Stereocontrolled Synthesis of Substituted Chiral Piperidines via One-Pot Asymmetric 6π-Azaelectrocyclization: Asymmetric Syntheses of (−)-Dendroprimine, (+)-7-Epidendroprimine, (+)-5-Epidendroprimine, and (+)-5,7-Epidendroprimine

The asymmetric one-pot 6π-azaelectrocyclization of alkenyl vinyl stannane, ethyl (<i>Z</i>)-2-iodo-4-oxobutenoate, and (−)-7-isopropyl-<i>cis</i>-aminoindanol in the presence of a Pd(0) catalyst stereoselectively produced the tetracyclic aminoacetal compounds, resulting from the four-bond formation accompanying by controlling the stereochemistry at the two asymmetric centers. The produced cyclic aminoacetals can be regarded as synthetic precursors of substituted chiral piperidines, and the syntheses of 2,4- and 2,4,6-substituted piperidines were realized from the obtained aminoacetals by the stereoselective hydrogenation of the double bond conjugated with the C-4 ester group and alkylation at the aminoacetal moiety. In addition, the stereoselective synthesis of an indolizidine alkaloid, (−)-dendroprimine, and its three stereoisomers, (+)-7-epidendroprimine, (+)-5-epidendroprimine, and (+)-5,7-epidendroprimine, were achieved

Stereocontrolled Synthesis of Substituted Chiral Piperidines via One-Pot Asymmetric 6π-Azaelectrocyclization: Asymmetric Syntheses of (−)-Dendroprimine, (+)-7-Epidendroprimine, (+)-5-Epidendroprimine, and (+)-5,7-Epidendroprimine

The asymmetric one-pot 6π-azaelectrocyclization of alkenyl vinyl stannane, ethyl (<i>Z</i>)-2-iodo-4-oxobutenoate, and (−)-7-isopropyl-<i>cis</i>-aminoindanol in the presence of a Pd(0) catalyst stereoselectively produced the tetracyclic aminoacetal compounds, resulting from the four-bond formation accompanying by controlling the stereochemistry at the two asymmetric centers. The produced cyclic aminoacetals can be regarded as synthetic precursors of substituted chiral piperidines, and the syntheses of 2,4- and 2,4,6-substituted piperidines were realized from the obtained aminoacetals by the stereoselective hydrogenation of the double bond conjugated with the C-4 ester group and alkylation at the aminoacetal moiety. In addition, the stereoselective synthesis of an indolizidine alkaloid, (−)-dendroprimine, and its three stereoisomers, (+)-7-epidendroprimine, (+)-5-epidendroprimine, and (+)-5,7-epidendroprimine, were achieved

Tpz1TPP1 prevents telomerase activation and protects telomeres by modulating the Stn1-Ten1 complex in fission yeast

In both mammalian and fission yeast cells, conserved shelterin and CST (CTC1-STN1-TEN1) complexes play critical roles in protection of telomeres and regulation of telomerase, an enzyme required to overcome the end replication problem. However, molecular details that govern proper coordination among shelterin, CST, and telomerase have not yet been fully understood. Here, we establish a conserved SWSSS motif, located adjacent to the Lys242 SUMOylation site in the fission yeast shelterin subunit Tpz1, as a new functional regulatory element for telomere protection and telomere length homeostasis. The SWSSS motif works redundantly with Lys242 SUMOylation to promote binding of Stn1-Ten1 at telomere and sub-telomere regions to protect against single-strand annealing (SSA)-dependent telomere fusions, and to prevent telomerase accumulation at telomeres. In addition, we provide evidence that the SWSSS motif defines an unanticipated role of Tpz1 in limiting telomerase activation at telomeres to prevent uncontrolled telomere elongation

The MluI Cell Cycle Box (MCB) Motifs, but Not Damage-Responsive Elements (DREs), Are Responsible for the Transcriptional Induction of the <i>rhp51</i>⁺ Gene in Response to DNA Replication Stress

<div><p>DNA replication stress induces the transcriptional activation of <i>rhp51</i>⁺, a fission yeast <i>recA</i> homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates <i>rhp51</i>⁺ transcription is not understood. The promoter region of <i>rhp51</i>⁺ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in <i>rhp51</i>⁺ transcription. The full-length <i>rhp51</i>⁺ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the <i>rhp51</i>⁺ promoter abolished the induction of <i>rhp51</i>⁺ transcription by DNA replication stress. Consistent with a role for MCB motifs in <i>rhp51</i>⁺ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded <i>rhp51</i>⁺ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated <i>rhp51</i>⁺ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected <i>rhp51</i>⁺ transcription. The transcription of <i>rhp51</i>⁺ and <i>cdc18</i>⁺, an MBF-dependent G1/S gene, peaked simultaneously in synchronized <i>cdc25-22</i> cells. Furthermore, DNA replication stress maintained transcription of <i>rhp51</i>⁺ similarly to <i>cdc18</i>⁺. Collectively, these results suggest that MBF and its regulators mediate <i>rhp51</i>⁺ transcription in response to DNA replication stress, and underlie <i>rhp51</i>⁺ transcription at the G1/S transition.</p></div

