Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY.

Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY's coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY's specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans-to-cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY's evolutionary origin and its recruitment to coumarin biosynthesis in eudicots.

Sporopollenin-inspired design and synthesis of robust polymeric materials.

Sporopollenin is a mechanically robust and chemically inert biopolymer that constitutes the outer protective exine layer of plant spores and pollen grains. Recent investigation of the molecular structure of pine sporopollenin revealed unique monomeric units and inter-unit linkages distinct from other previously known biopolymers, which could be harnessed for new material design. Herein, we report the bioinspired synthesis of a series of sporopollenin analogues. This exercise confirms large portions of our previously proposed pine sporopollenin structural model, while the measured chemical, thermal, and mechanical properties of the synthetic sporopollenins constitute favorable attributes of a new kind of robust material. This study explores a new design framework of robust materials inspired by natural sporopollenins, and provides insights and reagents for future elucidation and engineering of sporopollenin biosynthesis in plants.

Imine chemistry in plant metabolism.

Imine chemistry represents an important category of chemical reactions involved in the biosynthesis of plant natural products, ranging from the newly discovered mobile defense hormone N-hydroxy-pipecolic acid to the red-to-yellow tyrosine-derived betalain pigments. Spontaneous imine formation reactions have also served as the basis for the evolution of numerous plant metabolic enzymes, such as specialized Pictet-Spenglerases that produce the backbone structures of benzylisoquinoline and monoterpene indole alkaloids and pyridoxal 5'-phosphate-dependent enzymes of diverse functions. Here, we review occurrences of imine chemistry in plant metabolism and their chemical and biochemical mechanisms. A better understanding of plant imine chemistry will ultimately facilitate synthetic biology approaches to further expand the scope of imine natural product biosynthesis for broad biotechnological applications.

Selective N1/N4 1,4-Cycloaddition of 1,2,4,5-Tetrazines Enabled by Solvent Hydrogen Bonding.

An unprecedented 1,4-cycloaddition (vs 3,6-cycloaddition) of 1,2,4,5-tetrazines is described with preformed or in situ generated aryl-conjugated enamines promoted by the solvent hydrogen bonding of hexafluoroisopropanol (HFIP) that is conducted under mild reaction conditions (0.1 M HFIP, 25 °C, 12 h). The reaction constitutes a formal [4 + 2] cycloaddition across the two nitrogen atoms (N1/N4) of the 1,2,4,5-tetrazine followed by a formal retro [4 + 2] cycloaddition loss of a nitrile and aromatization to generate a 1,2,4-triazine derivative. The factors that impact the remarkable change in the reaction mode, optimization of reaction parameters, the scope and simplification of its implementation through in situ enamine generation from aldehydes and ketones, the reaction scope for 3,6-bis(thiomethyl)-1,2,4,5-tetrazine, a survey of participating 1,2,4,5-tetrazines, and key mechanistic insights into this reaction are detailed. Given its simplicity and breath, the study establishes a novel method for the simple and efficient one-step synthesis of 1,2,4-triazines under mild conditions from readily accessible starting materials. Whereas alternative protic solvents (e.g., MeOH vs HFIP) provide products of the conventional 3,6-cycoladdition, the enhanced hydrogen bonding capability of HFIP uniquely results in promotion of the unprecedented formal 1,4-cycloaddition. As such, the studies represent an example of not just an enhancement in the rate or efficiency of a heterocyclic azadiene cycloaddition by hydrogen bonding catalysis but also the first to alter the mode (N1/N4 vs C3/C6) of cycloaddition.

The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants.

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.

Precise Targeted Cleavage of a r(CUG) Repeat Expansion in Cells by Using a Small-Molecule-Deglycobleomycin Conjugate.

RNA repeat expansions cause more than 30 neurological and neuromuscular diseases with no known cures. Since repeat expansions operate via diverse pathomechanisms, one potential therapeutic strategy is to rid them from disease-affected cells, using bifunctional small molecules that cleave the aberrant RNA. Such an approach has been previously implemented for the RNA repeat that causes myotonic dystrophy type 1 [DM1, r(CUG)exp] with Cugamycin, which is a small molecule that selectively binds r(CUG)exp conjugated to a bleomycin A5 cleaving module. Herein, we demonstrate that, by replacing bleomycin A5 with deglycobleomycin, an analogue in which the carbohydrate domain of bleomycin A5 is removed, the selectivity of the resulting small-molecule conjugate (DeglycoCugamycin) was enhanced, while maintaining potent and allele-selective cleavage of r(CUG)exp and rescue of DM1-associated defects. In particular, DeglycoCugamycin did not induce the DNA damage that is observed with high concentrations (25 µM) of Cugamycin, while selectively cleaving the disease-causing allele and improving DM1 defects at 1 µM.

PBS3 and EPS1 Complete Salicylic Acid Biosynthesis from Isochorismate in Arabidopsis.

Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase, which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We found that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate, isochorismoyl-glutamate A. Moreover, we discovered that EPS1, a BAHD acyltransferase-family protein with a previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Synthesis, Characterization, and Rapid Cycloadditions of 5-Nitro-1,2,3-triazine.

