Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cyclization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1989

Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate.

The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.

Catalysis↗
PubMed2001

Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase.

The excised C-terminal thioesterase (TE) domain from the multidomain tyrocidine nonribosomal peptide synthetase (NRPS) was recently shown to catalyze head-to-tail cyclization of a decapeptide thioester to form the cyclic decapeptide antibiotic tyrocidine A [Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215-218]. The peptide thioester substrate was a mimic of the TE domain's natural, synthetase-bound substrate. We report here the synthesis of modified peptide thioester substrates in which parts of the peptide backbone are altered either by the replacement of three amino acid blocks with a flexible spacer or by replacement of individual amide bonds with ester bonds. Rates of TE domain catalyzed cyclization were determined for these substrates and compared with that of the wild-type substrate, revealing that some parts of the peptide backbone are important for cyclization, while other parts can be modified without significantly affecting the cyclization rate. We also report the synthesis of a modified substrate in which the N-terminal amino group of the wild-type substrate, which is the nucleophile in the cyclization reaction, is replaced with a hydroxyl group and show that this compound is cyclized by the TE domain to form a macrolactone at a rate comparable to that of the wild-type substrate. These results demonstrate that the TE domain from the tyrocidine NRPS can catalyze cyclization of depsipeptides and other backbone-substituted peptides and suggest that during the cyclization reaction the peptide substrate is preorganized for cyclization in the enzyme active site in part by intramolecular backbone hydrogen bonds analogous to those in the product tyrocidine A.

Amino Acid Substitution↗
PubMed2002

Laser flash photolysis kinetic studies of enol ether radical cations. Rate constants for heterolysis of alpha-methoxy-beta-phosphatoxyalkyl radicals and for cyclizations of enol ether radical cations.

Two series of enol ether radical cations were studied by laser flash photolysis methods. The radical cations were produced by heterolyses of the phosphate groups from the corresponding alpha-methoxy-beta-diethylphosphatoxy or beta-diphenylphosphatoxy radicals that were produced by 355 nm photolysis of N-hydroxypryidine-2-thione (PTOC) ester radical precursors. Syntheses of the radical precursors are described. Cyclizations of enol ether radical cations 1 gave distonic radical cations containing the diphenylalkyl radical, whereas cyclizations of enol ether radical cations 2 gave distonic radical cation products containing a diphenylcyclopropylcarbinyl radical moiety that rapidly ring-opened to a diphenylalkyl radical product. For 5-exo cyclizations, the heterolysis reactions were rate limiting, whereas for 6-exo and 7-exo cyclizations, the heterolyses were fast and the cyclizations were rate limiting. Rate constants were measured in acetonitrile and in acetonitrile solutions containing 2,2,2-trifluoroethanol, and several Arrhenius functions were determined. The heterolysis reactions showed a strong solvent polarity effect, whereas the cyclization reactions that gave distonic radical cation products did not. Recombination reactions or deprotonations of the radical cation within the first-formed ion pair compete with diffusive escape of the ions, and the yields of distonic radical cation products were a function of solvent polarity and increased in more polar solvent mixtures. The 5-exo cyclizations were fast enough to compete efficiently with other reactions within the ion pair (k approximately 2 x 10(9) s(-1) at 20 degrees C). The 6-exo cyclization reactions of the enol ether radical cations are 100 times faster (radical cations 1) and 10 000 times faster (radical cations 2) than cyclizations of the corresponding radicals (k approximately 4 x 10(7) s(-1) at 20 degrees C). Second-order rate constants were determined for reactions of one enol ether radical cation with water and with methanol; the rate constants at ambient temperature are 1.1 x 10(6) and 1.4 x 10(6) M(-1) s(-1), respectively.

Journal Article↗
PubMed2002

5-Endo-trig radical cyclizations: disfavored or favored processes?

Relative kinetic data were determined for the 5-endo-trig cyclization of radical 12 compared to hydrogen abstraction from (TMS)(3)SiH in the temperature range of 344-430 K, which allows for the estimation of a rate constant of 2 x 10(4) s(-)(1) at 298 K with an activation energy of ca. 9 kcal/mol for the cyclization process. The 5-endo-trig cyclization of a variety of radicals that afford five-membered nitrogen-containing heterocycles was addressed computationally at the UB3LYP/6-31G level. The 5-endo vs 4-exo mode of cyclication and the effect of delocalization of the unpaired electron in the transition state were investigated. Because the ring formed during cyclization contains five sp(2) centers, electrocyclization via a pentadienyl-like resonance form was also considered. For comparison, similar calculations were performed for 4-penten-1-yl and related radicals. The factors that affect the activation energies of homolytic 5-endo-trig cyclization were determined. In the absence of steric or conformational effects, the endo cyclization to form the five-membered ring was strongly favored over exo cyclization to form the four-membered ring not only on thermodynamic grounds but also kinetically. When a substituent on the double bond was able to delocalize the unpaired electron in the transition state of the 4-exo path, the two modes of cyclization became kinetically comparable. These results have an important bearing on the generalization of the Baldwin-Beckwith rules, which classified the 5-endo-trig radical cyclization as a "disfavored" process.

