and 2ODDs, and there was even a similar propensity for lineagespecific families of both bHLH and MYB genes. One of these lineage-specific MYB families includes thoseMYBs that control the expression of glucosinolates, amino acid derived defense compounds present in the Capparales. A recent investigation of how the glucosinolate enzymes and their affiliated MYBs evolved in the Capparales showed that early in their evolutionary history, a full biosynthetic pathway along with a specific MYB evolved to create a functioning glucosinolate system (Bekaert et al., 2012). During Capparales evolution, there were whole genome duplication events that duplicated the entire glucosinolate pathway and the MYBs. Following one of these duplications, the genes partitioned into largely two pathways, one for making glucosinolates from Tryptophan and the other using Methionine. Central to the issue of regulatory evolution, the MYBs also split along downstream pathway lines. Thus, as suggested by the phylogenetic analysis, chemical diversification also is likely driving regulatory factor diversification within plants. Intriguingly, recent work suggests that secondary metabolite transcription factors are not limited to a role in secondary metabolism. Analysis of volatile production and regulation within Petunia identified the missing link in a regulatory cascade of MYBs. In this system, three MYBs (EOBII, EOBI, andODO1) form a hierarchical regulatory cascade leading to the production of volatiles in Petunia (Spitzer-Rimon et al., 2013). These MYBs also directly regulated primary metabolite genes within the shikimate pathway, presumably controling precursor availability for the production of benzenoids. Importantly, these genes are not broadly evolutionarily conserved and appear to have arisen specifically in this lineage for secondary volatile production. This function in regulating secondary metabolism, however, did not prevent them from accruing regulatory control over a primary metabolite pathway like the shikimate pathway. Thus, secondary metabolite regulatory factors may also evolve roles in mediating primary metabolic or physiological processes as a part of coordinating secondary metabolite production, in this case regulating precursor availability. Together, these works suggest that the chemical diversification so central to the expansion of biochemical capacity is also driving an unrecognized expansion of regulatory capacity within vascular plants. More dramatically, if lineagespecific secondary metabolite transcription factors frequently acquire the capacity to control central physiological processes that enable the optimization of chemical defenses, then the chemical diversification central to Chemical Ecology may play a fundamental role in shaping areas outside the field’s traditional boundaries, such as core plant physiology and metabolism. In either case, studying how chemical diversification and regulatory evolution have proceeded in coordination will provide unique insight into how organisms and their networks evolve.

Synthesis of complex benzenoids via the intermediate generation of o-benzynes through the hexadehydro-Diels-Alder reaction

The hexadehydro-Diels-Alder (HDDA) cascade enables the synthesis of complex benzenoid products with various substitution patterns through aryne intermediates. The first stage of this cascade involves the generation of a highly reactive ortho-benzyne intermediate by a net [4+2] cycloisomerization of a triyne substrate. The benzyne can be rapidly 'trapped' either intramolecularly or intermolecularly with myriad nucleophilic or π-bond-donating reactants. As a representative example of a general procedure for synthesizing highly substituted benzenoids, this protocol describes the synthesis of a typical triyne substrate and its use as the reactant in an HDDA cascade to form a phthalide. The synthetic procedure detailed herein (four chemical reactions) takes 16-20 h of active effort over a period of several days for the preparation of the triyne precursor and ∼2 h of active effort over a 3-d period for the generation and trapping of the benzyne and isolation of the phthalide product.

The hexadehydro-Diels–Alder reaction

Summaryo-Benzynes (arynes) are among the most versatile of all reactive (short-lived) intermediates in organic chemistry. These species can be trapped to give products that are valuable from the perspective of both fine (pharmaceuticals) and commodity (agrochemicals, dyes, polymers, etc.) chemicals. Here we show a fundamentally new strategy that unites a de novo generation of benzynes, through the title hexadehydro-Diels–Alder (HDDA) reaction, with their in situ elaboration into structurally complex benzenoid products. In the HDDA reaction a 1,3-diyne is engaged in a [4+2] cycloisomerization with a third (pendant) alkyne–the diynophile–to produce the highly reactive benzyne intermediate. The metal- and reagent-free reaction conditions for this simple, thermal transformation are notable. The subsequent and highly efficient trapping reactions increase the power of the overall process. Finally, we provide examples of how this de novo benzyne generation approach allows new modes of intrinsic reactivity to be revealed.

