Catalytic Environmentally Friendly Protocol for Achmatowicz Rearrangement

The increasing interest in Achmatowicz rearrangement in organic synthesis calls for a more environmentally friendly protocol since the most popular oxidants <i>m</i>-CPBA and NBS produced stoichiometric organic side product (<i>m</i>-chlorobenzoic acid or succinimide). Mechanism-guided analysis enables us to develop a new catalytic method (Oxone/KBr) for AchR in excellent yield with K₂SO₄ as the only side product, which greatly facilitates the purification. This protocol was integrated with other transformations, leading to a rapid access to the highly functionalized dihydropyranones

Influence of TSA on the expression and histone H3 acetylation of pluripotent gene in hMSCs.

<p>Passage (P)1 hMSCs were grown in the presence of TSA (at 6.25 nM) or vehicle DMSO to passage 6. (A) Real-Time PCR analysis of Oct4, TERT, Sox2, Nanog, REX1 and CD133 genes of the cells in P1 and P6 (A, n=3, *<i>P</i><0.01 when P6-DMSO compared to P1 and to P6-TSA). (B) Acetylation of histone H3 in K9 and K14 in Oct4, Sox2 and TERT genes in DMSO- or TSA-treated hMSCs (*<i>P</i><0.01 when P6-DMSO compared to P1 and to P6-TSA). The experiments were repeated three times with similar results. </p

Differentiation of hMSCs.

<p>Passage (P)1 hMSCs were grown in the presence of TSA (at 6.25 nM) or vehicle DMSO to passage 6. Then the cells were incubated in adipogenic (A), osteogenic (B) and chondrogenic (C and D) induction media, respectively. MSCs differentiated into adipocytes (A, with oil red staining), osteoblasts (B, with Alizarin Red S staining) and chondrocytes (cultured in micromass and sectioned: C, with H&E staining; D, with toluidine blue staining showing pink extracellular matrix proteoglycans). The experiments were repeated three times with similar results and representative results from one experiment were shown. </p

TSA treatment of hMSCs.

<p>Human MSCs were grown in the presence of different concentrations of TSA (0^~300 nM) for 3 days and photographed under microscope. Reprehensive images were shown (A-C). Then the cells were detached and counted. Triplet wells were used for each experiment (D, n=4, <b>*</b><i>P</i><0.01).</p

Changes of MSCs in culture.

<p>Cultured in growth medium, MSCs in passage (P) 10 were larger and fatter compared to cells in P1 (A & B). Real-Time PCR analysis showed expressional changes of genes involved in stem cell pluripotency and osteogenic differentiation (C). The experiment was repeated three time with similar results, and representative results from one experiment were shown (*<i>P</i><0.01). ALP, asalkaline phosphatase; OPN, osteopontin.</p

Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.

<p>(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** <i>P</i><0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).</p

Fluorescence-activated cell sorting (FACS) analysis of MSCs.

<p>Passage 3 MSCs were analyzed by FACS after staining with FITC- or PE-conjugated control isotype IgG (gray peaks) or antibodies against indicated cell surface proteins (filled red or green peaks).</p

Morphological changes and spontaneous estrogenic differentiation of MSCs.

<p>Cultured in growth medium, (A & B) MSCs became larger and fatter upon expansion, (C) A few cells became extremely larger and flatter and were positive (in red) for alkaline phosphatase (ALP) stain. (D) Real-Time PCR analysis showed that the expression of genes associated with osteogenesis such as collagen type I (Col I), alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN) increased progressively with cell passages. P1 to P10 represents MSC passage number. Induction indicates MSCs after incubation with osteogenic induction medium for 14 days.</p

Differentiation of MSCs.

<p>Cultured in appropriate induction media, (A & B) MSCs differentiated into adipocytes (after oil red staining, A represents non-induced and B represents induced), (C & D) osteoblasts (after Alizarin Red S staining, C represents non-induced and D represents induced), and (E & F) chondrocytes (after Alcian Blue staining, E represents non-induced and F represents induced).</p

Effects of bFGF on MSC proliferation and spontaneous differentiation.

<p>(A) Growth curves of MSCs cultured in growth medium (control) and in growth medium supplemented with bFGF (bFGF). (B) MSCs cultured in growth medium (control) or in growth medium supplemented with bFGF (bFGF) for 3 days after Alizarin Red S staining. (C) Mean proportion of Alizarin Red S positive areas per field after quantification of 4 randomly selected fields (** <i>P</i><0.01).</p