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PubMed1991

Effects of temperature on excluded volume-promoted cyclization and concatemerization of cohesive-ended DNA longer than 0.04 Mb.

The 0.048502 megabase (Mb), primarily double-stranded DNA of bacteriophage lambda has single-stranded, complementary termini (cohesive ends) that undergo either spontaneous intramolecular joining to form open circular DNA or spontaneous intermolecular joining to form linear, end-to-end oligomeric DNAs (concatemers); concatemers also cyclize. In the present study, the effects of polyethylene glycol (PEG) on the cyclization and concatemerization of lambda DNA are determined at temperatures that, in the absence of PEG, favor dissociation of cohesive ends. Circular and linear lambda DNA, monomeric and concatemeric, are observed by use of pulsed field agarose gel (PFG) electrophoresis. During preparation of lambda DNA for these studies, hydrodynamic shear-induced, partial dissociation of joined cohesive ends is fortuitously observed. Although joined lambda cohesive ends progressively dissociate as their temperature is raised in the buffer used here (0.1 M NaCl, 0.01 M sodium phosphate, pH 7.4, 0.001 M EDTA), when PEG is added to this buffer, raising the temperature sometimes promotes joining of cohesive ends. Conditions for promotion of primarily either cyclization or concatemerization are described. Open circular DNAs as long as a 7-mer are produced and resolved. The concentration of PEG required to promote joining of cohesive ends decreases as the molecular weight of the PEG increases. The rate of cyclization is brought, the first time, to values that are high enough to be comparable to the rate observed in vivo. For double-stranded DNA bacteriophages that have a linear replicative form of DNA (bacteriophage T7, for example), a suppression, sometimes observed here, of cyclization mimics a suppression of cyclization previously observed in vivo. The PEG, temperature effects on DNA joining are explained by both the excluded volume of PEG random coils and an increase in this excluded volume that occurs when temperature increases.

Bacteriophage lambda↗

PubMed1982

High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces clavuligerus.

A thirteen-fold excess of dithiothreitol maintains delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) in its monomeric form under the conditions normally encountered in an ACV cyclization assay system, using Streptomyces clavuligerus. A reversed phase high performance liquid chromatographic (HPLC) system which separates ACV monomer from isopenicillin N, penicillin N and from other cyclization assay components has been developed as follows; mobile phase: 5% methanol-95% KH2PO4 (0.05 M adjusted to pH 4.0 with concentrated H3PO4; stationary phase: muBondapak-C18; flow rate: 2 ml/minute for 5 minutes, 3 ml/minute for the remainder; detection: 220 nm). Under these conditions, authentic samples of isopenicillin N and penicillin N elute with a retention time of 5.25 minutes, which coincides with a peak of newly-formed material observed in cyclization reaction mixtures. The combined concentration of isopenicillin N and penicillin N[(iso)penicillin N] in cyclization reaction mixtures corresponds closely to the concomitant decrease in the ACV monomer. Cyclization reaction mixtures, in which crude cell-free extract from S. clavuligerus NRRL 3585 is the enzyme source, contain (iso)penicillin N at a concentration of 43.3 micrograms/ml after a 1-hour incubation period. Cyclization reaction mixtures, in which salt-precipitated cell-free extract from S. clavuligerus is the enzyme source, contain 39.0 micrograms/ml (iso)penicillin N.

Anti-Bacterial Agents↗

PubMed1994

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.

Acetylation↗

PubMed1980

[Kinetic studies of the (1 linked to 4)-alpha-D-glucopyranosyltransferase reaction catalyzed by cyclodextrin glycosyltransferase, particularly the cyclization with (1 linked to 4)-alpha-D-glucopyranosyl chains (average polymerization of 16) as substrate].

The transfer reactions, particularly the cyclization reaction, catalyzed by the cyclodextrin glycosyltransferase ((1 leads to 4)-alpha-D-glucan:[(1 leads to 4)-alpha-D-glucopyranosyl]-transferase (cyclizing), EC 2.4.1.19; CGT) from Klebsiella pneumoniae M 5 al were studied with (1 leads to 4)-alpha-D-glucopyranosyl chains (d.p. 16). The initial rate of the cyclization reaction with substrate concentrations from 1 up to 16 mM indicated a V of 6.2 kat . kg-1 of protein and a molar catalytical activity of 421.6 kat . mol-1 of enzyme. Km was found to be 1.03 mM. In addition to the cyclization, CGT simultaneously catalyzed a disproportionation of the substrate, yielding shorter maltooligosaccharides and (1 leads to 4)-alpha-D-glucopyranosyl chains which were significantly longer than the substrate itself. Cyclohepta- and cycloocta-amylose were accumulated in the course of longer incubation. They arose mainly from coupling reactions with the initially formed cyclohexaamylose and corresponding disproportionation of these transfer products. The extremely low formation rates of the higher cyclodextrins point to a "mistake" of the enzyme, when cyclizing to cyclohepta- and cyclooctaamylose.