Activities of the full-length <i>rhp51</i>⁺ and 3xMCB reporters in Δ<i>yox1</i> and Δ<i>nrm1</i> cells treated with HU.

<p>Wild-type (wt) cells, Δ<i>yox1</i> cells, and Δ<i>nrm1</i> cells transformed with the full-length <i>rhp51</i>⁺ (A, “Rhp51”) or 3xMCB (B) reporter were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 3 for each group. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype using one-way ANOVA followed by Tukey's test. ###<i>P</i><0.001 compared with wild-type cells treated with the same HU concentration using one-way ANOVA followed by Tukey's test.</p

Chemical Synthesis of a Complex-Type <i>N</i>‑Glycan Containing a Core Fucose

A chemical synthesis of a core fucose containing <i>N</i>-glycan was achieved. Asparagine was introduced at an early stage of the synthesis, and the sugar chain was convergently elongated. As for the fragment synthesis, we reinvestigated α-sialylation, β-mannosylation, and <i>N</i>-glycosylation to reveal that precise temperature control was essential for these glycosylations. Intermolecular hydrogen bonds involving acetamide groups were found to reduce the reactivity in glycosylations: the protection of NHAc as NAc₂ dramatically improved the reactivity. The dodecasaccharide–asparagine framework was constructed via the (4 + 4) glycosylation and the (4 + 8) glycosylation using the tetrasaccharide donor and the tetrasaccharide–asparagine acceptor. An ether-type solvent enhanced the yields of these key glycosylations between large substrates. After the whole deprotection of the dodecasaccharide, the target <i>N</i>-glycan was obtained

Activities of the full-length <i>rhp51</i>⁺ and 3xMCB reporters in Δ<i>chk1</i>, Δ<i>cds1</i>, Δ<i>chk1</i>Δ<i>cds1</i>, and Δ<i>rad3</i> cells treated with HU.

<p>Cells transformed with the full-length <i>rhp51</i>⁺ (A, “Rhp51”) or 3xMCB (B) reporter were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 3 for each group. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype using one-way ANOVA. #<i>P</i><0.05, ##<i>P</i><0.01, and ###<i>P</i><0.001 compared with wild-type cells treated with the same HU concentration using one-way ANOVA. +<i>P</i><0.05 and ++<i>P</i><0.01 compared with Δ<i>chk1</i> cells treated with the same HU concentration using one-way ANOVA.</p

<i>rhp51</i>⁺ and <i>cdc18</i>⁺ transcription in synchronized <i>cdc25-22</i> cells treated with HU.

<p>Wild-type cells transformed with the full-length <i>rhp51</i>⁺ (A) or <i>cdc18</i>⁺ (B) reporter were cultured to mid-log phase at 25°C in EMM and shifted to 36°C for 4 h. The cells were then maintained at 36°C continuously for G2 block (“36°C→36°C”) or shifted to 25°C for block and release (“36°C→25°C”). The cells were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##<i>P</i><0.01 and ###<i>P</i><0.001 compared with G2 block at the same HU concentration using one-way ANOVA.</p

Real-time monitoring of <i>rhp51</i>⁺ gene transcription in wild-type cells treated with HU.

<p><i>A</i>. A schematic diagram of luciferase reporter vectors containing the full-length <i>rhp51</i>⁺ promoter or <i>rhp51</i>⁺ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the <i>rhp51</i>⁺ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the <i>rhp51</i>⁺ promoter. The following regions of the <i>rhp51</i>⁺ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51^MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51^DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51^ΔMCB). <i>B</i>. Effect of HU on promoter activation. Wild-type cells transformed with the full-length <i>rhp51</i>⁺ (Rhp51), Rhp51^MCB, Rhp51^DRE, or Rhp51^ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length <i>rhp51</i>⁺ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. <i>n</i> = 4 for each group. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.</p

<i>Schizosaccharomyces pombe</i> haploid strains used in this study.

<p><i>Schizosaccharomyces pombe</i> haploid strains used in this study.</p