Journal Article↗
PubMed2003

Mn(III)-based oxidative free-radical cyclizations of substituted allyl alpha-methyl-beta-ketoesters: syntheses, DFT calculations, and mechanistic studies.

The cyclizations of the substituted allyl alpha-methyl-beta-ketoester radicals 11, 14, and 18 were studied by the DFT method at the UB3LYP/6-31G level; the results show that the cis cyclization is easier than the corresponding trans cyclization, but the generated cis radicals are not necessarily more stable than the corresponding generated trans radicals after the cyclizations. The free-radical cyclizations of 11, 14, and 18 in the presence of Mn(OAc)(3) in acetic acid or acetonitrile are all reversible and operate under thermodynamic control, and stereoselectivity of the cyclizations depends on relative stability of the cyclization-generated radicals. Therefore, the oxidative free-radical cyclization of allyl alpha-methyl-beta-ketoester 5a with Mn(OAc)(3) gives a cis product as a major product, while the same oxidative free-radical cyclizations of substituted allyl alpha-methyl-beta-ketoesters 5b and 5c with Mn(OAc)(3) produce trans products as major products.

Journal Article↗
PubMed2006

Synthesis of 3,4-disubstituted piperidines by carbonyl ene and prins cyclizations: switching between kinetic and thermodynamic control with Brønsted and Lewis acid catalysts.

A novel approach to cis and trans 3,4-disubstituted piperidines is described. Carbonyl ene cyclization of aldehydes 4a-e catalyzed by MeAlCl(2) in refluxing chloroform afforded the trans piperidines 7a-e with diastereomeric ratios of up to 93:7, while aldehyde 4f afforded solely the cis product 6f, which was resistant to isomerization to the trans isomer. It was demonstrated for 4a that the cyclization catalyzed by a variety of Lewis acids at low temperature proceeded under kinetic control to afford predominantly the cis piperidine 6a, and this isomerized to the thermodynamically more stable trans piperidine 7a on warming. In contrast, Prins cyclization of 4a-e catalyzed by concentrated hydrochloric acid in CH2Cl2 at low temperature afforded cis piperidines 6a-e with diastereomeric ratios of up to >98:2. The yield and diastereoselectivity of these cyclizations could be improved by using HCl-saturated CH2Cl2 to form the corresponding chloride, followed by elimination of HCl effected by ammonia. Aldehydes 4f and 4galso cyclized in good yield under the latter conditions. Mechanistic studies supported by DFT calculations (B3LYP/6-31G(d)) suggest that the cyclizations proceed via a mechanism with significant carbocationic character, with the cis carbocation being more stable than the trans carbocation. DFT calculations (B3LYP/6-31G(d)) of the transition state energies for concerted cyclization show that the cis piperidine is also the favored product from cyclization through a more concerted mechanism.

Alkenes↗
PubMed1985

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis.

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.

Animals↗
PubMed1987

Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila.

We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction. This was accomplished by selectively removing the 3'-terminal nucleotide by oxidation and beta-elimination; the beta-eliminated IVS thereby is no longer capable of reacting with itself. However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site. We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis of the guanosine binding site involved in cyclization. The results indicate that functional groups on the guanine that are used in a G-C Watson-Crick base pair are important for the cyclization reaction. This is the same result that was obtained for the guanosine binding site involved in splicing [Bass, B. L., & Cech, T. R. (1984) Nature (London) 308, 820-826]. Unlike splicing, however, certain additional nucleotides 5' to the guanosine moiety make significant binding contributions. We conclude that the guanosine binding site in cyclization is similar to, but not identical with, the guanosine binding site in splicing. The same binding interactions used in cyclization could help align the 3' splice site of the rRNA precursor for exon ligation. We also report that the phosphodiester bond at the cyclization site is susceptible to a pH-dependent hydrolysis reaction; the phosphodiester bond is somehow activated toward attack by the 3'hydroxyl of a guanosine molecule or by a hydroxyl ion.

Animals↗
PubMed2002

On the regioselectivity of the cyclization of enyne-ketenes: a computational investigation and comparison with the Myers-Saito and Schmittel reaction.

