Cetalox and analogues: synthesis via acid-mediated polyene cyclizations

Using a novel, acid-mediated cyclization methodology, a direct access to Cetalox ((+/-)-1; a commercially important ambergris-type odorant) and various structurally related didehydro (i.e., 19, 26, and 30) and tetradehydro (i.e., 28 and 37/38) analogues is described. Treatment of either (E,E)-14 or (E)-15 with an excess of FSO(3)H in 2-nitropropane at -90 degrees stereospecifically afforded (+/-)-1 in 40 and 42% yield, respectively. Under similar conditions, cyclization of (E)-18 or 20 furnished 19 in 60 and 64% yield, respectively. Analogously, using an excess of ClSO(3)H in CH(2)Cl(2) at -80 degrees, 26 is formed with high stereoselectivity by cyclization of either (E)-24 or (Z)-25 (52 and 31% yield, resp.); in the same manner, 28 was prepared from 27 (22% yield). The same principle was applied to the synthesis of racemic Superambrox (30), via cyclization of 35, but only with poor selectivity (22%) and low yield (7%). Another approach via cyclization of (E)-40 under solvolysis conditions (excess TFA in CH(2)Cl(2) at -10 degrees) gave a higher yield (15%) with improved selectivity (43%). Finally, cyclization of 34 (1:1 diastereoisomer mixture) afforded 37/38 (10:1) in 27% yield. The qualitative organoleptic properties of 19, 26, 28, 30, and 37/38 (10:1) are briefly discussed.     

Exploring Solution-Phase Cyclization and Sulfamyl Safety-Catch Resin Strategies for the Total Synthesis of the Marine Antimicrobial Cyclic Tetrapeptide Cyclo(GSPE)

Head-to-tail cyclization of 12-membered tetrapeptides has been reported to be synthetically challenging due to their highly strained ring systems. Previously, we reported the total synthesis of the marine antimicrobial cyclic tetrapeptide cyclo(GSPE) using the glutamic acid side-chain Wang resin tethering strategy with a 5 % yield under microwave irradiation (Lim et al., Int J Pept Res Ther 16:145–152, 2010). Besides the low yield, this strategy was also constrained to glutamic acid-containing peptides. In this communication, we report the outcomes of two alternate strategies to synthesize cyclo(GSPE): (A) solution-phase cyclization and (B) solid-phase synthesis using the sulfamylbutyryl safety-catch resin. Experimental results using all four possible linear precursors revealed that the former strategy failed to produce the target cyclic peptide while the latter approach afforded an improved overall yield of 8–9 % using the linear precursor H-Ser(tBu)-Pro-Glu(OtBu)-Gly-resin. Interestingly, we discovered that the placement of proline at the N- or C-termini of the linear peptide precursors failed to produce the target cyclic peptide. We also concluded that strategy B could provide the versatility needed to make a cyclic tetrapeptide library for bioactivity screening.     

Synthesis of HC toxin and related cyclopeptides containing the (L-Ala-D-Ala-L-Ada-D-Pro) sequence

In studies leading to HC toxin synthesis, a phytotoxic cyclic tetrapeptide with the sequence cyclo (L-Ala-D-Ala-L-Aoe-D-Pro), we have determined optimal conditions for the cyclization which constitutes one of the most important steps in the synthesis of the toxin. All four possible sequences containing an optically active precursor, i.e. L-Ada = (2 S)-2-amino-9-decenoic acid instead of Aoe, have been prepared and subjected to cyclization. Owing to the differences in racemization risk during activation of the terminal carboxyl aminoacid different cyclization procedures have been applied. Cyclopeptide yields and selectivity between cyclomonomer and dimer both containing the title sequence are mainly controlled by the linear precursor sequence. The cyclic tetrapeptide is only obtained with D-proline in the C-terminal position, the best yield reached by the -ONSu activation method. Starting from the peptide, the (9S, 9R) HC toxin epimer on the epoxidic carbon atom has been further synthesized in two steps.     

Rebuilt 3D structure of the chloroplast f1 ATPase-tentoxin complex

The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.     

