Synthesis of a Potential Intermediate for TMC-95A via an Organocatalyzed Aldol Reaction

<i>N</i>-Prolinyl­anthranil­amide-based pseudopeptide organocatalyst <b>14</b> was shown to promote enantioselective direct aldol reaction of 7-iodoisatin and 2,2-dimethyl-1,3-dioxan-5-one with 90% conversion (75% isolated yield), 90% enantioselectivity, and 23:1 diastereoselectivity. To demonstrate the synthetic utility of this chemistry, the racemic aldol reaction product was converted in five steps to a potential intermediate for construction of the natural product TMC-95A

Synthesis of a Potential Intermediate for TMC-95A via an Organocatalyzed Aldol Reaction

<i>N</i>-Prolinyl­anthranil­amide-based pseudopeptide organocatalyst <b>14</b> was shown to promote enantioselective direct aldol reaction of 7-iodoisatin and 2,2-dimethyl-1,3-dioxan-5-one with 90% conversion (75% isolated yield), 90% enantioselectivity, and 23:1 diastereoselectivity. To demonstrate the synthetic utility of this chemistry, the racemic aldol reaction product was converted in five steps to a potential intermediate for construction of the natural product TMC-95A

Hepatocyte growth on fibroblast feeder layer.

<p>Hepatocyte proliferation curve represented by the absorbance at 540 nm of BrdU-labelled hepatocytes at different days of culture.</p

Western blot analysis of 5 days cultured buffalo hepatocytes.

<p>Blots of buffalo hepatocytes lysate by using antibodies against albumin (Panel A); lane 1: HepG2 (Positive control), Lane 2: buffalo hepatocytes (test); Lane 3: skin fibroblast (negative control); Lane 4: pre-stained protein marker; cytokeratin-18 (Panel B); Lane 1: pre-stained protein marker; Lane 2: HepG2 cells; Lane 3: buffalo hepatocytes; Lane 4: skin fibroblast; and α1-antitrypsin (Panel C), order of lanes is similar to that shown in panel B.</p

Immunostaining of 5 days old cultured buffalo hepatocytes with α1-antitrypsin antibody.

<p>Panel A shows light microscopic image of hepatocytes, and panel B shows immunostained hepatocytes with α1-antitrypsin antibody labelled with FITC (green) and nuclear stain DAPI (blue) dyes.</p

Immunostaining of 5 days old cultured buffalo hepatocytes with anti-cytokeratin-18 and anti-albumin.

<p>Immunostaining with (A) CY3 labelled anti-cytokeratin-18 antibodies (fluorescence signal in red); (B) FITC labelled anti-albumin antibodies (green); (C) staining of hepatocytes nuclei with DAPI (blue). Panel D shows the merged images from panels A, B and C. Panel E shows light microscopic image of hepatocytes; panel F shows negative control (Isotype control), and panels G shows staining with DAPI, while panel H shows images merged from panels F and G.</p

Agarose gel electrophoresis of RT-PCR products of hepatocyte-specific marker genes expressed in 5 days old cultured hepatocytes.

<p>Panel A shows 293 bp amplicon of albumin; B—130 bp amplicon of hepatocyte nuclear factor 4α; C—240 bp amplicon of glucose-6-phosphatase; D—136 bp amplicon of CYP1A1; E—164 bp amplicon of CYP3A4; F—142 bp amplicon of tyrosine aminotransferase. Lane 1: 100 bp ladder; Lane 2: RT-PCR of liver tissue (positive control) by using the gene-specific primers; Lane 3: RT-PCR of respective genes from cultured buffalo hepatocytes; Lane 4: RT-PCR from skin fibroblasts (negative control). Amplification of Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was used as housekeeping gene.</p