The Moore (C(2)-C(7)) cyclization and the alternative C(2)-C(6) cyclization of enyne-ketenes belong to the family of biradical cyclization reactions such as the Bergman reaction of ene-diynes, both the cyclizations of enyne-allenes and enyne-cumulenes. The latter garnered substantial interest due to their antitumor efficacy. The mechanisms of both cyclization modes of enyne-ketenes are still unclear, but as the enyne-ketenes can formally be regarded as heteroanalogues of enyne-allenes, both cyclizations are expected to react via biradical routes. Nevertheless, as shown recently for cyclic allenes, the substitution of a methylene group by oxygen can lead to different energetic ordering of the electronic states of the key intermediates. To elucidate the mechanism, the present work investigates the course of both cyclization modes for various model compounds. To reveal general motifs for the large family of biradical cyclizations, a comparison with enyne-allenes is performed.

Journal Article↗
PubMed2000

Study on the cyclization tendency of backbone cyclic tetrapeptides.

The cyclization kinetics of five backbone-cyclic tetrapeptides was investigated both experimentally and computationally. The aim was to both accurately measure the cyclization rates in solution and develop a method that efficiently estimates the relative cyclization tendencies computationally. Progression of the cyclization reaction was monitored directly, yielding the kinetics of changes in the amounts of the linear precursor and the products. These measurements were used to calculate the reaction rates; the results were consistent with a first-order reaction kinetics. In order to predict the cyclization rates computationally, the conformation space of the linear precursors was mapped and used to construct an approximate partition function. We assumed that the cyclization tendency was correlated with the relative probability of being found in a cyclization-prone conformation of the backbone, this probability was estimated from the partition function. The results supported this assumption and demonstrated that, within reasonable accuracy, we are able to predict the relative cyclization tendencies of the peptides measured.

Chromatography↗
PubMed2002

SmI(2)-mediated cyclizations of derivatized beta-lactams for the highly diastereoselective construction of functionalized prolines.

A series of C4-keto-functionalized 1-[(benzoyloxy)(ethoxycarbonyl)methyl]-2-azetidinones were prepared and studied for their tendency to undergo a Reformatsky-type cyclization to fused bicyclic or tricyclic beta-lactams with the single-electron reducing agent samarium diiodide. Whereas the azetidinone 21a underwent reductive cyclization, affording the potent antibiotic sanfetrinem's tricyclic [4.5.6] core structure as the major component, all other examples tested resulted in cyclization followed by an N to O acyl migration involving cleavage of the beta-lactam ring as the favored pathway. Highly functionalized proline derivatives were therefore accessed as single diastereomers through the reductive cyclization of benzoates 21b, 22, 23a,b, 24b, and 25-28. Pertinent for the success of these cyclizations was the addition of 1 equiv of tert-butyl alcohol, allowing for the protonation of the basic amide derivative obtained after the acyl migration step. The diastereoselectivities of these reactions deviate from those of similar cyclizations involving the corresponding lithium enolate. This divergence could be rationalized by the coordination of the metal ion of the samarium(III) enolate intermediate to the beta-lactam amide functionality in the cyclization step, which may not be possible for lithium enolates.

Azetidines↗
PubMed2005

8-endo versus 7-exo cyclization of alpha-carbamoyl radicals. A combination of experimental and theoretical studies.

Atom transfer radical cyclization reactions of N-(4-pentenyl)iodoacetamides were investigated. The reactions were efficiently promoted by BF3.OEt2. For N-alkenyl-substituted iodoamides, excellent regioselectivity in favor of 8-endo cyclization was observed, while both 7-exo and 8-endo cyclization products were formed with the 8-endo cyclization preferred in the cases of N-(2-allylphenyl)-substituted iodoamides. Density functional theory calculations at the B3LYP/6-31G level revealed that both the s-trans and the s-cis conformational transition structures were feasible for the 8-endo cyclization of N-alkenyl-substituted alpha-carbamoyl radicals while 7-exo transition structures were much less stable. For the cyclization of N-(2-allylphenyl)-substituted alpha-carbamoyl radicals, the transition structures for 8-endo and 7-exo cyclizations were of comparable energy. These results were in excellent agreement with the experimental observations.

Cyclization↗
PubMed2005

A study of vinyl radical cyclization using N-alkenyl-7-bromo-substituted hexahydroindolinones.

