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PubMed1985

Cyclization of RNA 3'-terminal phosphate by cyclase from HeLa cells proceeds via formation of N(3')pp(5')A activated intermediate.

RNA 3'-terminal phosphate cyclase has been partially purified from HeLa cells. In the presence of ATP and Mg2+, cyclase preparations catalyze conversion of RNA 3'-terminal phosphate to the 2',3'-cyclic phosphodiester. The mechanism of 3'-phosphate cyclization was studied with oligoribonucleotides containing terminal 2'-deoxy- or 2'-O-methylribose. Incubation of these substrates with cyclase and ATP results in formation of the corresponding activated 3'-terminal structures, dN(3')pp(5')A and Nm(3')pp(5')A. It is proposed that an intermediate step in cyclization is transfer of the adenylyl group from ATP to the 3' phosphate of RNA. Rapid attack of the adjacent 2'-OH normally follows, resulting in elimination of AMP and formation of the cyclic phosphodiester. Cyclase preparations can be covalently labeled with [alpha-32P]ATP, suggesting that an earlier step in the cyclization reaction involves formation of an adenylylated enzyme intermediate.

Adenosine Triphosphate↗

PubMed1992

Protein-induced bending and DNA cyclization.

We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.

Base Sequence↗

PubMed1985

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.

Acridine Orange↗

PubMed1983

Cyclization studies with a model pentapeptide.

A pentapeptide with the sequence Gly-Ala-D-Val-Leu-Ile was designed for a study of cyclization. Isoleucine was selected as the C-terminal residue in order to determine, from the amount of alloisoleucine in the cyclic product, the extent of racemization during activation and ring closure. The insolubility of cyclo (glycyl-alanyl-D-valyl-leucyl-isoleucyl) in the commonly used solvents facilitated its isolation and thus the evaluation of comparative experiments. Because of its thermal stability the cyclopentapeptide could be purified by sublimation in vacuo. The results of cyclization experiments carried out with this model suggest that separation of the steps of activation and coupling is preferable to cyclization with the help of coupling reagents, that is to the execution of activation and ring closure in a single operation.

Indicators and Reagents↗

PubMed1988

Chain reversals in model peptides: studies of cystine-containing cyclic peptides. II. Effects of valyl residues and possible i-to-(i + 3) attractive ionic interactions on cyclization of [Cys1], [Cys6] hexapeptides.

The synthesis of four N-acetyl N'-methylamide cystine-containing hexapeptides, CVPGVC, CGVVGC, CKPGEC, and CEPGKC, is described. These were used in disulfide-exchange reactions with the peptide CVPGGC as the formal oxidant. The relative propensities for peptide cyclization were thus deduced, and the tendency toward the formation of chain-reversal conformations was established quantitatively. An additional peptide, CVVVVC, was prepared but was never obtained as the cyclic monomer, demonstrating that the formation of chain-reversals in this peptide was of very low probability. Incorporation of pairs of valyl residues decreased the ease of cyclization, but it appeared that conformational flexibility in the cystine-containing hexapeptides may have compensated for substitutions which would have been expected to hinder the adoption of certain beta-turn conformations. The peptides containing ionic residues were cyclized more readily than expected, and this process was relatively insensitive to salt concentration. This observation is discussed with regard to the stabilization of beta-turns by i-to-(i + 3) ionic interactions in peptides and proteins. A method for blocking thiols was introduced as an improvement in the analysis of the equilibrium mixtures.

Amino Acid Sequence↗

PubMed1994

Side-product formation during cyclization with HBTU on a solid support.

The coupling reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was used in an attempt to prepare a highly strained 10-membered lactam ring on a solid support via side-chain to side-chain cyclization of the adjacent alpha,gamma-diaminobutyric (Dab) and D-glutamic acid residues in [Dab2,D-Glu3,Leu5]enkephalinamide. This attempted cyclization failed, however, and yielded linear products instead. Characterization by mass spectrometry, amino acid analysis, peptide sequencing and NMR indicated that the major products were the tetramethylguanidinium (Tmg) derivatives [Dab(Tmg)2,D-Glu3,Leu5]-enkephalinamide and the corresponding dimeric linear Tmg-containing peptide which resulted from the transfer of the tetramethyluronium moiety from HBTU to the amino side chain of Dab. The formation of these tetramethylguanidinium side products during cyclization reactions limits HBTU's usefulness for the formation of lactams.

Amino Acid Sequence↗

PubMed1996

Cyclic morphiceptin analogs: cyclization studies and opioid activities in vitro.

