Correction to Enantioselective Total Synthesis of (+)-Colletoic Acid via Catalytic Asymmetric Intramolecular Cyclopropanation of an α‑Diazo-β-keto Diphenylphosphine Oxide

Correction to Enantioselective Total Synthesis of (+)-Colletoic Acid via Catalytic Asymmetric Intramolecular Cyclopropanation of an α‑Diazo-β-keto Diphenylphosphine Oxid

Enantioselective Total Synthesis of (+)-Colletoic Acid via Catalytic Asymmetric Intramolecular Cyclopropanation of an α‑Diazo-β-keto Diphenylphosphine Oxide

The enantioselective total synthesis of (+)-colletoic acid, a potent naturally occurring 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor, is described. This total synthesis features a highly enantioselective catalytic asymmetric intramolecular cyclopropanation of an α-diazo-β-keto diphenylphosphine oxide and five highly stereoselective reactions (cyclopropane opening, Diels–Alder reaction, iodolactonization, alkene formation, and reduction of α,β-unsaturated carboxylic acid)

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

SEM and TEM images of TNAP-positive cells induced to differentiate into osteocyte-like cells.

<p>After isolation by FACS, TNAP-positive and -negative cells were cultured in OBM for 120 days. (a) SEM images of TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. (b) Images of toluidine blue-stained semi-thin sections of TNAP-positive cells are shown at low (left) and high (right) magnification with light microscopy. (c) TEM image of TNAP-positive cells. Arrowheads indicate cytoplasmic processes.</p

TNAP-positive cells express various osteocyte markers.

<p>(a) Osteogenic differentiation was confirmed by Alizarin Red staining after 40 days in OBM. The upper panels are whole-well images and lower panels are magnified images. (b) Phase-contrast images of TNAP-negative and -positive cells derived from hiPSCs at day 40 of culture in OBM (upper panel). The black box in the upper images represents the region shown in the middle and lower images. (c) Comparison of the expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by qRT-PCR. Osteogenic-differentiated HPDLCs (ODH) were used as a control. (d) TNAP-positive cells were treated with vehicle (white bars), 10 nM vitamin D3 (gray bars), or 50 nM vitamin D3 (black bars) for 6 days. (e) Comparison of expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by RT-PCR. Abbreviations: SOST, sclerostin; RELN, reelin; NPY, neuropeptide Y. Expression of these genes was analyzed by qRT-PCR, and mRNA levels of the genes were normalized to that of <i>GAPDH</i>. The experiments were performed in triplicate. Values represent mean ± S.D. (<i>n</i>  =  4). *<i>p</i><0.05, **<i>p</i><0.01.</p

Schematic representation of the protocol for differentiation of hiPSCs into osteoblast-like cells.

<p>EBs were prepared by culturing on low-attachment Petri dishes for 6 days and dissociated in 0.5 mg/ml collagenase type IV and 0.05% trypsin–EDTA. The trypsinized EBs were cultured in OBM on cell culture dishes. Next day, various cytokines were added to the dishes (day 0) and the OBM containing cytokines was changed every 3 days. After 14 days, the cells were analyzed and isolated by FACS.</p

Characterization of gene expression in TNAP-positive iPSCs.

<p>(a) Comparison of expression of ES cell markers in TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. qRT-PCR analysis of <i>OCT3/4</i>, <i>SOX2</i>, <i>NANOG</i>, <i>REX1</i>, <i>ESG1</i>, and <i>TERT</i> was performed in isolated cells using a FACSAria cell sorter. Parental iPSCs (hiPS) was used as a positive control. (b) Comparison of the expression of markers of osteoblast differentiation in TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. qRT-PCR analysis was performed in cells isolated by FACS. Parental iPSCs (hiPS) were used as a negative control and osteogenic-differentiated HPDLCs (ODH) as a positive control. (c) qRT-PCR analysis was performed with cells grown in OBM for 40 days. (d) After isolation by FACS, TNAP-positive cells were treated with vehicle (cont), 10 nM active vitamin D3 (VD10), or 50 nM active vitamin D3 (VD50) for 6 days. Abbreviations: RUNX2, runt-related transcription factor 2; TNAP, tissue-nonspecific alkaline phosphatase; COL1A1, type I collagen; OSX, osterix; BSP, bone sialoprotein; OCN, osteocalcin. The expression of these genes was analyzed by qRT-PCR, and the mRNA levels of the genes were normalized to that of <i>GAPDH</i> or 18S rRNA. The experiments were performed in triplicate. Values represent mean ± S.D. (<i>n</i>  =  4). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Characterization of ALP-positive cells derived from human iPSCs.

<p>(a) Single cells from hEBs were cultured with various cytokines for 2 weeks and stained for ALP activity. The cells were cultured in α-MEM containing 10% FBS (α-MEM); α-MEM containing 10% FBS, ascorbic acid, and β-glycerophosphate (β-GP) (OBM); OBM with FGF-2 and TGF-β1 (FGF2 + TGF); OBM with FGF-2, TGF-β1, and IGF-1 (FGF2 + TGF + IGF); OBM with FGF-2 and BMP-2/-7 (FGF2 + BMP); or OBM with FGF-2, BMP-2/-7, and IGF-1 (FGF2 + BMP + IGF). The percentages shown indicate the frequency of ALP-positive cells determined by FACS analysis. (b) FACS analysis for the isolation of ALP-positive cells (right) and isotype control (left). (c) Expression of ALP isoenzymes: germ cell-specific ALP (G), placenta-specific ALP (P), intestine-specific ALP (I), tissue-nonspecific ALP (T), and β-actin (B) in parental hiPSCs, isolated ALP-positive cells, and isolated ALP-negative cells. (d) FACS analysis of CD90 and E-cadherin in the TNAP-positive population. (e) Morphology of TNAP-positive (TNAP+) and TNAP-negative (TNAP−) cells.</p