Regio- and Stereoselective Cross-Coupling of Substituted Olefins and Imines. A Convergent Stereoselective Synthesis of Saturated 1,5-Aminoalcohols and Substituted Piperidines

A mild and efficient alkoxide-directed C−C bond-forming reaction is described that provides a regio- and stereoselective route to 1,5-aminoalcohols based on cross-coupling of substituted olefins and imines. Single and double diastereoselection is explored through the use of chiral imines and chiral homoallylic alcohols

Regio- and Stereoselective Cross-Coupling of Substituted Olefins and Imines. A Convergent Stereoselective Synthesis of Saturated 1,5-Aminoalcohols and Substituted Piperidines

A mild and efficient alkoxide-directed C−C bond-forming reaction is described that provides a regio- and stereoselective route to 1,5-aminoalcohols based on cross-coupling of substituted olefins and imines. Single and double diastereoselection is explored through the use of chiral imines and chiral homoallylic alcohols

Replacement of AA residues with functionally equivalent residues restores the properties of NFL-TBS.40-63.

<p>Using a similar experimental approach to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone-0049436-g001" target="_blank">Figure 1</a>, the amino-acids at position 1, 12, 14 and 16 were replaced by amino-acids with similar properties. While substitution of these AAs by alanine abolished their properties, replacement of these AAs by functionally equivalent residues restore the capacity to penetrate in cells and to alter the MT cytoskeleton. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone-0049436-g001" target="_blank">Figure 1A</a>, experiments were triplicated and a minimum of 200 cells was analyzed in each experiment.</p

CD spectra of wt and alanine-substituted peptides.

<p>CD spectra of 20 µM biotinylated wt and alanine-substituted NFL-TBS.40-63 peptides were measured in KF/phosphate buffer (Panel A), 2 mM SDS (Panel B) and pure water (Panel C). The spectra of wt peptide are in black. The spectra of peptides with a substitution by alanine at positions from 2 to 20 (except at position 6 where the original AA is alanine) are similar and represented by red lines. The spectra of wild-type (black), Tyr1Ala (magenta), Ser21Ala (dark blue), Ser22Ala (olive), Gly23Ala (light blue) and Ser24Ala (wine) are also presented. The number corresponds to the position of the replaced AA residue. CD spectra of 20 µM NFL-TBS.40-63 peptides phosphorylated at positions 17 (wine), 19 (magenta) and the two positions (cyan) were measured in KF/phosphate buffer (Panel D).</p

CD spectra of NFL-TBS.40-63 peptide.

<p>(A): CD spectra of 20 µM biotinylated NFL-TBS.40-63 peptide were measured in pure water (black line), 2 mM SDS (red line) and KF/phosphate buffer (blue line). The spectrum of peptide with a shuffled sequence (green) is also shown. (B): Change in CD signal at 220 nm of 20 µM biotinylated NFL-TBS.40-63 in KF/phosphate buffer (blue line) and 2 mM SDS (red line) with increasing temperature was measured and is presented as a function of temperature. The change with decreasing temperature measured in KF/phosphate buffer (light blue line) is also shown.</p

The 31-nt Y4-RNA fragment.

<p>(A) The amount of the 31-nt Y4-RNA fragment in plasma. A percentage of a read number of the 31-nt Y4-RNA fragment to a total read number of the 5–40-nt RNAs in plasma are graphed for three healthy persons (N1–N3) and three myeloma patients (MM1–MM3). (B) The structures of human 94-nt Y4-RNA and its 31-nt fragment. The Y4-RNA fragment can form a 5′-half-tRNA-like structure and bind a target RNA to form a pre-tRNA-like structure. An arrowhead and an arrow denote a cleavage site to generate the 31-nt fragment and a potential cleavage site by tRNase Z^L, respectively.</p

Phosphorylation of NFL-TBS.40-63 affects its properties.

<p>Replacement of Ser-17 or Ser-19 by a chemically phosphorylated serine, or by Asp strongly affected the capacity of the peptide to penetrate in cells and to destroy the MT network. The experimental conditions are similar to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone-0049436-g001" target="_blank">Figure 1A</a>.</p

Deep sequencing analysis of small RNA from human PBMC.

<p>(A) Frequencies of 5–40-nt RNAs. Read numbers of 5–40-nt RNAs in PBMC from three healthy persons (N4–N6) and three myeloma patients (MM4–MM6) are presented. (B) Relative frequencies of the 10 RNA categories. Each read RNA sequence was assigned to the categories mRNA, rRNA, scRNA, snRNA, srpRNA, miRNA, lncRNA, piRNA, snoRNA, and/or tRNA, and their relative frequencies are graphed for the above six persons.</p

Predicted fold of NFL-TBS.40-63 peptide.

<p>A. The 3D structure of NFL-TBS.40-63 predicted by PEP-FOLD shows a β-hairpin involving the N-terminal section (1–5) and a central section (14–18) of the peptide. The C-terminal end shows the initiation of an α-helix. B. Alternative 3D structure predicted by PEP-FOLD, where the peptide mainly presents an α-helix fold. The figures were prepared using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone.0049436-Humphrey1" target="_blank">[49]</a>.</p

Potential Small Guide RNAs for tRNase Z^L from Human Plasma, Peripheral Blood Mononuclear Cells, and Cultured Cell Lines

<div><p>Several pieces of evidence suggest that small RNA degradation products together with tRNase Z^L appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5′-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase Z^L, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5–40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5–40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5′-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase Z^L and sgRNA may be extended intercellularly.</p></div