Phosphine-Catalyzed (3 + 2) Annulation of δ‑Acetoxy Allenoates with 2‑Sulfonamidomalonate: Synthesis of Highly Substituted 3‑Pyrrolines and Mechanistic Insight

A mild and efficient synthetic protocol for 3-pyrrolines via the phosphine-catalyzed (3 + 2) annulation of δ-acetoxy allenoates with 2-sulfonamidomalonate is reported. The asymmetric version (up to 83% ee) is also achieved by using phosphine (<i>R</i>)-SITCP as the catalyst. Mechanistic experiments disclose that the involved deprotonation of amide N–H and aza-addition to vinyl phosphonium might proceed in a concerted manner

Design of gene targeting for <i>dArf6</i>, <i>Dscam-N</i> and <i>Dscam-C</i> founder Knock-out lines.

<p>*: 5′+3′ Arms: the lengths of 5′ and 3′ homology arms in targeting construct.</p><p>**: According to <i>Drosophila</i> genome release FB2011.07 at <a href="http://www.flybase.org" target="_blank">www.flybase.org</a>.</p

Expression of <i>W::Neo</i> confers both <i>w+</i> and G418-resistance in flies.

<p><b>A</b>. Eye color in representative males from (clockwise from top right): <i>w¹¹¹⁸</i>, <i>y w; pArf6^GX22/TM3</i>, <i>y w; pDscam-N^GX113/TM3</i>, and <i>y w; pDscam-C^GX1/TM3</i>. <b>B</b>. G418-resistance of <i>W::Neo</i> transgenic donor lines and founder knock-out flies. Arrowheads indicate 0% survival rate. <i>FRT^[Neo]</i>: <i>y w; FRT-42D ubi::GFP^NLS/+</i>; <i>P{donor}</i>: transgenic donor insertion; <i>KO</i>: targeting allele; <i>w^[−]</i>: <i>TM3/+</i> and <i>CyO/+</i> cross progeny. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#s3" target="_blank">Materials and Methods</a> for detail genotypes.</p

Gene targeting of <i>Dscam-N</i> and <i>Dscam-C</i>.

<p><b>A</b>. Targeting design and PCR verification of <i>Dscam-N</i> and <i>Dscam-C</i> founder lines. Boxed are the genomic DNA (gDNA) structure and alternative-splicing patterns of <i>Dscam</i> locus. <i>Dscam</i> locus contains four alternative-splicing exons: 4, 6, 9 and 17 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Schmucker1" target="_blank">[13]</a>. Green boxes are gDNA regions used for 5′ and 3′ homology arms in the targeting constructs. In the <i>Dscam-N</i> founder knock-out line, a 5.7 kb genomic DNA covering the alternatively spliced exon 4 are deleted. In the <i>Dscam-C</i> founder knock-out line, a 7.6 kb genomic DNA covering the alternatively spliced exon 17 plus all the remaining downstream exons and 3′UTR are deleted. <i>Dscam-N</i> and <i>Dscam-C</i> founder knock-out lines carrying <i>W::Neo</i> marker are verified by 5′ and 3′ PCRs. 5′ or 3′ PCR is designed with one primer annealing within the W::Neo, while another primer anneals outside the gDNA region used for homology arms (“5′ gDNA” or “3′ gDNA”) in targeting constructs. Thus, only the correct targeting events will yield PCR products of expected size. <i>Dscam-N</i> and <i>Dscam-C</i> founder lines with <i>W::Neo</i> removed are further verified by dPCR-1 and dPCR-2. dPCR-1 is located within, while dPCR-2 spans over, the deleted region of <i>Dscam-N</i> or <i>Dscam-C</i>. <b>B</b>. 5′ and 3′ PCR-1 (red and yellow arrowheads, respectively) results from adults of <i>Dscam-N^GX07[w+]/CyO, Dscam-N^GX01[w+]/CyO, Dscam-C^GX101[w+]/CyO</i> and <i>Dscam-C^GX06[w+]/CyO</i>. <i>w¹¹¹⁸</i> was used as wild type control. White arrowheads pointing to non-specific PCR products. <b>C</b>. dPCR-1 (yellow arrowhead) and dPCR-2 (red arrowhead) results from embryos of <i>Dscam-N^GX01[w−]</i> and <i>Dscam-C^GX101[w−]</i> with <i>W::Neo</i> removed. <i>Dscam-N^GX01[w−]</i> and <i>Dscam-C^GX101[w−]</i> were balanced on <i>CyO, twi-GAL4, UAS-2xEGFP</i> (“CyO twiGFP”) chromosome so homozygous embryos could be distinguished by the absence of GFP. <i>w¹¹¹⁸</i> was used as the wild type control. For each PCR reaction genomic DNA was prepared by pooling approximately ten embryos together. For each sample, dPCR-1 and dPCR-2 reactions were carried out separately and were pooled before loading on the gel. MW: 1kb-plus DNA ladder (from Invitrogen); 5′ and 3′: the 5′ and 3′ homology arms of <i>Dscam-N</i> and <i>Dscam-C</i> targeting construct.</p