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.     

Examination of methodology for the synthesis of cyclic thioether peptide libraries derived from linear tripeptides.

Work was undertaken to examine methodology for the cyclization of linear tripeptides on the solid phase via intramolecular S-alkylation using the Multipin(trade mark) Solid-Phase Peptide Synthesis platform. While previous work had shown that this chemistry could be used to efficiently cyclize linear tetrapeptide libraries, application of this synthetic strategy to the model linear tripeptide sequence Leu-Ser-Lys resulted in significant cyclic dimer formation. Ultimately, it was found that the addition of a large excess of lithium in the form of LiCl to the cyclization solution, significantly reduced cyclic dimer formation affording highly pure crude cyclic monomer. The application of this modified cyclization protocol to the preparation of cyclic peptide libraries was successfully demonstrated with the synthesis of a 20-membered library 4{1-20} based on the linear tripeptide sequence Leu-Xxx-Lys in which the position Xxx was varied with the standard 20 proteogenic residues.     

Syntheses of oligomannosides in solution and on a soluble polymer support: a comparison

The alpha-(1-->6)-linked and the alpha-(1-->2)-linked linear mannotetraose glycosides and, respectively, and the branched mannopentaoside [R=CH2(CH2)2CH2Cl] were synthesised by conventional methods in solution, using trichloroacetimidate donors, and the products were obtained in 39%, 42% and 40% overall yield, respectively. For comparative purposes, the same two linear tetrasaccharides were prepared by use of MPEG as a soluble polymer support, the yields being 34% and 14%, respectively. An attempted MPEG-supported synthesis of the branched pentasaccharide was unsuccessful. The merits and shortcomings of oligosaccharide syntheses on MPEG are discussed.     

Tentoxin effects on variable fluorescence and P515 electrochromic absorbance changes in tentoxin-sensitive and -resistant plant species

The effects of the cyclic tetrapeptide phytotoxin, tentoxin, on variable chlorophyll fluorescence quenching and the P515 electrochromic absorbance change were examined in tentoxin-sensitive and -resistant species and an interspecies hybrid. Immediately after infiltration of leaf discs, up to 100 μM tentoxin had no effect on the O, I, D, or P portions of the fluoresence transients of either tentoxin-sensitive or -resistant plants. However, the quenching of fluorescence following maximal fluorescence was reduced by approximately fourfold in tentoxin-treated tissues of tentoxin-sensitive plants, but was unaffected in resistant plants. Tentoxin significantly increased the magnitude of the P515 electrochromic absorbance change in all tentoxin sensitive plants by an average of approximately twofold. However, it was unaffected by tentoxin in resistant species. These data suggest that there is a close correlation between interaction of tentoxin with CF₁ ATPase in vivo and the ability to cause chlorosis in developing chloroplasts.     

Phenylglycine as a novel P2 scaffold in hepatitis C virus NS3 protease inhibitors

Molecular modeling and inhibitory potencies of tetrapeptide protease inhibitors of HCV NS3 proposed phenylglycine as a new promising P2 residue. The results suggest that phenylglycine might be capable of interacting with the NS3 (protease-helicase/NTPase) in ways not possible for the common P2 proline-based inhibitors. Thus, a series of tripeptides, both linear and macrocyclic, based on p-hydroxy-phenylglycine in the P2 position were prepared and their inhibitory effect determined. When the p-hydroxy group was replaced by methoxy, isoquinolin-, or quinolinyloxy functions, inhibitors with improved potencies were obtained. The P2 phenylglycine-based inhibitors were further optimized by C-terminal extension to acyl sulfonamides and by P1-P3 cyclization, which gave products with inhibition constants in the nanomolar range ( approximately 75nM).     

Convenient synthesis of bifunctional tetraaza macrocycles.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.     