Cyclization↗

PubMed2000

Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase.

BACKGROUND: The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers. RESULTS: Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5. CONCLUSIONS: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes.

Alkenes↗

PubMed2000

Isothermal Fourier transform infrared microspectrosopic studies on the stability kinetics of solid-state intramolecular cyclization of aspartame sweetener.

A novel Fourier transform infrared (FT-IR) microspectrophotometer equipped with differential scanning calorimetry (DSC) was used to investigate the kinetics of intramolecular cyclization of aspartame (APM) sweetener in the solid state under isothermal conditions. The thermal-dependent changes in the peak intensity of IR spectra at 1543, 1283, and 1259 cm(-1) were examined to explore the reaction. The results support that the intramolecular cyclization process in APM proceeded in three steps: the methoxyl group of ester was first thermolyzed to release methanol, then an acyl cation was attacked by the lone pair of electrons available on nitrogen by an S(N)1 pathway, and finally ring-closure occurred. The intramolecular cyclization of APM determined by this microscopic FT-IR/DSC system was found to follow zero-order kinetics after a brief induction period. The bond cleavage energy (259.38 kJ/mol) of thermolysis for the leaving group of -OCH(3), the bond conversion energy (328.88 kJ/mol) for the amide II NH band to DKP NH band, and the CN bond formation energy (326.93 kJ/mol) of cyclization for the DKP in the APM molecule were also calculated from the Arrhenius equation. The total activation energy of the DKP formation via intramolecular cyclization was 261.33 kJ/mol, calculated by the above summation of the bond energy of cleavage, conversion, and formation, which was near to the value determined by the DSC or TGA method. This indicates that the microscopic FT-IR/DSC system is useful as a potential tool not only to investigate the degradation mechanism of drugs in the solid state but also to directly predict the bond energy of the reaction.

Aspartame↗

PubMed1992

The utility of side-chain cyclization in determining the receptor-bound conformation of peptides: cyclic tripeptides and angiotensin II.

The effect of side-chain cyclization on accessible backbone conformations of tripeptides, X-Ala-Y (X and/or Y = Cys, Hcy (Hcy: homocysteine), cis 4-mercaptoproline (MPc), and trans 4-mercaptoproline (MPt)), was elucidated using two variants of systematic conformational search. In addition to cyclization through a disulfide bond, the thioether (-S-CH2-) and amide (-CO-NH-) side-chain analogues of Cys-Ala-Cys and Hcy-Ala-Hcy were evaluated. The number of valid backbone conformations and the allowed phi, psi space were evaluated for each compound, and the ability of the cyclic tripeptides to accommodate beta-turn conformations was examined in order to assess the value of cyclization in limiting conformational freedom. Based on the number of conformations, cyclization was highly effective in reducing the backbone degree of freedom: in order of decreasing number of conformations, Ala-Ala-Ala 1 >> Hcy-Ala-Hcy 2 >> Cys-Ala-Hcy 3 approximately equal to Hcy-Ala-Cys 4 >> MPc-Ala-Hcy 5, 7 > Cys-Ala-Cys 6 > MPc-Ala-Cys 8 > Hcy-Ala-MPt 9 > Cys-Ala-MPt 10 approximately equal to MPc-Ala-MPt 11. Although Hcy-Ala-Hcy 2 had the greatest number of conformations of the cyclic peptides studied, it was still greatly constrained relative to its linear analogue 1. The bicyclic ring system introduced by MP was even more effective in constraining the cycle, having greater impact at position 3 than at position 1. Under the conditions of the study, cyclization of MP-containing analogues could be effected only with the cis isomer (MPc) at position 1 and/or the trans isomer (MPt) at position 3. Sterically allowed conformations of Ala2 for the cyclic tripeptides 2-4 were generally similar to those of the linear tripeptide 1, while those of Cys-Ala-Cys 6 and MPc-Ala-Hcy 7 were restricted to a smaller region of phi 2, psi 2 space: the right- and left-handed alpha-helical conformation and the beta-conformation. This trend was even more pronounced for Hcy-Ala-MPt 9, Cys-Ala-MPt 10, and MPc-Ala-MPt 11, in which Ala2 was severely restricted to a very small region of phi, psi space: the left-handed alpha-helical conformation for 9-11, plus the beta conformation for 9. This suggests that MP at the 3-position is incompatible with a right-handed alpha-helical conformation at position 2.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1998

Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics.

