tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Purine-containing oligonucleoside methylphosphonates may be depurinated by treatment by hydrochloric acid at 65° C. The methylphosphonate linkage is resistant to hydrolysis by these conditions as shown by the stability of d-TpT and d-TpTpT. On the other hand, dimers or trimers containing purine bases such as d-ApA, dApT, dTpA or d-ApApA have half-lives between 92 and 144 min in 0.01 M hydrochloric acid. Following neutralization with ammonium hydroxide, the products of these reactions were characterized by reversed phase HPLC. d-ApA gave adenine, while both d-ApT and d-TpA gave adenine and thymidine as the sole products of the reaction. Adenine and d-ApA were the only products observed when d-ApApA was partially hydrolyzed. When d-CpCpApT was completely hydrolyzed in acid three products, d-CpC, adenine and thymidine, were observed in a molar ratio of 1:1:1.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Cells were reprogrammed according to Example 11, and were then cultured in DMEM/F12, 100 ng/ml activin A, 25 ng/ml Wnt3a, 0.01% recombinant HSA, 1×ITSE for 1 day, followed by DMEM/F12, 100 ng/ml activin A, 0.01% recombinant HSA, 1×ITSE for 2 days, followed by DMEM/F12, 50 ng/ml FGF10, 0.25 μM KAAD-cyclopamine, 0.01% recombinant HSA, 1×ITSE for 3 days, followed by DMEM/F12, 1% B27, 2 μM all-trans retinoic acid, 50 ng/ml FGF10, 0.25 μM KAAD-cyclopamine for 4 days, followed by DMEM/F12, 1% B27, 1 μM γ-secretase inhibitor DAPT, 50 ng/ml exendin-4, 10 nM betacellulin, 10 mM nicotinamide for 2 days, followed by DMEM/F12, 50 mg/L ascorbic-acid-2-phosphate, 1% B27, 1 μM γ-secretase inhibitor DAPT, 50 ng/ml exendin-4, 50 ng/ml IGF-1, 50 ng/ml HGF, 10 nM betacellulin, 10 mM nicotinamide for 6 days to generate glucose-responsive insulin-producing cells (FIG. 5A). The resulting cells can be used in vitro or in vivo for screening bioactive molecules for the study of diabetes or for the development of therapeutics for diabetes. for 1 day for 2 days