Through-Space, Lone-Pair Promoted Aromatic Substitution: A Relay Mechanism Can Beat Out Direct Activation.

We report a detailed experimental and theoretical analysis of through-space arene activation with halogens, tetrazoles and achiral esters and amides. Contrary to previously assumed direct activation through σ-complex stabilization, our results suggest that these reactions proceed by a relay mechanism wherein the lone pair-containing activators form exothermic π-complexes with electrophilic nitronium ion before transferring it to the probe ring through low barrier transition states. Noncovalent interactions (NCI) plots and Quantum Theory of Atoms in Molecules (QTAIM) analyses depict favorable interactions between the Lewis base (LB) and the nitronium ion in the precomplexes and the transition states, suggesting directing group participation throughout the mechanism. The regioselectivity of substitution also comports with a relay mechanism. In all, these data pave the way for an alternate platform of electrophilic aromatic substitution (EAS) reactions.

Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid-base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure-function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

1-BENZYLSPIRO[PIPERIDINE-4,1'-PYRIDO[3,4-b]indole] 'co-potentiators' for minimal function CFTR mutants.

We previously identified a spiro [piperidine-4,1-pyrido [3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro [piperidine-4,1-pyrido [3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6'-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC50 ∼600 nM representing an ∼17-fold improvement over the original compound identified in a small molecule screen.

Crystal Structure and Mechanistic Molecular Modeling Studies of Mycobacterium tuberculosis Diterpene Cyclase Rv3377c.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.

Trapping and Electron Paramagnetic Resonance Characterization of the 5'dAdo• Radical in a Radical S-Adenosyl Methionine Enzyme Reaction with a Non-Native Substrate.

S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5'-deoxyadenosyl radical (5'dAdo•), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5'dAdo• radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe-S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe-Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5'dAdo• radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C-H bond dissociation free energy as 5'dAdo•, providing a possible explanation for our ability to trap an appreciable fraction of 5'dAdo• in this specific rSAM reaction. The radical's EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5'dAdo• radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5'dAdo• radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.

Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds.

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

Synthesis of Highly Stereodefined Tetrasubstituted Acyclic All-Carbon Olefins via a Syn-Elimination Approach.

An efficient synthesis of stereodefined tetrasubstituted acyclic all-carbon olefins has been developed via a bis(2,6-xylyl)phosphate formation of stereoenriched tertiary alcohols, followed by in situ syn-elimination of the corresponding phosphates under mild conditions. This chemistry tolerates a wide variety of electronically and sterically diverse substrates and generates the desired tetrasubstituted olefins in high yields and stereoselectivities (>95:5) in most cases. This stereocontrolled olefin synthesis has been applied to the synthesis of anticancer drug tamoxifen in three steps from commercially available 1,2-diphenylbutan-1-one in 97:3 stereoselectivity and 78% overall yield.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase.

The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.

Decarboxylation Facilitated by Carbocation Formation and Rearrangement during Steam Distillation of Vetiver Oil.

Density functional theory (DFT) calculations are used to probe the validity of mechanistic proposals for the conversion of isozizanoic acid to 12-norisoziza-5-ene, a reaction that occurs during steam distillation of vetiver oil. While this conversion corresponds overall to a simple decarboxylation, a multistep mechanism involving carbocation intermediates is supported by the computational results.

The chemical biology of hydropersulfides (RSSH): Chemical stability, reactivity and redox roles.

Recent reports indicate the ubiquitous prevalence of hydropersulfides (RSSH) in mammalian systems. The biological utility of these and related species is currently a matter of significant speculation. The function, lifetime and fate of hydropersulfides will be assuredly based on their chemical properties and reactivity. Thus, to serve as the basis for further mechanistic studies regarding hydropersulfide biology, some of the basic chemical properties/reactivity of hydropersulfides was studied. The nucleophilicity, electrophilicity and redox properties of hydropersulfides were examined under biological conditions. These studies indicate that hydropersulfides can be nucleophilic or electrophilic, depending on the pH (i.e. the protonation state) and can act as good one- and two-electron reductants. These diverse chemical properties in a single species make hydropersulfides chemically distinct from other, well-known sulfur containing biological species, giving them unique and potentially important biological function.

A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Biomimetic total synthesis of santalin†Y.

A biomimetic total synthesis of santalin Y, a structurally complex but racemic natural product, is described. The key step is proposed to be a (3+2) cycloaddition of a benzylstyrene to a "vinylogous oxidopyrylium", which is followed by an intramolecular Friedel-Crafts reaction. This cascade generates the unique oxafenestrane framework of the target molecule and sets its five stereocenters in one operation. Our work provides rapid access to santalin Y and clarifies its biosynthetic relationship with other colorants isolated from red sandalwood.

Quantum chemical study of the isomerization of 24-methylenecycloartanol, a potential marker of olive oil refining.

Quantum chemical calculations on the isomerization of 24-methylenecycloartanol are described. An energetically viable mechanism, with a rate-determining protonation step, is proposed. This rearrangement may find applicability in tests for determining if an olive oil has been refined.

ΔF508-CFTR correctors: synthesis and evaluation of thiazole-tethered imidazolones, oxazoles, oxadiazoles, and thiadiazoles.

The most common mutation causing cystic fibrosis (CF) is deletion of phenylalanine residue 508 in the cystic fibrosis transmembrane regulator conductance (CFTR) protein. Small molecules that are able to correct the misfolding of defective ΔF508-CFTR have considerable promise for therapy. Reported here are the design, preparation, and evaluation of five more hydrophilic bisazole analogs of previously identified bithiazole CF corrector 1. Interestingly, bisazole ΔF508-CFTR corrector activity was not increased by incorporation of more H-bond acceptors (O or N), but correlated best with the overall bisazole molecular geometry. The structure activity data, together with molecular modeling, suggested that active bisazole correctors adopt a U-shaped conformation, and that corrector activity depends on the molecule's ability to access this molecular geometry.

Constrained bithiazoles: small molecule correctors of defective ΔF508-CFTR protein trafficking.

Conformationally constrained bithiazoles were previously found to have improved efficacy over nonconstrained bithiazoles for correction of defective cellular processing of the ΔF508 mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. In this study, two sets of constrained bithiazoles were designed, synthesized, and tested in vitro using ΔF508-CFTR expressing epithelial cells. The SAR data demonstrated that modulating the constraining ring size between 7- versus 8-membered in these constrained bithiazole correctors did not significantly enhance their potency (IC50), but strongly affected maximum efficacy (Vmax), with constrained bithiazoles 9e and 10c increasing Vmax by 1.5-fold compared to benchmark bithiazole corr4a. The data suggest that the 7- and 8-membered constrained ring bithiazoles are similar in their ability to accommodate the requisite geometric constraints during protein binding.

Enzyme inhibition by hydroamination: design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor.

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,ß-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

Identification and optimization of short helical peptides with novel reactive functionality as catalysts for acyl transfer by reactive tagging.

Herein we describe the screening and subsequent optimization of peptide catalysts for ester activation. A combinatorial methodology using dye-tagged substrate analogs is described for determining which components of a His-containing helical library display acyl transfer activity. We found that helical peptides display high activity, and amino acids that reinforce this propensity are advantaged. Through this approach two new structural motifs have been discovered that are capable of activating esters in organic solvents. Unlike most acyl transfer catalysts functioning in organic solvents, these catalysts are histidine- rather than N-alkyl histidine-based. Longer peptides with localization of reactive groups on the C-terminal end of the peptide were found to further enhance catalytic activity up to ∼2800-fold over background.