A Monte Carlo simulation method for studying DNA cyclization (or ring-closure) has been extended to the case of protein-induced bending, and its application to experimental data has been demonstrated. Estimates for the geometric parameters describing the DNA bend induced by the catabolite activator protein (CAP or CRP) were obtained which correctly predict experimental DNA cyclization probabilities (J factors), determined for a set of 11 150 to 166 bp DNA restriction fragments bearing A tracts phased against CAP binding sites. We find that simulation of out-of-phase molecules is difficult and time consuming, requiring the geometric parameters to be optimized individually rather than globally. A wedge angle model for DNA bending was found to make reasonable predictions for the free DNA. The bend angle in the CAP-DNA complex is estimated to be 85 to 90 degrees, in agreement with estimates from gel electrophoresis and X-ray co-crystal structures. Since the DNA is found to have a pre-existing bend of 15 degrees, the change in bend angle induced by CAP is 70 to 75 degrees, in a agreement with an estimate from topological measurements. We find evidence for slight (approximately 10 degrees) unwinding by CAP. The persistence length and helical repeat of the unbound portion of the DNA are in accord with literature-cited values, but the best-fit DNA torsional modulus C is found to be 1.7 (+/- 0.2) x 10(-19) erg. cm, versus literature estimates and best-fit values for the free DNA of 2.0 x 10(-19) to 3.4 x 10(-19) erg.com. Simulations using this low value of C predict that cyclization of molecules with out-of-phase bends proceeds via undertwisting or overtwisting of the DNA between the bends, so as to align the bends, rather than through conformations with substantial writhe. We present experiments on the topoisomers formed by cyclization with CAP which support this conclusion, and thereby rationalize the surprising result that cyclization can actually be enhanced by out-of-phase bends if the twist required to align the bends improves the torsional alignment of the ends. The relationship between the present work and previous studies on DNA bending by CAP is discussed, and recommendations are given for the efficient application of the cyclization/simulation approach to DNA bending.

Computer Simulation↗

PubMed1996

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Amino Acid Sequence↗

PubMed1994

The protective effect of superoxide dismutase and catalase against formation of reactive oxygen species during reduction of cyclized norepinephrine ortho-quinone by DT-diaphorase.

Norepinephrine was oxidized by the Mn(3+)-pyrophosphate complex to the corresponding o-quinone at pH 6.5. Cyclized norepinephrine ortho-quinone showed an absorption maximum at 289 and 483 nm. No oxygen consumption was observed during oxidation of norepinephrine to o-quinone by Mn3+ and subsequent cyclization. The reduction of cyclized norepinephrine ortho-quinone to the corresponding hydroquinone was catalyzed by DT-diaphorase. However, the hydroquinone formed proved to be unstable in the presence of oxygen, since reduction of cyclized norepinephrine o-quinone by DT-diaphorase was accompanied by continuous oxidation of NADH and oxygen consumption. Addition of the chelator DETAPAC or SOD to the incubation mixture during reduction of cyclized norepinephrine ortho-quinone by DT-diaphorase strongly inhibited NADPH oxidation and oxygen consumption, suggesting that manganese and superoxide radicals were involved in hydroquinone autoxidation. Elimination of the effects of superoxide radicals, manganese and H2O2 on autoxidation of hydroquinone by addition of SOD, catalase and DETAPAC to the incubation mixture resulted in a 79% inhibition of NADH oxidation, suggesting that 21% of the autoxidation is oxygen-dependent. However, the effect of these additions on oxygen consumption was even more pronounced (93% inhibition).

Animals↗

PubMed2001

Primary cyclization in the polymerization of bis-GMA and TEGDMA: a modeling approach to understanding the cure of dental resins.

An optimal dental restorative polymeric material would have a homogeneous cross-linking density giving it consistent mechanical strength throughout the material. When multifunctional monomers are polymerized, a pendant double bond can react intramolecularly with the radical on its propagating chain to form a loop, which results in a primary cyclization reaction. Primary cyclization does not contribute to overall network structure, causes microgel formation, and leads to heterogeneity in the polymer. Knowledge of how cure conditions control the degree of primary cyclization and cross-linking in the polymer is important in developing better dental materials. To gain more understanding about the evolving polymer network, the photopolymerization of a typical dental resin (75/25 wt% bis-GMA/TEGDMA) is modeled using a first principals approach. The overall polymerization rate behavior of 75/25 wt% bis-GMA/TEGDMA is predicted using experimentally obtained propagation and termination kinetic rate constants. The effect of chain stiffness and light intensity on the polymerization kinetics is also explored. Furthermore, the model predicts the extent of cross-linking and primary cyclization in the growing polymer network. At 45% conversion, the fraction of bis-GMA and TEGDMA pendant double bonds created that have cycled is 11 and 33%, respectively. The model shows that using a stiff monomer, like bis-GMA, in dental resins diminishes the extent of cyclization and increases the cross-linking density of the polymer. Therefore, better mechanical properties are obtained than if more flexible monomers were used.