The synthesis, characterization, and a study of the cycloaddition reactions of 5-nitro-1,2,3-triazine (3) are reported. The electron-deficient nature of 3 permits rapid cycloaddition with a variety of electron-rich dienophiles, including amidines, enamines, enol ethers, ynamines, and ketene acetals in high to moderate yields. 1H NMR studies of a representative cycloaddition reaction between 3 and an amidine revealed a remarkable reaction rate and efficiency (1 mM, <60 s, CD3CN, 23 °C, >95%).

Direct Synthesis of ß-Aminoenals through Reaction of 1,2,3-Triazine with Secondary Amines.

Simple and direct nucleophilic addition of secondary amines, including imidazole, to 1,2,3-triazine under mild reaction conditions (THF, 25-65 °C, 12-48 h), requiring no additives, cleanly provides ß-aminoenals 4 in good yields (21 examples, 31-79%). The reaction proceeds by amine nucleophilic addition to C4 of the 1,2,3-triazine, in situ loss of N2, and subsequent imine hydrolysis to provide 4.

Catalysis of Heterocyclic Azadiene Cycloaddition Reactions by Solvent Hydrogen Bonding: Concise Total Synthesis of Methoxatin.

Although it has been examined for decades, no general approach to catalysis of the inverse electron demand Diels-Alder reactions of heterocyclic azadienes has been introduced. Typically, additives such as Lewis acids lead to nonproductive consumption of the electron-rich dienophiles without productive activation of the electron-deficient heterocyclic azadienes. Herein, we report the first general method for catalysis of such cycloaddition reactions by using solvent hydrogen bonding of non-nucleophilic perfluoroalcohols, including hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE), to activate the electron-deficient heterocyclic azadienes. Its use in promoting the cycloaddition of 1,2,3-triazine 4 with enamine 3 as the key step of a concise total synthesis of methoxatin is described.

Indolidenes and Indolidenium Intermediates in the Synthesis of Cyclopent[b]indoles: Mechanistic Studies on Intramolecular Cyclizations.

Tetracyclic products featuring predominantly a trans-hexahydroindane unit annelated onto the C(2)/C(3) positions of indole can be accessed by intramolecular cyclocondensation of tethered alkenyl sulfides with either indolidene or indolidenium cation intermediates. Studies with geometrically pure E- and Z-alkenyl sulfide isomers reveal a likely dichotomy of reaction paths that provide mixtures of both regioisomers and stereoisomers of the hexahydroindane adducts.

Total synthesis of dihydrolysergic acid and dihydrolysergol: development of a divergent synthetic strategy applicable to rapid assembly of D-ring analogs.

The total syntheses of dihydrolysergic acid and dihydrolysergol are detailed based on a Pd(0)-catalyzed intramolecular Larock indole cyclization for the preparation of the embedded tricyclic indole (ABC ring system) and a subsequent powerful inverse electron demand Diels-Alder reaction of 5-carbomethoxy-1,2,3-triazine with a ketone-derived enamine for the introduction of a functionalized pyridine, serving as the precursor for a remarkably diastereoselective reduction to the N-methylpiperidine D-ring. By design, the use of the same ketone-derived enamine and a set of related complementary heterocyclic azadiene [4 + 2] cycloaddition reactions permitted the late stage divergent preparation of a series of alternative heterocyclic derivatives not readily accessible by more conventional approaches.

Cycloadditions of 1,2,3-Triazines Bearing C5-Electron Donating Substituents: Robust Pyrimidine Synthesis.

The examination of the cycloaddition reactions of 1,2,3-triazines 17-19, bearing electron-donating substituents at C5, are described. Despite the noncomplementary 1,2,3-triazine C5 substituents, amidines were found to undergo a powerful cycloaddition to provide 2,5-disubstituted pyrimidines in excellent yields (42-99%; EDG = SMe > OMe > NHAc). Even select ynamines and enamines were capable of cycloadditions with 17, but not 18 or 19, to provide trisubstituted pyridines in modest yields (37-40% and 33% respectively).

Intramolecular [3 + 2] cyclocondensations of alkenes with indolidenes and indolidenium cations.

C(2)-C(3) cyclopentannelated indole constructs are prepared by either (a) a cyclization cascade of an alkenyl sulfide tethered to a 2-azido-1-allenylbenzene core or (b) cationic cyclization of a tethered alkenyl sulfide with a putative 2-indolidenium cation. In both cases, issues of C-C versus C-N bond formation emerge, and the results indicate that the former is favored.

Cycloadditions of noncomplementary substituted 1,2,3-triazines.

The scope of the [4 + 2] cycloaddition reactions of substituted 1,2,3-triazines, bearing noncomplementary substitution with electron-withdrawing groups at C4 and/or C6, is described. The studies define key electronic and steric effects of substituents impacting the reactivity, mode (C4/N1 vs C5/N2), and regioselectivity of the cycloaddition reactions of 1,2,3-triazines with amidines, enamines, and ynamines, providing access to highly functionalized heterocycles.