Single molecules put a ring on it

A variant of a classical reaction has been used to generate short-lived chemical species called arynes, allowing the one-step synthesis of structurally complex benzene derivatives from simple precursors. See Article p.208 Arynes are reactive intermediates derived from aromatic systems, which can be 'trapped' to give products that find use as chemical reagents and in pharmaceuticals, agrochemicals, dyes and polymers. This study explores a new synthetic strategy that combines de novo generation of benzynes — through a hexadehydro-Diels–Alder reaction — with their in situ elaboration into structurally complex benzenoid products. The reaction is metal-free and reagent-free, and the authors provide examples of how this approach allows new modes of intrinsic reactivity to be revealed.

A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. ‘Mitchell Diploid’ flower

Floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is a complex and coordinate cellular process executed by petal limb cells of a Petunia×hybrida cv. ‘Mitchell Diploid’ (MD) plant. In MD flowers, the majority of benzenoid volatile compounds are derived from a core phenylpropanoid pathway intermediate by a coenzyme A (CoA) dependent, β-oxidative scheme. Metabolic flux analysis, reverse genetics, and biochemical characterizations of key enzymes in this pathway have supported this putative concept. However, the theoretical first enzymatic reaction, which leads to the production of cinnamoyl-CoA, has only been physically demonstrated in a select number of bacteria like Streptomyces maritimus through mutagenesis and recombinant protein production. A transcript hasbeen cloned and characterized from MD flowers that shares high homology with an Arabidopsis thaliana transcript ACYL-ACTIVATING ENZYME11 (AtAAE11) and the S. maritimus ACYL-COA:LIGASE (SmEncH). In MD, the PhAAE transcript accumulates in a very similar manner as bona fide FVBP network genes, i.e. high levels in an open flower petal and ethylene regulated. In planta, PhAAE is localized to the peroxisome. Upon reduction of PhAAE transcript through a stable RNAi approach, transgenic flowers emitted a reduced level of all benzenoid volatile compounds. Together, the data suggest that PhAAE may be responsible for the activation of t-cinnamic acid, which would be required for floral volatile benzenoid production in MD.

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production

The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both beta-oxidative and non-beta-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers. Using functional genomics, we identified a 3-ketoacyl-CoA thiolase, PhKAT1, which is involved in the benzenoid biosynthetic pathway and the production of BA. PhKAT1 is localised in the peroxisomes, where it is important for the formation of benzoyl-CoA-related compounds. Silencing of PhKAT1 resulted in a major reduction in BA and benzenoid formation, leaving the production of other phenylpropanoid-related volatiles unaffected. During the night, when volatile benzenoid production is highest, it is largely the beta-oxidative pathway that contributes to the formation of BA and benzenoids. Our studies add the benzenoid biosynthetic pathway to the list of pathways in which 3-ketoacyl-CoA thiolases are involved in plants.

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.

Regulation of terpenoid and benzenoid production in flowers

The production and emission of fragrant molecules by flowers are strictly regulated during the floral lifespan and often peak when pollinators are active. The best-studied classes of floral volatiles are benzenoids and terpenoids. The production of these molecules appears to be primarily regulated at the level of precursor biosynthesis. The genes from the petunia floral shikimate pathway, which provides the precursors for the formation of benzenoids, have recently been shown to be regulated by a MYB transcription factor. The floral terpenoids of snapdragon appear to be derived exclusively from the methyl-erythritol-phosphate pathway in plastids. This pathway controls precursor levels for geranyl diphosphate synthase, which in turn is transcriptionally regulated.

Regulation of volatile benzenoid biosynthesis in petunia flowers

The petunia flower has served as a model for the study of several physiological processes including floral development, self-incompatibility, anthocyanin biosynthesis and ethylene signalling during senescence. More recently, Petunia hybrida 'Mitchell' has been used to understand the complex regulation of volatile benzenoid biosynthesis, which occurs predominantly in flower petal tissues. Benzenoid biosynthesis is temporally and circadian controlled and is tightly down-regulated by ethylene during floral senescence. Using targeted transcriptomics and gene knockouts, both biosynthetic genes and a transcription factor regulating benzenoid synthesis have been recently discovered and characterized. It appears that benzenoid production is regulated predominantly by transcriptional control of the shikimate pathway, benzenoid biosynthesis genes and S-adenosyl-methionine cycle genes.