Tentoxin sensitivity of citrus: cotyledon-dependency of growth inhibition and reversibility of chlorosis

Tentoxin is a cyclic tetrapeptide, produced by the fungus Alternariaalternata, that induces chiorosis in germinating seedlings ofsome angiosperms. Since the most pronounced chiorotic effectof tentoxin is at the initial stages of germination most studieshave evaluated the effects of tentoxin on cotyledons. In thispreliminary work a unique biological system was establishedfor the study of the mechanism of tentoxin induced chiorosisin developing citrus seedlings. This system was used to comparethe effects of tentoxin on the in vitro germination of intactversus decotyle donized embryos. It is demonstrated here thatthe chlorotic effect of tentoxin is reversible and that ten-toxin blocks the ability of decotyledonized embryos to utilizenutrients from the growth medium and, there fore, to compensatefor the lack of cotyledons. The citrus system offers a uniqueway to study the relation between the effect of tentoxin onthe activity of choloplast ATPase and the induction of chlorosis. Key words: Tentoxin, citrus, chiorosis     

Cyclic peptides. XXVI. Synthesis of AM-toxin II analogs by cyclization through ester bond formation

In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation.     

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.     

Potassium transport through lipid bilayer membranes facilitated by tentoxin dimers: A new mechanism of ion carrier transport?

The cyclic tetrapeptide tentoxin at concentrations greater than 5 X 10(-7) M selectively increases the ion conductivity for potassium of lipid bilayer membranes, while the naturally occurring derivative dihydrotentoxin has no influence on this property. Current-voltage curves, zero-current potential and charge-pulse measurements were used to characterize the action of tentoxin. The results suggest that a new mechanism of facilitated ion transport operates. The model of tentoxin dimerization and tentoxin-K+ association developed is in contradiction to the model of tentoxin pore formation described recently by Heitz et al. (Biophys. Chem. 23 (1986) 245).     

Anaerobic batch co-digestion of sisal pulp and fish wastes

Co-digestion of various wastes has been shown to improve the digestibility of the materials and biogas yield. Batchwise digestion of sisal pulp and fish waste was studied both with the wastes separately and with mixtures in various proportions. While the highest methane yields from sisal pulp and fish waste alone were 0.32 and 0.39 m3 CH4/kg volatile solids (VS), respectively, at total solid (TS) of 5%, co-digestion with 33% of fish waste and 67% of sisal pulp representing 16.6% of TS gave a methane yield of 0.62 m3 CH4/kg VS added. This is an increase of 59-94% in the methane yield as compared to that obtained from the digestion of pure fractions at 5% TS.     

Aggregation and ion transfer induced by tentoxin

It is shown that tentoxin, a cyclic tetrapeptide with two N-methylated residues, is able, when added to lipid bilayers, to increase the transmembrane current through discrete events. Conformational investigations involving 1H-NMR, infrared and circular dichroism studies show that, at concentrations above 7 X 10(-5) M, the cyclic tetrapeptide aggregates in chloroform. We suggest that the aggregates could form a pore through a stacking of cycles.     

Growth and internal ion concentrations in seedlings of winter wheat are affected by 1 pM tentoxin

The long-term effect of tentoxin on K^+;, Ca²⁺ and total phosphorus (P) concentrations in the roots and shoots of 7- and 14-day-old seedlings of winter wheat ( Triticum aestivum L. cv. Martonvásári-8) was studied. Growth (dry weight) and shoot to root ratios (dry weight and mineral concentrations) were also estimated. One p M tentoxin increased the shoot to root ratio for dry weight after a 14-day period of application. The concentration of Ca²⁺ slightly increased in the shoot. In roots, tentoxin caused a 30% higher accumulation of Ca²⁺ after 7 days, which did not change with treatment during the following 7 days. The accumulation of Ca²⁺ was enhanced by increasing concentrations of tentoxin. K⁺ and total P levels increased in roots but decreased in shoots after 7 days. However, they were redistributed between root and shoot during days 8–14 of tentoxin treatment. The effect of tentoxin is explained as a stimulation of ion transport mainly into the vacuoles of the immature metaxylem elements. It is suggested that tentoxin and other microbial products effective at very low concentrations may have a general significance in promoting plant infection or symbiosis via the modification of physiological or biochemical processes.     