Generation of founder knock-out lines by ends-out targeting.

<p><b>^a.</b>Total estimated number of screening cross progeny screened in each targeting experiment. Because progeny of multiple vials or bottles were pooled and screened together, we did not register the clonality of the preliminary candidates. We assumed that each targeting mutant obtained was due to a distinct targeting event, based on the low HR frequency observed.</p><p><b>^b.</b>Since all female candidates were discarded in targeting experiments, the adjusted HR frequency should be twice higher than listed here.</p><p><b>^c.</b>Screening crosses were set up on the normal food first, then transferred to G418 food after two days.</p><p><b>^d.</b>A <i>dArf6^ΔKG#1</i> deletion allele generated by P-excision was used for complementation assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Huang4" target="_blank">[7]</a>.</p><p><b>^e.</b>Null allele of <i>P{PZ}Dscam⁰⁵⁵¹⁸</i> (BL#11412) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Schmucker1" target="_blank">[13]</a> was used for complementation assays.</p

Application of multiple selections in gene targeting.

<p><b>A</b>. Genetic crosses of ends-out targeting based on the dual positive screening of <i>w+</i> and <i>Neo+</i> for targeting candidates, together with the negative selection of UAS-Rpr (Rpr+) for eliminating false positives. “X”: genotypes eliminated or greatly reduced in numbers by G418 selection or UAS-Rpr counter-selection. <b>B</b>. Map of pGX-attP-WN. pGX-attP-WN is a P-element based transforming vector. <i>5′P</i> and <i>3′P</i>: 5′ and 3′ P-element sequences; Amp^R: ampicillin-resistant gene.</p

Comparisons between the reference and modeled fractions.

<p>(<b>a</b>) Vegetation fraction scatter plot for 2010, (<b>b</b>) vegetation fraction residuals for 2010, (<b>c</b>) vegetation fraction scatter plot for 2002, and (<b>d</b>) vegetation fraction residuals for 2002. Perfect agreement, represented by the 1∶1 line, is displayed in the scatter plots. The best-fit line is displayed in the residual plots to indicate the general trends of overestimation and underestimation.</p

Total percent vegetated area and changes from 1990 to 2010 for the study area and the two scenic spots.

<p>TPVA is the total percent of vegetated area, XNWP is the Xixi National Wetland Park, and WLSS is the West Lake Scenic Spots.</p><p>Total percent vegetated area and changes from 1990 to 2010 for the study area and the two scenic spots.</p

Concentric vegetation coverage analysis in each belt for each year.

<p>(a) Average vegetation fraction (AVF), (b) percentage of area of high coverage pixels, (c) percentage of area of middle-high coverage pixels, (d) percentage of area of middle coverage pixels, and (e) percentage of area of low coverage pixels.</p

Spatial characteristics of the vegetation fraction change from the urban center over the two periods.

<p>(a) 1990–2002 and (b) 2002–2010. The vegetation change categories are shown by the different colors in the legend.</p