Increasing complexity of a diterpene synthase reaction with a single residue switch

Terpene synthases often catalyze complex reactions involving intricate series of carbocation intermediates. The resulting, generally cyclical, structures provide initial hydrocarbon frameworks that underlie the astonishing structural diversity of the enormous class of terpenoid natural products (>50,000 known), and these enzymes often mediate the committed step in their particular biosynthetic pathway. Accordingly, how terpene synthases specify product outcome has drawn a great deal of attention. In previous work, we have shown that mutational introduction of a hydroxyl group at specific positions within diterpene synthase active sites can "short circuit" complex cyclization and/or rearrangement reactions, resulting in the production of "simpler"' diterpenes. Here we demonstrate that the converse change, substitution of an Ile for Thr at the relevant position in a native pimaradiene synthase, leads to a dramatic increase in reaction complexity. Product outcome is shifted from the tricyclic pimaradiene to a rearranged tetracycle, aphidicol-15-ene. Thus, the nature of the residue at this position acts as a true switch for product outcome. In addition, the ability of aliphatic residue substitution to enable a more complex reaction emphasizes the importance of substrate conformation imposed by a largely inert active site. Furthermore, the profound plasticity of diterpene synthases exemplified by this single residue switch for product outcome is consistent with the screening/diversity-oriented hypothesis of natural products metabolism.     

Electrostatic effects on (di)terpene synthase product outcome

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to largely control their reactions via steric effects, serving as templates. However, recent results highlight electrostatic effects also exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may steer carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues has been shown to be sufficient to "short-circuit" complex cyclization and/or rearrangement reactions, with the converse change further found to be sufficient to increase reaction complexity. The mechanistic hypothesis for the observed effects is hydroxyl dipole stabilization of the specific carbocation formed by initial cyclization, enabling deprotonation of this early intermediate, whereas the lack of such stabilization (i.e. in the presence of an aliphatic side chain) leads to carbocation migration towards the pyrophosphate co-product, resulting in a more complex reaction. This is further consistent with the greater synergy exhibited between pyrophosphate and aza-analogs of late, relative to early, stage carbocation intermediates, and crystallographic analysis of the monoterpene cyclase bornyl diphosphate synthase wherein mechanistically non-relevant counter-ion pairing between aza-analogs of early stage carbocation intermediates and pyrophosphate is observed. Thus, (di)terpene synthases seem to mediate specific reaction outcomes, at least in part, by providing electrostatic effects to counteract those exerted by the pyrophosphate co-product.     

Origins of Large Rate Enhancements in the Nazarov Cyclization Catalyzed by Supramolecular Encapsulation

The self‐assembled supramolecular host [Ga₄L₆]^12? ( 1 ; L=N,N‐bis(2,3‐dihydroxybenzoyl)‐1,5‐diaminonaphthalene) catalyzes the Nazarov cyclization of 1,3‐pentadienols with extremely high levels of efficiency. The catalyzed reaction proceeds at a rate over a million times faster than that of the background reaction, an increase comparable to those observed in some enzymatic systems. A detailed study was conducted to elucidate the reaction mechanism of both the catalyzed and uncatalyzed Nazarov cyclization of pentadienols. Kinetic analysis and ¹⁸O‐exchange experiments implicate a mechanism, in which encapsulation, protonation, and water loss from substrate are reversible, followed by irreversible electrocyclization. Although electrocyclization is rate determining in the uncatalyzed reaction, the barrier for water loss and for electrocyclization are nearly equal in the assembly‐catalyzed reaction. Analysis of the energetics of the catalyzed and uncatalyzed reaction revealed that transition‐state stabilization contributes significantly to the dramatically enhanced rate of the catalyzed reaction.     

Proposed mechanism for the reaction catalyzed by a diterpene cyclase,aphidicolan-16beta-ol synthase: experimental results on biomimetic cyclization and examination of the cyclization pathway by ab initio calculations

To examine the mechanism of the cyclization reaction catalyzed by aphidicolan-16beta-ol synthase (ACS), which is a key enzyme in the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, skeletal rearrangement of 2a and biomimetic cyclization of 4b were employed. The structures of the reaction products, which reflect penultimate cation intermediates, allowed us to propose a detailed reaction pathway for the Lewis acid-catalyzed cyclizations and rearrangements. Isolation of these products in an aphidicolin-producing fungus led us to speculate that the mechanism of the ACS-catalyzed cyclization reaction is the same as that of a nonenzymatic reaction. Ab initio calculations of the acid-catalyzed reaction intermediates and the transition states indicate that the overall reaction catalyzed by ACS is an exothermic process though the reaction proceeds via an energetically disfavored secondary cation-like transition state. In conjunction with the solvent effect in the acid-catalyzed reactions, this indicates that the actual role of ACS is to provide a template which enforces conformations of the intermediate cations leading to the productive cyclization although it has been believed that the cation-pi interaction between cation intermediates and aromatic amino acid residues in the active site is important for the enzymatic catalysis. This study provided important information on the role of various cationic species, especially secondary cation-like structures, in both nonenzymatic and enzymatic reactions.     