A new method for the synthesis of the octahydropyrrolo[3,2,1-ij]quinoline ring system that possesses the characteristic skeleton of the aspidosperma family of alkaloids has been developed. The method utilizes an intramolecular Diels-Alder reaction of an amido-substituted furan across a tethered indole pi-bond. To apply this strategy to the synthesis of the indole alkaloid spegazzinidine, it was necessary to address the problem of assembling the final D-ring of the pentacyclic skeleton. Radical cyclization of a model N-allyl-7-bromo-3a-methylhexahydroindolinone system was found to preferentially lead to the 6-endo-trig cyclization product, with the best yield being obtained under high dilution conditions. The six-membered cyclized product is generated through two reaction pathways: (a) 6-endo-trig ring closure and (b) rearrangement of an intermediate methylene-cyclopentyl radical obtained by 5-exo-trig cyclization. A number of related 7-bromo-substituted hexahydroindolinones containing tethered olefinic groups were prepared and found to undergo efficient cyclization under both radical and palladium-mediated reaction conditions. Vinyl radical cyclization with several N-butenyl-substituted systems afforded a mixture of 6-exo and 7-endo cyclization products. A protocol to introduce an ethyl substituent into the C20-position of the aspidospermidine skeleton was also developed.

Alkaloids↗
PubMed2006

Stereoselectivity of intramolecular SN' cyclizations of alkyllithium reagents on methoxy alkenes.

The cyclization of alkyllithium reagents onto methoxy alkenes has been investigated. The alkyllithium reagent was generated by reductive lithiation of an alkyl nitrile. In an unbiased substrate, a methoxy leaving group attached to a stereogenic secondary carbon atom led to the cyclization product with high optical purity. The configuration of the product demonstrated that the cyclization had proceeded with high syn-S(N)' selectivity. Previously we have shown that 2-lithiotetrahydropyran reagents cyclize to form spirocycles with the alkene cis to the pyran oxygen. Substrates were prepared to evaluate the importance of the configuration of the secondary allyl methyl ether against the alpha-alkoxy alkyllithium configuration. In the matched case (cyano acetal 38), a very selective cyclization ensued. In the mismatched case (cyano acetal 39), the spiro ether selectivity dominated. The syn-S(N)' selectivity overcame the normal E selectivity in the cyclization and accounted for the major product, Z-alkene 45. Thus the stereochemical preference in these alkyllithium cyclizations is dominated by the spiroether effect, followed by the syn-S(N)' selectivity and finally the preference for E-alkene formation.

Acetals↗
PubMed1990

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes.

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.

Acyclic Monoterpenes↗
PubMed2002

Asymmetric cyclization of omega-formyl-1,3-dienes catalyzed by a zerovalent nickel complex in the presence of silanes.

A nickel(0)-catalyzed asymmetric cyclization of omega-formyl-1,3-diene in the presence of silanes in which five- or six-membered carbocycles or pyrrolidine derivatives were afforded up to 86% ee by the use of (2R,5R)-2,5-dimethyl-1-phenylphospholane as a monodentate chiral ligand was investigated. The reaction course of this asymmetric cyclization can be explained for by two possible mechanisms: one in which the cyclization proceeds via a pi-allylnickel intermediate to produce a cyclized product having an internal olefin in the side chain, and one in which the cyclization proceeds via a sigma bond metathesis of silane and the nickel-oxygen bond of oxanickelacycle to produce a cyclized compound having a terminal olefin and/or an internal olefin in the side chain. It was speculated that both of these mechanisms operate in this asymmetric cyclization depending upon the nature of silanes and the reaction conditions.

Journal Article↗
PubMed1997

Tandem Dipolar Cycloaddition-Mannich Cyclization as an Approach to Tricyclic Nitrogen Heterocycles.

A series of 2-diazo-N-[2-(3,4-dimethoxyphenyl)ethyl]-N-hept-6-enoylmalonamides were prepared and treated with a catalytic amount of rhodium(II) perfluorobutyrate. The resultant carbenoids undergo facile cyclization onto the neighboring amide carbonyl oxygen atom to generate isomünchnone-type intermediates. Subsequent 1,3-dipolar cycloaddition across the pendant olefin affords intramolecular cycloadducts in high yield. Exposure of these cycloadducts to boron trifluoride etherate results in a Lewis acid-induced ring opening to generate N-acyliminium ions which then undergo Mannich cyclization onto the neighboring pi-framework attached to the amide nitrogen atom. The cis stereochemistry of the resulting A/B ring fusion is analogous to similar erythrinane products obtained via a Mondon-enamide-type cyclization. The stereochemical assignment of the final cyclized products was determined by X-ray crystallography. Molecular mechanics calculations show that the ground state energy of the cis-fused diastereomer is 4.6 kcal lower than that of the trans diastereomer, and presumably some of this thermodynamic energy difference is reflected in the transition state for cyclization. In certain cases, proton loss from the initially formed N-acyliminium ion occurs prior to cyclization to give acyl enamides which subsequently cyclize producing epimeric products.

Journal Article↗
PubMed2000

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

Bacillus↗