Stimulation of Tentoxin Synthesis by Aged-Culture Filtrates and Continued Synthesis in the Presence of Protein Inhibitors

Tentoxin, a cyclic tetrapeptide produced by Alternaria alternata (Fries) Keissler, induces chlorosis in certain seedling plants. It can be extracted from culture filtrates of the fungus. Tentoxin production is stimulated and increased by using a mixture of aged culture filtrates and modified Richards solution. Aged culture filtrates can be obtained from 3-week-old or older cultures of A. alternata in modified Richards solution or Pratts solution. A mixture of aged culture filtrate and fresh medium in the ratio 2:3 gives the maximal enhancement of tentoxin production. This growth system provided us with a model for studying the effects of protein synthesis inhibitors on tentoxin production. Two antibiotics which inhibit protein synthesis at the ribosomal level were tested on growth, protein synthesis, and tentoxin production in A. alternata cultures. Cycloheximide at concentrations of 500 μg/ml or emetine at concentrations of 250 μg/ml did not inhibit tentoxin synthesis, although they stopped mycelial growth and protein synthesis of the fungus at the logarithmic growth stage in the enhancement medium. These results led us to conclude that tentoxin, like certain other bioactive cyclic peptides, is synthesized by a nonribosomal peptide synthesis mechanism.     

Synthesis of four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide via the asymmetric transformation (combined isomerization-preferential crystallization) of 1,4-thiazane-3-carboxylic acid

In order to synthesize four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide (TCA SO), (S)-1,4thiazane-3-carboxylic acid [(S)-TCA], which is one of the precursors, was prepared by the asymmetric transformation (combined isomerization-preferential crystallization) of (RS)-TCA. This asymmetric transformation was used (2R, 3R)-tartaric acid [(R)-TA] as a resolving agent and salicylaldehyde as the epimerization catalyst in propanoic acid at 110 degrees C to afford a salt of (S)-TCA with (R)-TA in 100% de with a yield of over 90%. Optically pure (S)-TCA was obtained by treating the salt with triethylamine in methanol in a yield of over 80%, based on (RS)-TCA as the starting material. In addition, asymmetric transformation of (R)-TCA gave (S)-TCA in a yield of 60-70%. (S)-TCA was oxidized by hydrogen peroxide in dilute hydrochloric acid to selectively crystallize (1S, 3S)-TCA.SO. (1R, 3S)-TCA SO of 70% de from the filtrate was allowed to form a salt with (R)-TA after a treatment with triethylamine to give (1R, 3S)-TCA SO as a single diastereoisomer. (1R, 3R)- and (1S, 3R)-TCA.SO were also prepared by starting from (R)-TCA that had been synthesized from L-cysteine.     

The NMR-derived conformation of neuropeptide AF,an orphan G-protein coupled receptor peptide

The tertiary structure of the pain modulating and anti-opiate neuropeptide, human neuropeptide AF (NPAF) (the sequence is AGEGLNSQFWSLAAPQRF-NH(2)), was determined by (1)H-NMR. The structure of NPAF was determined in two solvent systems, namely 50%/50% trifluoroethanol-d(3)/H(2)O (TFE/H(2)O) and in the cell membrane mimetic micelle, sodium dodecylsulfate-d(25) (SDS). The receptor for NPAF is an orphan G-protein coupled receptor, and the micellar SDS solvent system was used to emulate the cell membrane surface in line with the Cell Membrane Compartments Theory proposed by R. Schwyzer (Biopolymers, 1995, Vol. 37, pp. 5-16). In both solvent systems, NPAF was found to be primarily alpha-helical within the central portion of the molecule, from Asn(6) to Ala(14). The N-terminus was random in both solvent systems. In the SDS solution, the C-terminal tetrapeptide was structured and formed a type I beta-turn, whereas in TFE/H(2)O it was unstructured, showing the importance of the C-terminal tetrapeptide in receptor recognition. NPAF was found to associate with SDS, and was shown to be near the surface of the micelle by spin label studies with 5-doxyl-stearic acid.