Structure and Mechanism of the Monoterpene Cyclolavandulyl Diphosphate Synthase that Catalyzes Consecutive Condensation and Cyclization

We report the three‐dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclization to form a cyclic monoterpene, CLPP. The structures of apo‐CLDS and CLDS in complex with Tris, pyrophosphate, and Mg²⁺ ion were refined at 2.00 Å resolution and 1.73 Å resolution, respectively. CLDS adopts a typical fold for cis‐prenyl synthases and forms a homo‐dimeric structure. An in vitro reaction using a regiospecifically ²H‐substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure‐based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase‐catalyzed reactions.     

Rose Oxides: A Facile Chemo and Chemo‐Enzymatic Approach

Nonracemic rose oxides were synthesized from racemic or nonracemic monoterpene alcohol citronellol by a novel chemo and chemo‐enzymatic route. After the key step of bromomethoxylation of citronellol, the intermediate was dehydrobrominated by a base, followed by an acid catalyzed demethoxylation along with cyclization to furnish rose oxides in high yield. The racemic dehydrobrominated precursor was also kinetically resolved using various lipases to produce nonracemic rose oxides.     

Copper(I)-catalyzed chlorine atom transfer radical cyclization reactions of unsaturated alpha-chloro beta-keto esters

Copper(I) chloride catalyzed chlorine atom transfer radical cyclization reactions of a series of olefinic alpha-chloro beta-keto esters were investigated. It was found that alpha-dichlorinated beta-keto esters were suitable substrates; the chlorine transfer mono or tandem radical cyclization reactions catalyzed by CuCl complex with bis(oxazoline) or bipyridine proceeded smoothly in dichloroethane at room temperature or 80 degrees C, providing cyclic and bicyclic compounds in moderate to high yield. [reaction: see text]     

Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase: A chemistry–biology interdisciplinary study of the protein's structure–function–reaction mechanism relationships

The oxidosqualene cyclases (EC 5.4.99‐) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)‐2,3‐oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure–function relationships of the Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase‐catalyzed cyclization/rearrangement reaction. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302–325; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20157     

Electrophilic cyclization of (Z)-selenoenynes: synthesis and reactivity of 3-iodoselenophenes

We present here our results of the electrophilic cyclization reaction of (Z)-selenoenynes with different electrophiles such as I(2), ICl, PhSeBr, and PhSeCl. The cyclization reaction proceeded cleanly under mild reaction conditions, and 3-substituted selenophenes were formed in moderate to excellent yields. We observed that the nature of solvent and structure of (Z)-selenoenyne were important to the cyclization reaction. In addition, the obtained 3-iodoselenophenes were readily transformed to more complex products using a metal-halogen exchange reaction with n-BuLi and trapping the intermediate formed with aldehydes, furnishing the desired secondary alcohols in good yields. Conversely, using the palladium or copper catalyzed cross-coupling reactions with terminal alkynes or alkyl alcohols, we were able to convert 3-iodoselenophene to Sonogashira or Ullmann type products, respectively, in good yields.     

Enantioselective carbonyl-ene-type cyclization of α-ketoester and 2-substituted vinylsilane catalyzed by a chiral Cu-BOX complex

We developed an enantioselective carbonyl-ene-type cyclization using 2-substituted vinylsilane as a nucleophilic ene moiety catalyzed by a chiral copper-BOX complex. This reaction is the first example of enantioselective carbonyl-ene cyclization using a 1,2-disubstituted olefin. This methodology gave chiral indenols with a tetrasubstituted carbon.     

Enzymatic cyclization of linear peptide to plant cyclopeptide heterophyllin B

Cyclopeptide is an important family of natural products.Over the past decade,about100cyclopep-tides have been isolated from higher plants by our group[1].The biosynthesis of cyclopeptides from mi-crobes such as tyrocidine A has been reported re-cently[2-4…     

Catalytic enantioselective pyrrole alkylations of alpha,beta-unsaturated 2-acyl imidazoles

[reaction: see text] Enantioselective additions of pyrroles to alpha,beta-unsaturated 2-acyl imidazoles catalyzed by the bis(oxazolinyl)pyridine-scandium(III) triflate complex (1) have been accomplished. The alpha,beta-unsaturated 2-acyl imidazoles were synthesized in high yields through Wittig olefination. A short, enantioselective synthesis of the alkaloid (+)-heliotridane has been accomplished utilizing this methodology and a 2-acyl imidazole cleavage and cyclization. This methodology was then extended to the one-pot asymmetric synthesis of 2-substituted indoles.     