Attempts were undertaken to develop cyclic beta-casomorphin-5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH2). The tetrapeptide sequence Boc-Tyr(tBu)-D-Xaa-Phe-Yaa-OH (Xaa = Lys, Orn, A2bu; Yaa = Pro in L- or D-configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the omega-amino group of Xaa and the C-terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and 1H-NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L-configuration could a cyclic monomer be detected, and the cyclodimer (7-9) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11-membered ring of the cyclic [D-A2bu2, D-Pro4]-morphiceptin analog 12, since only traces of the monomer were found. In the case of both the [D-Lys2, D-Pro4]-analog 10 and the [D-Orn2, D-Pro4]-analog 11, the cyclomonomer/cyclodimer ratio was about 80:20. The cyclic monomers 10 and 11 showed high opioid activity in the mu-receptor-representative guinea pig ileum assay (IC50 = 2-5 nM) and in the delta-receptor representative mouse vas deferens assay (IC50 = 50-60 nM), whereas the potency of the cyclodimers was 2-3 orders of magnitude lower in both in vitro bioassays.

Analgesics↗

PubMed1998

Solid-phase synthesis and on-resin cyclization of a disulfide bond peptide and lactam analogues corresponding to the major antigenic site of HIV gp41 protein.

A cyclic peptide that spans the major antigenic determinant of the human immunodeficiency virus (HIV) glycoprotein 41 (gp41) has been synthesized according to various strategies. For immunodiagnostic applications, biotin was added at the N-terminus of the peptide and aminohexanoic acid was used as a spacer. Polymer-supported oxidations were carried out in a variety of ways with thallium (III) trifluoroacetate. The biotinylcyclic peptide was released from the support using trimethylsilyl trifluoromethane sulfonate and various scavengers. The efficacy of these different cyclization and cleavage procedures was compared. Side reactions were studied, and a simple and efficient procedure was set up to monitor peptide cyclization by mass spectrometry. In a second series of syntheses the disulfide bridge was replaced by an amide bond. For this purpose, an aspartic acid derivative and a diaminopropionic acid were introduced during the synthesis in place of the two cysteine residues in the parent sequence. On-resin cyclization was performed and led to a major side-product identified as a piperidide. This undesired base-mediated side reaction was prevented when, instead of piperidine, 1,8-diazabicyclo-[5.4.0]undec-7-ene was used for fluorenylmethyl ester deprotection. Reactivity of these peptides with different patients' sera and with a monoclonal antibody directed against the whole gp41 was tested using an enzyme-linked immunosorbent assay.

Antibodies, Monoclonal↗

PubMed1999

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.

Antibodies, Monoclonal↗

PubMed1996

The carotenoid 7,8-dihydro-psi end group can be cyclized by the lycopene cyclases from the bacterium Erwinia uredovora and the higher plant Capsicum annuum.

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc-crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8-dihydro-beta-carotene, via monocyclic beta-zeacarotene. zeta-Carotene was also found to be cyclized to bicyclic 7,8,7',8'-tetrahydro-beta-carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8-dihydro-psi end group to a 7,8-dihydro-beta end group, in addition to the usual cyclization of the psi end group to the beta end group. Furthermore, beta-carotene hydroxylase from Erwinia (CrtZ) was able to add a hydroxyl group to the 7,8-dihydro-beta end group and the beta end group.

Bacterial Proteins↗

PubMed1999

Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis experiments: initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially cyclized A-ring.

In order to clarify the function of the DXDDTA motif in squalene-hopene cyclase and to identify the acidic amino acid residues crucial for the catalysis, site-directed mutagenesis experiments were carried out. The following results were found: (1) residues D374 and D376 work for the initiation of polyolefin cyclization which arises from the proton attack on the terminal double bond; (2) residue D377 stabilizes C-10 carbocation of the initially cyclized A-ring intermediate, leading to subsequent B-ring closure, which was further verified by isolating the partially cyclized monocyclic product; (3) residues D313 and D447 outside the DXDDTA motif were identified as new active sites; (4) the H451 residue is likely to work in the protonated form to enhance the acidity of the carboxyl groups of D374 and/or D376.

Amino Acid Sequence↗

PubMed1991

Oxidative cyclization of 2',3'-O-isopropylideneadenosines to 5'-O,8-cycloadenosines: considerations of N6-substituent effects and mechanism.

Oxidative cyclization of 2',3'-O-isopropylideneadenosines to the corresponding 5'-O,8-cyclo-2',3'-O-isopropylideneadenosines was achieved by using by lead tetraacetate, cupric chloride, and N-halogeno-succinimide as an oxidant, and by irradiation with a uv-visible light in the presence of pyrimido[5,4-g]pteridinetetrone 5-oxide. The reaction modes for the oxidative cyclization were investigated and discussed. In particular, N6-substituent effects on the oxidative cyclization provided a positive proof supporting the respective reaction mode.