Algorithms↗

PubMed2000

Concurrent induction of two chiral centers from symmetrical 3, 4-disubstituted and 3,3,4-trisubstituted 4-pentenals using Rh-catalyzed asymmetric cyclizations

Asymmetric cyclization of symmetrical 3,4-disubstituted and 3,3, 4-trisubstituted 4-pentenals was studied using Rh-complexes with chiral ligands. The cyclization of symmetrical 4-pentenals 4a,b by a neutral Rh[(R)-BINAP]Cl afforded cis-3,4-disubstituted (4R)-cyclopentanones 9a,b of >95% ee in 25-31% yields; on the other hand, the cyclization of 4a-c by a cationic Rh[(R)-BINAP]ClO(4) afforded trans-3,4-disubstituted (4S)-cyclopentanones 10a-c of >95% ee in 70-81% yields. All stereoisomers could be stereoselectively made by the selection of a neutral or cationic Rh-complex, and (R)- or (S)-BINAP ligand. The Rh-catalyzed cyclization could be applied to the construction of cyclopentanones 17 and 18 bearing a chiral quaternary carbon. The cyclization by the cationic Rh[(R)-BINAP]ClO(4) afforded the optically active trans-3,3, 4-trisubstituted cyclopentanones 18a-c of 92-95% ee in 75-83% yields. The catalytic cycle was also studied by using deuterium aldehyde, and the tentative mechanisms of the enantio- and diastereoselection were proposed.

Journal Article↗

PubMed1999

Synthesis and kinetics of cyclization of MHC class II-derived cyclic peptide vaccine for diabetes.

Conformationally constrained cyclic peptides are known to be better vaccines because of their ability to mimic the native structure of a protein against which an immune response is sought. To test the hypothesis of using conformationally constrained, disease-associated, MHC-derived peptides as vaccines for the prevention of type I diabetes, a 22 amino acid nonobese diabetic(NOD) mouse MHC class II-derived synthetic peptide was cyclized by the formation of end-to-end disulfide bonds and used to prevent diabetes and insulitis in NOD mice. The peptide was synthesized by Fmoc chemistry and cyclized using two methods: a commercially available cyclizing resin (Ekathiox) and air oxidation. When a 10 m excess of resin was used, the Ekathiox yielded a substantial amount of cyclic peptide with few or no side reactions. The kinetics of cyclization by air oxidation at different temperatures indicated that increasing both temperature and pH decreased the cyclization time significantly. Air oxidation at pH 10 at 37-55 degrees C yielded the desired product within 2 h.

Amino Acid Sequence↗

PubMed2001

The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase.

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-2H](E, E)-FDP to >98% [5-2H]and [11-2H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-CDN, 15.8% [6,6,15,15,15-2H5]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The kcat/Km values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-bisabolol in the reaction mixtures was a Mg2+-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)-beta-farnesene, and beta-bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two alpha-helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.

Binding Sites↗

PubMed1983

Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium.

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive. In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Acremonium↗

PubMedDate not supplied

Design, synthesis and characterization of bradykinin antagonists via cyclization of the modified backbone.

With the aim of synthesizing cyclic antagonists of the nonapeptide hormone bradykinin with minimal side chain modification, we performed backbone to backbone and backbone to side chain cyclization. To probe and compare different strategies for this new kind of cyclization, the branched peptide bonds were formed by both reductive alkylation on the solid phase and by using preformed building units. Lactam bridges between the modified amide groups were formed by the use of the phenylalanine derivatives N(CH2COOH)Phe and N(CH2CH2NH2)Phe. The best results in the formation of the N-alkylamide bond were obtained with the coupling reagent PyBrop. The coupling rate was monitored by estimation of the N-terminal Fmoc-group. The cyclization was performed on the solid support. Unexpected difficulties resulted from the instability of the N-alkylamide bond under strong acidic conditions, as used for deprotection and for removal from the resin. We synthesized peptides with backbone to backbone cyclization between positions 2 and 5, as well as backbone to side chain cyclizations between positions 0 and 5, and between 2 and 6. The relatively high biological activities of some of the cyclic analogues support the supposed receptor-bound conformation of bradykinin antagonists with a beta-turn in the N-terminal sequence.

Animals↗

PubMed1986

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction.

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.

Catalysis↗

PubMed2000

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.

Acetylation↗