Plants within the Caryophyllaceae, and certain other families, produce cyclic peptides (CPs) which generally consist of 5–12 proteinogenic amino acids. Until recently, very little was known about the biosynthesis of CPs in the Caryophyllaceae. Recently, in the Covello lab, two enzymes in Saponaria vaccaria were found to be involved in the processing of ribosome-derived linear precursors, giving rise to cyclic peptides. Thus, oligopeptidase 1 (OLP1) and peptide cyclase 1 (PCY1) are involved in the biosynthesis of segetalin A (a six-membered CP) from a 32 amino acid linear peptide precursor called presegetalin A1. PCY1 carries out the unusual cyclization reaction to form mature segetalin A from a linear intermediate. The purified recombinant PCY1, the first cloned plant enzyme whose function is peptide cyclization, was identified as a homologue of a prolyl-oligopeptidase from the S9 serine protease family. In principle, PCY1 performs an intra-molecular transpeptidation reaction to produce a CP. A homology-based structural model of PCY1 suggests that it has two domains, a catalytic α/β hydrolase domain and an unusual β- propeller domain. In an effort to define the substrate specificity of PCY1, a wide variety of synthetic peptide precursors were tested in assays and the results are discussed.

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No description available.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21777号 / 工博第4594号 / 新制||工||1716(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 木村 俊作, 教授 瀧川 敏算, 教授 藤田 晃司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Celogentin C and theonellamide F are a class of natural products that have potential antimitotic behavior. They both contain interesting bicyclic structures with unusual linkages within a central moiety. Celogentin C's highly functionalized tryptophan moiety has two unusual linkages, a β-substituted Leu connection to the C6 of the indole structure that makes up the left-hand ring, and a τ-N connection of the imidazole to the C2 of the indole constructing the right-hand ring. This right-hand ring connection was solved via a novel oxidative coupling procedure developed in our group and the left-hand ring was initially constructed via a radical conjugated addition of an isopropyl group. Due to stereoselective concerns, our group explored hydrogen bond donors as potential catalyst candidates. Unfortunately, there were challenges in limiting the background reaction and obtaining reproducible results. We then designed an alternative route to solve this left-hand ring connection which would have utilized MacMillan asymmetric hydrogenation and α-chlorination procedures. Further work towards a second generation synthesis of the β-Leu-(C6)Trp connection was halted with the publication of two formal syntheses of celogentin C. Theonellamide F contains a τ-L-histidino-D-alanine (τ-HAL) bridging unit that separates the left-and right-hand rings. Previous efforts in the synthesis of this natural product were hindered due to an inefficient regioselective synthesis of τ-HAL. Our proposed synthesis of τ-HAL began with commercially available L- and D-Ser methyl esters which were then chemically transformed and coupled to one another to create a bis-amino subunit. Further preparations afforded us with an important cyclic intermediate which should readily lead to the first regioselective synthesis of a τ-HAL.

No description available.