Adenosine↗

PubMed1989

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene.

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.

Bicyclic Monoterpenes↗

PubMed1988

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (-)-endo-fenchol.

The conversion of geranyl pyrophosphate to (-)-endo-fenchol is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with a preparation of (-)-endo-fenchol cyclase (synthase) from common fennel (Foeniculum vulgare) gave labeled product of unchanged 3H:14C ratio in both cases, and each was dehydrated to a mixture of alpha- and beta-fenchene which were oxidized to the corresponding alpha- and beta-fenchocamphorones, again without change in isotope ratio. 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 findings indicated that the configuration at C1 of the substrate was retained in the enzymatic transformation to (-)-endo-fenchol which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to (3R)-linalyl pyrophosphate and cyclization of the latter via the anti-endo-conformer. These absolute stereochemical elements of the reaction sequence were confirmed by the enzymatic conversion of (3R)-1Z-[1-3H]linalyl pyrophosphate to (-)-endo-fenchol and by the location of the tritium in the derived fenchocamphorones as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to (-)-endo-fenchol.

Camphanes↗

PubMedDate not supplied

Influence of solid support, solvent and coupling reagent on the head-to-tail cyclization of resin-bound peptides.

Head-to-tail cyclization of peptides attached to insoluble supports by means of the side chains of aspartic acid or glutamic acid allows the rapid synthesis of cyclic peptides of widely varied amino acid sequence. However, two side reactions dramatically reduce the yield of the desired compound; these are (1) interpeptide condensation that creates dimers, trimers and higher-order oligomers and (2) racemization of the C-terminal residue. A comparison was made between benzotriazoyloxy-tris-(dimethyl-amino)phosphonium hexafluorophosphate (BOP) and diisopropylcarbodiimide (DIPCDI) as cyclization reagents on kieselguhr-supported polydimethylacrylamide, chloromethyl-PolyHipe, and two commercially available polystyrene supports using DMF, CH2Cl2 and THF as cyclization solvents. Both of the side reactions are dependent on the amino acid sequence, the reagent used to couple the ends of the peptide and the solid support.

Amino Acid Sequence↗

PubMed2004

Titanocene-catalyzed cascade cyclization of epoxypolyprenes: straightforward synthesis of terpenoids by free-radical chemistry.

The titanocene-catalyzed cascade cyclization of epoxypolyenes, which are easily prepared from commercially available polyprenoids, has proven to be a useful procedure for the synthesis of C(10), C(15), C(20), and C(30) terpenoids, including monocyclic, bicyclic, and tricyclic natural products. Both theoretical and experimental evidence suggests that this cyclization takes place in a nonconcerted fashion via discrete carbon-centered radicals. Nevertheless, the termination step of the process seems to be subjected to a kind of water-dependent control, which is unusual in free-radical chemistry. The catalytic cycle is based on the use of the novel combination Me(3)SiCl/2,4,6-collidine to regenerate the titanocene catalyst. In practice this procedure has several advantages: it takes place at room temperature under mild conditions compatible with different functional groups, uses inexpensive reagents, and its end step can easily be controlled to give exocyclic double bonds by simply excluding water from the medium.

Catalysis↗

PubMed2005

Intramolecular, reductive cyclization of beta-ketoisothiocyanates promoted by using samarium diiodide.

A novel samarium diiodide (SmI2) promoted intramolecular cyclization of beta-ketoisothiocyanate, derived from alpha,beta-unsaturated esters and ammonium thiocyanate led to alpha-hydroxythiolactams and/or thiolactams in high yields. Treatment of beta-ketoisothiocyanate with two equivalents of SmI2 gave a mixture of alpha-hydroxythiolactam and thiolactam. Four equivalents of SmI2 afforded only thiolactam in high yields. The intramolecular cyclization took place with high to complete stereoselectivity. A mechanism to explain this transformation is proposed.

Cyclization↗

PubMed2005

Palladium(0)-catalyzed tandem cyclization of allenenes: direct construction of tricyclic heterocycles through aromatic C--H activation.

Palladium(0)-catalyzed tandem cyclization of allenenes is described. Treatment of allenenes with an aryl halide, potassium carbonate, and catalytic [Pd(PPh(3))(4)] in dioxane afforded tri- or tetracyclic heterocycles in moderate to good yields through insertion of arylpalladium(II) halide into the allenic moiety, intramolecular carbopalladation, and aromatic C--H bond activation. The substituent on the olefin terminus has proven to be essential for the success of the tandem cyclization. The reaction with heterocyclic aryl halides such as iodopyrazine or 4-bromo-1-methylindole afforded tri- or tetracyclic heteroaromatic products in good yields.

Alkenes↗