Eight cyclic analogs and corresponding linear homologs of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae were synthesized using solid-phase procedures on a phenylacetamidomethyl support. On-resin lactamization of the side chains of residues 7 and 10 to form rings containing from 14 to 18 atoms was effected by the BOP reagent. All peptides were highly homogeneous and gave expected molecular ions by FAB mass spectrometry. The constrained analogs had biological activities varying from 10% to less than 0.1% of that of [Nle12]-alpha-factor. In all cases, cyclic analogs with Glu in position 10 were more active than the homolog with Asp at this position. This trend was also found with the corresponding linear pheromones, suggesting that a gamma-carbonyl in position 10 is an important determinant of pheromone potency. The cyclic peptides had from 50- to 20000-fold lower affinities for the alpha-factor receptor than for [Nle12]-alpha-factor, as judged using a competition binding assay. Circular dichroism studies indicate that the cyclic lactam-containing region of cyclo7.10[Orn7, Glu10,Nle12]-alpha-factor retains a beta-turn-like structure similar to that found in the corresponding model tetrapeptide. The results show that covalently constrained analogs of the linear pheromone can maintain biological activity, despite binding poorly to the receptor, and indicate that a beta-turn-like structure in the center of the pheromone allows signal transduction.
Creative problem solving (CPS) is an approach for identifying solutions to problems within a structured, facilitated process. In the current studies, CPS was customized for general education intervention (GEI) teams in elementary schools. In the first study, 24 GEI teams were randomly assigned either to a CPS for GEI training condition or to a control group. Team outcome measures were tracked over the course of a school year, and the CPS for GEI teams consistently demonstrated superior performance relative to controls across all measures. One year later, a second study investigated 2 approaches to delivering training in CPS for GEI teams. Five teams received CPS for GEI training directly from university-based staff, and 9 teams received training from employees in their district who had previously received CPS for GEI training from the university-based staff (a “train-the-trainers” approach). Schools receiving training from their own staff performed as well as the independently trained schools, thereby providing support for the train-the-trainers approach. Discussion focuses on the results from both studies and identifies areas for future research and practice with CPS for GEI teams.
Analogs of the Saccharomyces cerevisiaeα‐mating factor, Trp‐His‐Trp‐Leu‐Gln‐Leu‐Lys‐Pro‐Gly‐Gln‐Pro‐Met‐Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid‐phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]α‐factor, where X = D‐Val, D‐Ala, l‐Ala and Gly, were prepared by on‐resin cyclization using thallic trifluoroacetate in yields of 20–30%. Linear sulfhydryl‐containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100‐fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5–10‐fold more active than their sulfhydryl‐contaihing homologs, and cyclo7,10[Cys7,L‐Ala9,Cys10,Nle12] α‐factor was 50‐fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the α‐factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5‐aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form β‐turns spanning residues 7–10 than their less active linear counterparts. The results provide strong evidence that this β‐turn is important for optimal signal transduction by α‐factor.
Analogues of α‐factor, the Saccharomyces cerevisiue tridecapeptide mating pheromone (H‐Trp‐His‐Trp‐Leu‐Gin‐Leu‐Lys‐Pro‐Gly‐Gln‐Pro‐Met‐Tyr‐OH), containing both p‐benzoyl phenylalanine (Bpa), a photoactivatable group, and 3‐(mono‐ or di‐iodo‐4‐hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid‐phase methodologies on a [phenylacetamidol‐methyl (PAM) resin. Bpa was introduced into the peptides using Bpa‐hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated α‐factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton‐Hunter reagent (3‐[diiodo‐4‐hydroxyphenyl]propanoic acid hydroxysuccinimide ester) in N,N‐dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high‐performance liquid chromatography on a reversed‐phase μ‐Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, α‐(diiodotyrosine)‐His‐Bpa‐Leu‐Gln‐Leu‐Arg‐Pro‐Gly‐Gln‐Pro‐Nle‐Tyr‐OH, and ε‐(diiodo‐HPP)‐Lys‐His‐Bpa‐Leu‐Gln‐Leu‐Arg‐Pro‐Gly‐Gln‐Pro‐Nle‐Tyr‐OH, were one‐twentieth to one‐fortieth as active as α‐factor, and exhibited approximately one order of magnitude lower affinity to the α‐factor receptor. The results suggest that these two analogues are α‐factor agonists and that they can be used as probes of the α‐factor receptor. © Munksgaard 1995.
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