The interaction of rhodium carbenoids with carbonyl compounds as a method for the synthesis of tetrahydrofurans

The transition metal catalyzed reaction of α-diazo carbonyl compounds has found numerous applications in organic synthesis, and its use in either heterocyclic or carbocyclic ring formation is well precedented. In contrast to other catalysts that are suitable for carbenoid reactions of diazo compounds, those constructed with the dirhodium(II) framework are most amenable to ligand modification that, in turn, can influence reaction selectivity. The reaction of rhodium carbenoids with carbonyl groups represents a very efficient method for generating carbonyl ylide dipoles. Rhodium-mediated carbenoid–carbonyl cyclization reactions have been extensively utilized as a powerful method for the construction of a variety of novel polycyclic ring systems. This article will emphasize some of the more recent synthetic applications of the tandem rhodium carbenoid cyclization/cycloaddition cascade for natural product synthesis. Discussion centers on the chemical behavior of the rhodium metal carbenoid complex that is often affected by the nature of the ligand groups attached to the metal center.     

Cu（I）-Catalyzed Synthesis of 2-Substituted Benzimidazoles from 2-lodoanilines and Amides

A novel and efficient approach to the synthesis of 2-substituted benzimidazoles has been developed via CuI/DMEDA-catalyzed coupling reaction and post-cyclization with glacial acetic acid from readily available 2-iodoanilines and amides. This method is suitable for the construction of a variety of benzimidazoles in moderate to good yields under short reaction times.     

[Ru(DMSO)4]Cl2 catalyzes the α-alkylation of ketones by alcohols

The electrophilic α-alkylation of ketones with alcohols was accomplished by a [Ru(DMSO)₄]Cl₂ catalyzed process, water being the only wasted material. The reaction can be successfully governed to produce either the expected ketones or their related alcohols only by changing the reaction conditions. When 2-aminobenzyl alcohol was used, a cyclization process took place to yield 2,3-disubstituted quinolines.     

Rhodium‐Catalyzed Cascade Reaction of 1,6‐Enynes Involving Addition,Cyclization, and β‐Oxygen Elimination

The reaction of arylboronic acids with 1,6‐enynes that contain an allylic ether moiety is catalyzed by a rhodium(I) complex to produce cyclopentanes with a tetrasubstituted exo olefin and a pendant vinyl group. The reaction is initiated by the regioselective addition of an arylrhodium(I) species to the carbon–carbon triple bond of the 1,6‐enyne. The resulting alkenylrhodium(I) compound subsequently undergoes intramolecular carborhodation of the allylic double bond in a 5‐exo‐trig mode. β Elimination of the methoxy group affords the cyclization product and the catalytically active methoxorhodium(I) species. The use of alkyl Grignard reagents instead of arylboronic acids as organometallic nucleophiles was also examined.     

Divergent Enantioselective Total Synthesis of Siphonarienal,Siphonarienone, and Pectinatone

A divergent synthesis of siphonarienal, siphonarienone, and pectinatone has been achieved from a common precursor 4 , which was synthesized by using an enzymatic desymmetrization approach. The major key steps involved were Grignard reaction, Wittig reaction, Evans' asymmetric alkylation, and base‐catalyzed cyclization.     

Catalytic Asymmetric Torgov Cyclization: A Concise Total Synthesis of (+)‐Estrone

An asymmetric Torgov cyclization, catalyzed by a novel, highly Brønsted acidic dinitro‐substituted disulfonimide, is described. The reaction delivers the Torgov diene and various analogues with excellent yields and enantioselectivity. This method was applied in a very short synthesis of (+)‐estrone.     

A catalytic antibody against a tocopherol cyclase inhibitor

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.     

Kinetic simulations for cyclization of α,ω‐telechelic polymers

In this work, we conducted kinetic simulations to examine the effects of various experimental conditions on cyclization during the feed of α,ω‐telechelic polymers into a reaction mixture. The simulations showed that the interplay between the feed rate and rate coefficients for cyclization and multiblock formation were the dominant and controlling parameters. The simulations were in good agreement with previously published results on cyclization of α,ω‐telechelic polystyrene with different molecular weights by the Cu‐catalyzed azide/alkyne cycloaddition (CuAAC) reaction. They also showed that high dilution was not a necessary condition for cyclization and that high percentages of monocyclic could be rapidly produced in solutions that are more concentrated. Previously reported work demonstrated that cyclic polystyrene could be prepared in less than 9 min at 25 °C using the CuAAC “click” reaction. These simulations allow for optimization and better experimental design, leading to the possibility of large scale production of cyclic polymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010